studies on the microbial degradation of homocholine …

STUDIES ON THE MICROBIAL

DEGRADATION OF HOMOCHOLINE

(~g: ~ IC: ct Q 7.1\ =E ::] IJ ~CD~~ IC: ~ -r Q fin~)

ISAM ALI MOHAMED AHMED ALI

Ph.D. Dessertation

Submitted to the United Graduate School of Agricultural Sciences, Tottori University in partial fulfilhnent of the requirements for the degree of

Doctor of Philosophy in Bioresources Sciences

2010

DEDICATION

I wou!dlllie to dedicate this humfite thesis to:

My beloved wife Khalda and my sweet son Elmujtaba

My family: Ali, Zeinab, Buthina, Fatihia, Mohamed Ahmed, Hajer, N awal, Eghbal, Entisar and Ekhlass

for their generous Love antfszg;yort

TABLE OF CONTENTS

TABLE OF CONTENTS ............................................................................................... i LIST OF FIGURES ...................................................................................................... v LIST OF TABLES ...................................................................................................... vii ABBREVIATIONS ................................................................................................... viii

CHAPTERl:GENERALINTRODUCTION 1.1 INTRODUCTION ................................................................................................... I 1.2 SYNTHETIC LONG CHAIN QUATERNARY AMMONIUM COMPOUNDS

(sQACs) ................................................................................................................. 4 1.3 NATURALLY OCCURRING QUATERNARY AMMONIUM COMPOUNDS

(nQACs) ................................................................................................................ 5 1.3.1 Choline .............................................................................................. .

1.3.2 Homocholine

1.3.3 Glycine Betaine

1.3.4 B-Alanine Betaine.

1.3.5 L-Carnitine

1.4 MICROBIAL DEGRADATION OF QUATERNARY AMMONIUM

........ 9

. ....... 9

11

COMPOUNDS .................................................................................................... 13 1.4.1 Biodegradation of Long Chain QAC by Microorganisms 13

1.4.2 Biodegradation of Naturally Occurring QAC by Microorganisms ...... . 16

1.4.2.1 Bacterial metabolism of choline

1.4.2.2 Bacterial metabolism of 4-N-trimethylamino-1-butanol... ................................................ 19

1.4.2.3 Bacterial metabolism ofL-carnitine

1.5 RESEARCH OBJECTIVES .................................................................................. 21

CHAPTER 2: SCREENING, ISOLATION AND IDENTIFICATION OF HOMOCHOLINE DEGRADING MICROORGANISIMS

2.1 INTRODUCTION ................................................................................................. 23 2.2 MATERIALS AND METHODS ........................................................................... 24

2.2.1 Materials

2.2.2 General Methods

2.2.3 Chemical Synthesis

...... 24

2.2.4 Screening and Isolation of Homocholine Degrading Bacteria ....................................... 27

2.2.5 Morphological, Biochemical and Physiological Characterization ................................. 28

2.2.6 Sequencing of 16S rRNA Gene

2.2.6.1 Polymerase chain reaction (PCR)

2.2.6.2 Agarose gel electrophoresis of DNA and visualization

2.2.6.3 PCR product purification and sequencing .......................................................................... 32

2.2.7 Phylogenetic Analysis

2.2.8 Creation of Phylogenetic Trees

2.2.9 Analytical Methods ..........

2.2.9.1 Thin layer chromatography (TLC)

2.2.9.2 Capillary electrophoresis

2. 3 RESULTS AND DISCUSSION ............................................................................ 35 2.3 .1 Screening of Homocholine Degrading Microorganisms ........... 35

2.3.2 Isolation and Characterization of Homocholine Degrading Arthrobacter sp. Strain E5

.............. 37

2.3.2.1 Morphological and physiological characteristics of strain E5 .....

2.3.2.2 16S rR.l\TA gene sequence analysis

2.3.2.3 Utilization of several organic compounds by strain

2.3 .3 Isolation and Characterization of Homocholine Degrading Rhodococcus sp. Strains

A2

2.3.3.1 Enrichment and isolation of strain A2

2.2.3.2 Morphological and physiochemical characterization of strain A2

2.3.3.2 Phylogenetic analysis of strain

2.3.3.3 Growth of strain A2 on homocholine and other structurally related compounds ............ 47

2.3.4 Isolation and Characterization of Homocholine Degrading Pseudomonas sp. Strains

A9 and B9b ............... 50

2.3.4.1 Morphological and physiological characteristics of the isolated strains

2.3.4.2 16S rRNA gene sequence analysis of strains A9 and B9b ................................................ 51

2.3.4.3 Utilization of several C.N. sources by strains A9 and B9b ............................................... 54

2.4 CONCLUSIONS ................................................................................................... 56

CHAPTER 3: DEGRADATION OF HOMOCHOLINE BY RESTING CELL SUSPENSIONS AND GROWING CELL CULUTRES OF THE ISOLATED STRAINS


3. 2.1 Materials



3.2.4 Growth and Homocholine Degradation by the Isolated Strains .......... .

3.2.5 Homocholine Degradation by Resting Cells of the Isolated Strains ............................. 62

3.2.6 Isolation and Quantification of the Metabolites ofHomocholine .................................. 62

3.2.6.1 Homocholine

3.2.6.2 Trimethylaminopropionaldehyde (TMAPaldehyde)

3.2.6.3 P-alanine betaine and trimethylamine (TMA) .................................................................... 63

3.2.6.4 Trimethylamine

3.2.7 Analytical Methods

11

3. 2. 7.1 Capillary electrophoresis

3.2.7.2 Gas chromatography-mass spectrometry (GC-MS) .......................................................... 65

3 .2. 7.3 Fast atom bombardment- mass spectrometry (F AB-MS)

3.3 RESULTS AND DISCUSSION ............................................................................ 65 3. 3.1 Identification of Homocholine Degrading Metabolites .................................................. 65

3.3.2 Degradation of Homocholine by Resting Cells of the Isolated Strains ......................... 69

3.4 CONCLUSIONS ................................................................................................... 74

CHAPTER 4: ENZIMATIC ACTIVITIES IN THE DEGRADATION PATHWAY OF HOMOCHOLINE BY THE ISOLATED STRAINS


4.2.1 Materials .... 79


4.2.3 Screening for Homocholine degrading Enzymatic Activities ........................................ 80

4.2.3.1 Plate replica staining method

4.2.3.2 Cell free extract preparation ................................................................................................ 81

4.2.3.3 NAD+-dependent homocholine dehydrogenase

4.2.3.4 NAD+ -dependent TMAPaldehyde dehydrogenase ........................................................... 82

4.2.3.5 Membrane-bound homocholine dehydrogenase

4.2.3.6 Homocholine oxidase

4.2.3.7 NAD+ -dependent 3-hydroxypropionate dehydrogenase .................................................... 83

4.2.4 Time Course of the Growth and Enzyme Formation of Pseudomonas sp. strains A9 .84

4.2.5 Stability of Homocholine Dehydrogenase Activity of Strain A9

4.2.6 Formation ofHomocholine Dehydrogenase on Various Media

4.2.7 Measurement of Protein Content

4.2.8 Measurement of Molecular

............... 84

................. 85

.86

4.2.9 Polyacrylamide Gel Electrophoresis ................................................................................ 86

4.2.1 0 Intact and Dry Cell Reaction of Pseudomonas sp. Strain A9

4.2.11 Detection of 3-Hydroxypropionate by LC/MS/MS

4.3 RESULTS AND DISCUSSION ............................................................................ 89 4.3.1 Screening for Homocholine degrading Activity ....... 89

4.3.2 Time Course of the Growth and Enzyme Formation of Pseudomonas sp. Strains A9 90

4.3.3 Formation ofHomocholine Dehydrogenase on Various Media

4.3.4 Stability ofHomocholine Dehydrogenase of Pseudomonas sp. Strain A9

4.3.5 Intact and Dry Cell Reaction of Pseudomonas sp. Strain A9

4.3.6 Substrate Specificity of Homocholine Dehydrogenase

.94

4.3. 7 Measurement of the Native Molecular Mass of Homocholine Dehydrogenase ......... 10 1

4. 3. 8 Detection of 3 -hydroxypropionate Dehydrogenase Activity 102

4.4 CONCLUSIONS ................................................................................................. 105

111

CHAPTER 5: GENERAL CONCLUSIONS ......................................................... 107

REFERENCES ......................................................................................................... 111

ACKNOWLEDGMENT ........................................................................................... 127

ABSTRACT ............................................................................................................. 129

iii~ .......................................................................................................................... 133

LIST OF ORIGINAL PUBLICATIONS ................................................................... 135

lV

Fig. 1. 1 Metabolic fate of choline

Fig. 1. 2 Metabolism of choline

LIST OF FIGURES

Fig. 1. 3 The synthetic pathway to ~-alanine betaine in plant.... 10

Fig. 1. 4 Function of L-camitine in fatty acid metabolism ............................................................. 11

Fig. 1. 5 General degradation pathway of cationic surfactants ...................................................... 15

Fig. 1. 6 Choline oxidation pathway 18

Fig. 1. 7 Degradation pathway of 4-N-trimethylamino-1-butanol in Pseudomonas sp. 13 CM ... 20

Fig. 2. 11 H NMR spectrum of chemically synthesized 3 -N-trimethylamino-1-propanol ............ 26

Fig. 2. 2 Histogram showed the cell growth of isolated microorganisms.

Fig. 2. 3 Morphology of Arthrobacter sp. strain E5 as seen by light microscopy under

optical and phase contrast conditions ........ .

Fig. 2. 4 Phylogenetic tree based on 16S rRNA sequence showing the relationship between

strain E5 and most closely related species of the genusArthrobacter ............................. 41

Fig. 2. 5 Morphology of Rhodococcus sp. strain A2 as seen by phase contrast microscopy and

transmission electron microscopy

Fig. 2. 6 Phylogenetic tree based on 16S rRNA sequence showing the relationship between

strain A2 and most closely related species of the genus Rhodococcus . ......................... .47

Fig. 2. 7 Time course study of the growth and metabolism of homocholine by Rhodococcus sp.

strain A2 ................................................................................................................................... 49

Fig. 2. 8 Morphology of isolated strains, Pseudomonas sp. strain A9 and B9b as seen by phase

contrast microscopy and transmission electron microscopy ................................................ 51

Fig. 2. 9 Phylogenetic tree based on 16S rRNA sequence showing the relationship between the

isolated strains A9 and B9b and most of the closely related P~eudomonas species ......... 53

Fig. 3. 11 H NMR spectrum of chemically synthesized 3 -N-trimethylamino-1-propionic acid (~

Alanine betaine)

Fig. 3. 2 Capillary electrophoresis chromatogram showing the generated metabolite peaks from

the degraded homocholine by the isolated strains alongside with authentic standards peaks

of trimethylamine and ~-alanine betaine

Fig. 3. 3 GC-MS spectra of the metabolite trimethylamine formed during the degradation of

homocholine by the isolated strains ..................................................................................... 68

Fig. 3. 4 F AB-MS spectra of metabolites in the degradation pathway of homocholine by the

isolated strains ......................................................................................................................... 69

v

Fig. 3. 5 Time course degradation of homocholine and the generation of the metabolites,

trimethylamine and beta-alanine betaine by strains E5, A2 and A9

Fig. 3. 6 Proposed pathway for the aerobic biodegradation of homocholine by the isolated

strains

Fig. 4. 1 Bovine serum albumin standard curve as determined by Lowry method ................... 86

Fig. 4. 2 Screening of NAD+ -dependent dehydrogenase activity in the isolated strains by (A)

replica staining and (B) spectrophotometric assays ........... .

Fig. 4. 3 Time course of the growth and homocholine dehydrogenase activity formation of

Pseudomonas sp. strain

Fig. 4. 4 Effect of stabilizers on the stability of homocholine dehydrogenase of

Pseudomonas sp. strain A9 ................................................................................................... 96

Fig. 4. 5 Evaluation of the stability ofhomocholine dehydrogenase during storage at 4°C ........ 96

Fig. 4. 6 Degradation ofhomocholine by resting cells of Pseudomonas sp. strain A9 ................ 97

Fig. 4. 7 Degradation ofhomocholine by dried cells of Pseudomonas sp. strain A9 ................... 98

Fig. 4. 8 Native-PAGE analysis of crude homo choline dehydrogenase ..................................... 10 1

Fig. 4. 9 Estimation of the native molecular mass of homocholine dehydrogenase by size

exclusion chromatography on TSK-gel G3000 SW column ............................................... 102

Fig. 4. 10 Native-PAGE analysis of crude 3-hydroxypropionate dehydrogenase ...................... 103

Fig. 4. 11 LC/MS/MS analysis of 3 -hydroxypropionate in the degradation pathway of

homocholine by the intacted cells of Pseudomonas sp. strain A9 ...................................... 104

Fig. 4. 12 Proposed degradation pathway of homocholine by Pseudomonas sp. strain A9 ...... 1 06

Vl

LIST OF TABLES

Table 1. 1 Structural features of synthetic sQACs and their industrial usage

Table 2. 1 Basal-homocholine (basal-HC) liquid medium

Table 2. 2 Basal-homocholine (basal-HC) agar medium ............................................................... 28

Table 2. 3 Nutrient broth (meat extract) media

Table 2. 4 Primers used in PCR amplification

Table 2. 5 Morphological and physiological characteristics of Arthrobacter sp. strain E5 ....... .42

Table 2. 6 Morphological and physiological characteristics of Rhodococcus sp. strain A2 ...... .44

Table 2. 7 Utilization of homo choline and other structurally related compounds by

Rhodococcus sp. strain A2 ..................................................................................................... .49

Table 2. 8 Phenotypic characteristics of Pseudomonas sp. strains A9 and B9b .......................... 55

Table 4. 1 Protease inhibitor cocktail

Table 4. 2 Reaction mixture for activity staining on native-PAGE gels

Table 4. 3 Formation of Homo choline dehydrogenase on various media

Table 4. 4 Substrate specificity of homocholine dehydrogenase from strain A9 ....................... 1 00

Table 4. 5 NAD+-dependent 3-hydroxypropionate dehydrogenase activity in the cell extracts of

homocholine-, choline- and glucose-growing cells 103

Vll

~-AB

Basal-He

CTAB

DCIP

DMA-Propanol

DNPH

DTT

FAB-MS

GC-MS

GMC

MLA

nAChR

nQACs

NTB

PMS

sQACs

TMA

TMABA

TMA-Butanol

TMAP

TMAPaldehyde

QACs

ABBREVIATIONS

~-Alanine betaine

Basal-homocholine medium

Cetyl trimethyl ammonium bromide

2,6-dichlorophenol-indophenol

3-N-dimethy larnino-1-propanol

2, 4-dinitropheny hy drazine

Dithiothreitol

Fast atom bombardment- mass spectrometry

Gas chromatography-mass spectrometry

Glucose-methanol-choline oxidoreductases

Methyllycaconitine

Nicotinic acetylcholine receptor

Naturally occurring quaternary ammonium compounds

Nitro tetrazolium blue

Phenazine methosulfate

Synthetic quaternary ammonium compounds

Trimethy I amine

4-N-trimethylamino-1-butyraldehyde

4-N-trimethylarnino-1-butanol

3-N-trimethylarnino-1-propanol (homocholine)

Trimethylarninopropionaldehyde

Quaternary ammonium compounds

Vlll

CHAPTERl

GENERAL INTRODUCTION

1.1 INTRODUCTION

Quaternary ammonium compounds (QACs) are organic compounds that contain four

functional groups attached covalently to a positively charged central nitrogen atom

(~N+). These functional groups (R) include either long chain alkyl, benzyl or methyl

groups. The term quaternary ammonium compounds refer to either chemically

synthesized long-chain quaternary ammonium compounds (sQACs) or naturally

occurring quaternary ammonium compounds (nQACs). Long-chain sQACs have been

important since their bactericidal properties were recognized in the 1930s. Since then,

they are extensively used in domestic and industrial applications as surfactants,

emulsifiers, fabric softeners, disinfectants and corrosion inhibitors (Garcia et al., 2004;

Patrauchan and Oriel, 2003). Such extensive use of sQACs in various branches of

industry has resulted in increased presence of these compounds in wastewater,

waterways, lakes, sludges and soils (Gerike et al., 1978). These compounds are toxic to

aquatic plant and animal organisms. With their surface-active properties, they facilitate

solubility of many dangerous rnicropollutants, such as pesticides, of which toxins can

easily penetrate living organisms (Grabinska-Sota 2004). On the other hand, nQACs

are widely distributed in the biosphere: there are more than 100 reported examples

including well-known representatives such as choline, glycine betaine and ~-alanine

1

betaine (Anthoni et al., 1991) which have different biological functions, such as

adaptation of organisms to environmental stress and transportation of chemical groups

in many metabolic processes (Anthoni et al., 1991). Choline is an essential nutrient that

is widely distributed in foods, principally in form of phosphatidylcholine. Choline or its

metabolites are required for synthesis of phospholipids in cell membranes, methyl group

metabolism and cholinergic neurotransrnission and transmembrane signaling, as well as

for lipid-cholesterol transport and metabolism (Fig. 1.1). This quaternary amme IS

present in tissues predominantly as one of the choline-phospholipids such as

phosphatidylcholine and sphingomyelin (Zeisel et al., 1994). The osmoprotective role

of glycine betaine, which derived from choline via an oxidation reaction, is evident in a

number of diverse microbial systems, including enteric bacteria (Andresen et al., 1988),

soil bacteria (Smith et al., 1988), halophilic bacteria (Galinski et al., 1982),

cyanobacteria (Mackay et al., 1984) and methanogenic archaebacteria (Robertson et al.,

1990). Besides being an osmoprotectant, glycine betaine also has a role in general

metabolism where one methyl group of glycine betaine is incorporated into methionine

in mammals (Finkelstein et al., 1972, Millian et al., 1998) and microorganisms (White

et al., 1971), or into cobalamin (Vitamin B12) in microorganisms (White et al., 1971).

Recently, glycine betaine was proven to be an effective cryoprotectant for a wide range

of prokaryotic organisms during freeze-drying, liquid-drying and liquid nitrogen

freezing (Cleland et al., 2004). L-Camitine a trimethylated amino acid that is similar in

structure to choline is highly polar zwitterionic quaternary amine carboxylic acid and

present in some prokaryotes and all eukaryotes (Ramsay et al., 2001). In prokaryotic

cells, L-carnitine serves either as a nutrient, such as a carbon and nitrogen source

(Kleber, 1997), or as an osmoprotectant (Jung et al., 1990; Robert et al., 2000).

However, in eukaryotic cells, it serves exclusively as a carrier of acyl moieties through

2

various subcellular compartments (Ramsay et al., 1993). Moreover, it has an important

role in the transport of activated long-chain fatty acids across the inner mitochondrial

membrane (Bremer, 1983). 3-N-Trimethylamino-1-propanol (homocholine) an analogue

of choline, in which the amino alcohol group is lengthened by one CH2-group, has been

shown to resemble choline in many aspects of cholinergic metabolisms (Boksa and

Collier, 1980). It has been reported that when homocholine transported into the rat

brain synaptosome, it acetylated and released as acetylhomocholine from a superior

cervical ganglion and minces of mouse forebrain by a calcium-dependent process

during depolarization (Collier et al., 1977; Carroll and Aspry, 1980). Moreover, it was

found to be effective in preventing fat infiltration both in fat and cholesterol fatty livers

(Channon et al., 1937). From the choline-like structure, one would expect that

homocholine is degraded similarly and at a rate comparable to that of choline. To date,

no microorganism degrading homocholine as a sole source of carbon and nitrogen has

been isolated and consequently, this catabolic pathway remains unclear.

~<,

<:• H,•: --0·---C --r.~ CH,:OPO,CHPfl;t"{CH~' I I ~---<1-(,-.t..o-tH <) ,' ''<, in

! !I ! 1 ~ ! NH

phospha;;~~~dl~~~~"~-,Hz-t,•-·. -~ o f S=uctural membrane phospholipids

HO,_ ,' H, ' I_,CH; • ·p~ C. '.,..N~

phosphochohne,jl . o· -~ 'c><,.

\~.c;) ···t/ Neurotransmitterlr----1&w.aw =~?--~ethyl group donor 1 'm • ·- m1 o

0

1• Crls , II / i

'y~ ~~ ~ I _.-CHs 10Sif1 ' H,c.._ ";"~\ ~ ll C , N'+ , I \

1

' ·c :

0

acetylcholine II HO/ -......._c"(' ~GH, / Jl "-" ,/ >;U

· ~ ch~li~e 11 6etaine

platelet-activating fac~pr ;'"!l

Signaling phospholipids

sphingosylphosphorylcholine

Fig. 1. 1 Metabolic fate of choline (Degani et al., 2006)

3

1.2 SYNTHETIC LONG CHAIN QUATERNARY AMMONIUM

COMPOUNDS (sQACs)

Synthetic quaternary ammonium compounds (sQACs) containing a long chain alkyl

group or a benzyl group are cationic surfactants (Table 1.1) that play an important role

in many industrial fields due to their versatile physico-chemical properties. Such

widespread uses as disinfectants, fabric softening agents, foam depressants, and

antistatic agents lead to massive discharge into the environment with its associated

concerns (Wee and Kennedy, 1982).

Potential risks have been reported in many previous studies that repeated exposure

to long-chain sQACs can induce microbial resistance against antibiotics in many

pathogenic microorganisms (McDonnell and Russell, 1999). In addition, discharge of

long-chain sQACs can disturb the purifying activities of natural aquatic systems or

public wastewater treatment plants because of their toxicity to microbial life

(Laopaiboon et al., 2002; Tubbing and Admiraal, 1991). The possibility to eliminate

sQACs-related pollution effectively has been investigated in many ways. As one of the

most economical means, biological treatment found to be an effective way to remove

sQACs. In particular, aerobic treatment processes can provide rapid biodegradation via

a consortium of bacteria (Scott and Jones, 2000).

4

Table 1. 1 Structural features of synthetic sQACs and their industrial usage

QAC Group

Alkyl trimethyl ammonium

Dialkyl dimethyl ammonium

Alkyl benzyl dimethyl ammonium

Alkyl pylidinium

Dierhylester dimethyl ammonium (Esterqn::1t)

Molecular Structure

CH,

1 x-R-~....,.-cH3

I CH3

CH,

I ~-R-l'r-cH, I -

R

CH-

I ~- --R-::~--cri;

I H''D R

x·

0

II CH,cH,OCR I - -

CE,-N""-CH, . x·l ---CH,CH,OCR

- - II 0

ApplicatiollS

Orgnnoclays, phase tran;;fer catalyst,

oilfield application>.

Biocides. wood preservatives. oilfield applications

Biocide::,. cosmetics. wood preservation.

phase-transfer catalyst. organoclnys

Plw~e transfer cataly,t. pesticides

Active ingredient in falnic softeners

1.3 NATURALLY OCCURRING QUATERNARY AMMONIUM

COMPOUNDS (nQACs)

Naturally occurring quaternary ammonium compounds constitute a class of metabolites

with more than 100 reported examples, such as choline, glycine betaine, ~-alanine

betaine, y-butyrobetaine and L-camitine (Anthoni et al., 1991). They form a structurally

heterogeneous class of compounds with a unifYing character of a polar and fully methyl

substituted nitrogen atom, creating a permanent positive charge on the N moiety

5

(Rhodes and Hanson, 1993). Their occurrence apparently reflects several independent

evolutionary patterns ranging from highly specialized functions for each nQAC to a

general strategy for adaptation of the organisms to fluctuating or stressing

environmental conditions (Anthoni et al., 1991). Moreover, it has been suggested that

nQACs may be involved in transporting various chemical moieties within plants, acts as

macromolecular components, and have role in overcoming water and salt stresses

(Blunden and Gordon, 1986). In addition, the presence or absence of these compounds

could be used as taxonomic indicator as well as they could used in the pharmacology to

prevent artherosclerosis (Hoppe, 1979), to reduce plasma cholesterol levels (Kaneda

and Abe, 1984), smooth muscle relaxant (Baker and Murphy, 1976) and to inhibit

mammalian neuromuscular transmission (Hosein and McLennan, 1959).

1.3.1 Choline

Choline is an essential nutrient that is widely distributed in foods, principally in the

form of phosphatidylcholine. Organ meats, eggs, soybeans, nuts, fish and broccoli are

particularly good sources (Howe et al., 2004). The only source of choline other than

diet is de novo biosynthesis of phosphatidylcholine from phosphatidyl-ethanolamine

(Zeisel, 1990). Choline is required to make the phospholipids such as

phosphatidylcholine, lysophosphatidylcholine, choline plasmalogen and sphingomyelin

essential components of all cell membranes. It is a precursor for the biosynthesis of the

neurotransmitter acetylcholine and in biological methylation reactions. Choline

phospholipids in cell membranes play a role in generating various second messengers

during signal transduction (Zeisel, 1990; Zeisel and Blusztajn, 1994). Choline can be

acetylated, phosphorylated and oxidized (Fig. 1.2). Only a small fraction of dietary

choline is acetylated by choline acetyltransferase (EC.2.3.1.6) (Zeisel, 1990; Zeisel and

6

Blusztajn, 1994). This enzyme is highly concentrated in the terminals of cholinergic

neurons, but also present in non-nervous tissue such as the placenta. Phosphorylation

of choline is the first step in the mqjor pathway for phosphotidylcholine synthesis,

catalyzed by choline kinase (EC. 2.7.1.32), using Mi+ and ATP. This enzyme is

widely distributed in mammalian tissues, including liver, brain, kidney and lung.

Choline deficiency results in liver dysfunction, fatty liver, decreased in growth,

infertility, Alzheimer's disease, abnormal kidney function, decreased red blood cell

synthesis, high blood pressure and liver cancer (Zeisel, 1990; Zeisel and Canty, 1993;

Zeisel and Blusztajn, 1994).

S-adenosyl-homocysteine

Dimethylglycine Phosphatidylethanolamine

Methionine __..,. S-adenosylmethionine

Homocysteine

Betaine .. Betaine aldehyde dehydroge:;Jase

Betaine aldehyde

! Trimethylamine

.... Choline

dehydrogenase

Phosphatidylcholine

i CDP-choline :diacylglycerol cholinephosphotransfease

CDP-choline

i CTP :phosphocholine cytidy!ytram:.ferase

Phosphocholine

i Choline

Choline .l.jnase

--IIl-li- Acetylcholine Choline

acetyltrar1.lerase

Fig. 1. 2 Metabolic pathway of choline (Hassan, 2008)

1.3.2 Homocholine

Homocholine (3-N-trimethylamino-1-propanol) an analogue of choline, in which the

amino alcohol group is lengthened by one CH2-group, has been shown to resemble

choline structurally and in many aspects of cholinergic metabolisms (Boksa and Collier,

7

1980). After it is transported into the rat brain synaptosome, it is acetylated and

released as acetylhomocholine from a superior cervical ganglion and minces of mouse

forebrain by a calcium-dependent process during depolarization (Collier et al., 1977;

Carroll and As pry, 1980). Homocholine, however, differs dramatically from choline in

a very important aspect. That is not acetylated by solubilized cholineacetyltransferase,

but is acetylated by intact rat brain synaptosomes (Collier et al., 1977), possibly because

homocholine is acetylated by cholineacetyltransferase that is associated with vesicular

and/or neuronal membranes (Carroll and Aspry, 1980). Nelson et al. (1980) assumed

that the extracellular precursors of choline and homocholine may be directly

accumulated by a crude vesicular fraction of mouse forebrain, independently of the

cytoplasm, and may be utilized to replace the loss in acetylcholine. Additionally, they

suggested that the extracellular products, acetylcholine and acetylhomocholine, may not

be capable of replacing acetylcholine lost from a crude vesicular fraction. Moreover,

acetylhomocholine is released from brain slices both spontaneously and in response to

stimulation via mechanism similar to those that released acetylcholine, but some

differences in specificity of the acetylcholine storage and/or release processes might be

present (Nelson et al., 1980). Previous studies on acetylhomocholine synthesis and

release have partly classified these false transmitter criteria by showing that

homocholine is acetylated in tissues containing cholinergic nerve terminals (Collier et

al., 1977), that acetylhomocholine is released by a calcium-dependent mechanisms

during nerve stimulation (Collier et al., 1977), and that acetylhomocholine has actions

qualitatively similar to but quantitatively different from those of acetylcholine on both

muscarinic and nicotinic receptors (Hunt and Renshaw, 1934; Curtis and Ryall, 1966;

Barrass et al., 1970). In pharmacology, choline analogues such as homocholine,

methylcholine and ethylcholine are used to study the chemical specificity of the various

8

processes involved in neurotransmission and in particular it has been used to investigate

the subcelular origin of the transmitter released by stimulation.

1.3.3 Glycine Betaine

Glycine betaine is found in microorganisms, plants and animals, and is a significant

component of many foods including wheat, shellfish, spinach and sugar beets (Craig,

2004). Glycine betaine is compatible osmolyte that increases the water retention of

cells, replaces inorganic salts and protect intracellular enzymes against osmotically

induced or temperature induced inactivation. In human, glycine betaine is catabolized

via a series of enzyme reactions that occur mainly in the mitochondria of liver and

kidney cells. Glycine betaine also acts as a catabolic source of methyl groups via

transmethylation for use in many biochemical pathways. The formation of methionine

from homocysteine can occur either via glycine betaine or 5-methyltetrahydrofolate.

The conversion of homocysteine to methionine is important to conserve methionine,

detoxify homocysteine and produce S-adenosylmethionine (Millian et al., 1998).

Glycine betaine is synthesized by a two-step oxidation of choline or by methylation

reaction from glycine. Two N-methyltransferases genes from halotolerant

cyanobacteriumAphanothece halophytica have been isolated (Wadi tee et al., 2003).

1.3.4 P-Alanine Betaine

Many plants, bacteria, and marine algae accumulate quaternary ammonium compounds

(QACs) in response to abiotic stresses such as drought and salinity (Rhodes and

Hanson, 1993; Gorham, 1995). Zwitterionic compounds such as glycine betaine, ~

alanine betaine and proline betaine, are known to be the most effective osmoprotectants

9

(Yancey, 2005). In most members of the highly stress-tolerant plant family

Plumbaginaceae, B-alanine betaine was reported to replace glycine betaine (Hanson et

al., 1991, 1994). B-alanine betaine synthesis is not constrained by choline availability,

because it is derived by theN-methylation of the non-protein amino acid, B-alanine (Fig.

1.3). Unlike glycine betaine synthesis, B-alanine betaine synthesis does not require

oxygen, and hence it was suggested to be suitable for osmoprotection under saline and

hypoxic conditions (Hanson et al., 1994; Rathinasabapathi, 2000). Accordingly, B-

alanine betaine was distributed among species of the Plumbaginaceae, adapted to a

wide range of adverse stress environments including saline and hypoxic conditions

(Hanson et al., 1994). Although B-alanine betaine accumulation has been intensely

studied for its role in osmoprotection (Hanson et al., 1994; Rathinasabapathi et al.,

2000, 2001 ), early work on this compound in marine algae also suggested that it had a

cholesterol-reducing effect in animal feeding experiments (Abe and Kaneda, 1973).

1~:\Tan.ineJ

'\; -M~Ihyl P-alaninc

N.N-Dimclhyl P-alanine

N. N,N-Tnm<:thyl P-alaninc: =~-Alanine betaine

H.N~COOII

l .\' ·<l<it'IW\ l'f .f. ·IIJ('flltrJJII/1<' dt'f'<'l!ci<.•tlf

\'-mcFhl·lrrarrs[f'rc/sc

II C ' 's~lUOH H

l S ·mkmHrl·l. ·mcrhwmnc dt'f'll'ndem S-merhlltnm.l{i:ra.w

II,C, ~COUll H

1C/N

j S·adt•Jiold·l.·lllc'tlltmll/1<.' dc'f)Cfld<'lll + .\'-mctln·lcraru{erasc

II,C tr;e.=::N~coo H,C/

Fig. 1. 3 The synthetic pathway to B-alanine betaine in plant (Duhaze et al., 2003)

10

,.

1.3.5 L-Camitine

A trimethylated amino acid roughly similar in structure to choline, L-carnitine is a highly

polar zwitterionic quaternary amine carboxylic acid present in some prokaryotes and all

eukaryotes (Ramsay et al., 2001). In prokaryotic cells, L-carnitine serves either as a

nutrient, such as a carbon and nitrogen source (Kleber, 1997), or as an osmoprotectant

(Jung et al. , 1990; Robert et al. , 2000). However, in eukaryotic cells, it serves

exclusively as a carrier of acyl moieties through various subcellular compartments

(Ramsay et al. , 1993). It has an important role in the transport of activated long-chain

fatty acids across the inner mitochondrial membrane (Bremer, 1983). Cytosolic long-

chain fatty acids, which are present as CoA esters, are transesterified to L-carnitine at

the mitochondrial outer membrane (Fig. 1.4).

0

II S-CoA R-C -Fatty acyl ~~

Co A

cooe I CH, I -

HS- CoA

cooe I

0 CH, II I -

HO-C-H R-C-0-C-H I

C:H, I -

<i!> N(CH,J ,

L-Camitine

I CH, I -

<XlN (CH J 3

Acylcarnitine

"l ) r-

( LC1~3L'''"'J 0 II

~- Curnitine ~ J/' =-~y ltransft>rn.~ "'-.

R-C - S-CoA [!

HS-CoA

Fnlly acyl CoA

!NTERMEMBRANE SPACE

MATRIX

Fig. 1. 4 Function ofL-carnitine in fatty acid metabolism (Nelson and Cox, 2004)

11

In this reaction, the acyl moiety of the long chain fatty acids is transferred from

CoA to the hydroxyl group of carnitine. The resulting long chain acylcarnitine esters are

transported over the inner mitochondrial membrane via a specific carrier, camitine

acylcamitine translocase. In the mitochondrial matrix, the enzyme carnitine

acetyltransferase is able to reconvert short and medium chain acyl-CoAs into

acylcamitines using intrarnitochondrial carnitine. Through this mechanism of reversible

acylation, carnitine is able to modulate the intracellular concentrations of free CoA and

acyl-CoA. Furthermore, it is involved in the transfer of the products of peroxisomal B -

oxidation, including acetyl-CoA, to the mitochondria for oxidation to C02 and H20 in

the Krebs cycle (Jakobs and Wanders, 1995).

In humans, approximately 75% of body L-carnitine sources comes from the diet

and 25% from de novo biosynthesis (Tein et al., 1996). In general, L- carnitine is low in

foods of plant origin and high in animal foods (Rebouche, 1992). L-Carnitine ultimately

is synthesized from lysine and methionine. L-Lysine provides the carbon chain and

nitrogen atom of carnitine, and L-methionine provides the methyl group. In the

pathway of L-camitine in mammals, 8-N-trimethyl-L-lysine is synthesized only as a

post-translational modification of protein synthesis and is released for L-carnitine

biosynthesis by normal processes of protein turnover. A number of proteins contain

one or more 8-N-trimethyl-L-lysine residues, including histones, actin, myosin, and

calmodulin. The majority of 8-N-trimethyl-L-lysine synthesized in the body probably

is formed in skeletal muscle (Rebouche, 1992).

12

1.4 MICROBIAL DEGRADATION OF QUATERNARY

AMMONIUM COMPOUNDS

1.4.1 Biodegradation of Long Chain QAC by Microorganisms

The susceptibility of cationic surfactants to biodegradation was first reported in the

1950s. Gerike (1982) confirmed the study done before using activated sludge and cetyl

trimethyl ammonium bromide (CTAB), dodecyl benzyl dimethyl ammonium chloride

and didecyl dimethyl ammonium chloride.

Studies on the biodegradation pathways have been conducted for various sQACs.

The utilization of tetramethyl ammonium chloride by Pseudomonas sp. was described

by Hampton and Zatman (1973). According to their work, the oxidation of this

compound is initiated by splitting the C-N bond, therefore producing methanal and

trimethylamine. The intermediate trimethylamine is oxidized to trimethylamine N-oxide

(Large et al., 1972) or transformed to methanal and dimethylamine (Colby and Zatman,

1973; Meiberg and Harder, 1978). The trimethylamine N-oxide is further converted to

dimethylamine (Large, 1971; Myers and Zatman, 1971), followed by the generation of

methanal and methylamine (Colby and Zatman, 1973). Methylamine is then converted

to methanal and ammonium. Based on an investigation of the utilization of alkyl

trimethyl ammonium bromides by microorganisms derived from sewage and soil, Dean

Raymond and Alexander (1977) demonstrated that the first step of degradation occurs

with hydroxylation of the terminal carbon of the alkyl group and the resulting carboxylic

acid undergoes P-oxidation. Another study dealing with hexadecyl trimethyl ammonium

chloride suggested a central fission of Calky1-N bond as the first step of degradation (Van

Ginkel et al., 1992). Not only hexadecyl trimethyl ammonium chloride, but also

13

hexadecanal and hexadecanoate produced as metabolites, were all utilized by

Pseudomonas species. It is believed that the central Calkyi-N bond fission is first carried

out by an oxygen dependent dehydrogenase as suggested for alkyl trimethyl ammonium

compounds (Van Ginkel et al., 1992) and then intermediates are formed consecutively

by N-demethylation reactions.

In contrast to the previous findings, a recent study proposed the degradation of

dodecyl trimethyl ammonium chloride by Pseudomonas sp. strain 7-6, isolated from a

wastewater treatment plant, via dual pathways, which besides an initial attack on the

Caikyi-N bond also initiates degradation via cleavage of a Cmethy1-N bond, hence producing

dodecyl dimethylarnine as an intermediate product (Takenaka et al., 2007).

Overall, general degradation pathways of cationic sw-factants shown in Fig. 1.5

indicated that biodegradation is mostly commenced with cleavage of the Calkyi-N bond

irrespective of the type of sQACs and the degradation of the produced alkanals

proceeds via B-oxidation for complete mineralization (Van Ginkel, 2004).

On the other hand, because of the sw-factants character and the limited enzymatic

ability of individual microorganisms, only a few known surfactants, alkane sulphonates,

alkyl sulphates, and alkyl arnines, are completely degraded by a single microorganism

(Van Ginkel, 1996). For this reason, consortia of microorganisms are highly efficient, as

well as necessary for the complete degradation of surfactants (Scott and Jones, 2000;

Van Ginkel, 1996). The lack of the isolation of microbes degrading quaternary

ammonium surfactant to completion may be due to the fact that in all those studies

nitrogen was provided in form of ammonium in addition to the nitrogen-containing

14

surfactants. In this way, no selective pressure to use nitrogen deriving from the

surfactant itself was imposed during enrichment and isolation.

(CI-b}~OH I

CH,(CI-J.,)c-N -' - . \

(C:I-b)...OH

(0-l,.):,OH '

(O-I£1:PH

l:!-b H;C~CH.::),-f\j

\

CH3

;;,~

\ ,)

CH., I ~

%(CH.::l. -~.t-(Cl-h)CH3

CH3

0 /f

CH3(C~t:-1-C\ H

~ 0

/f CH3(CH2)x-1- C~

(JH

t CC~+~O

CH., ,.=-... I ~ I \

1--1f''(1-H-N'"-Cl-h-<x\ /; , '3 -·\- - . I \L__5f

CI-t,

CH.,

\4-CH,J~·) c~ ... '\__j

{:::1-1:3 I

Clll( / ~C(CH.:;),-r-Cf-1.;

l"E:J·. a-lz a-~z-rJ::ris :. \

C!:-!:,1

Fig. 1. 5 General degradation pathway of cationic surfactants.

The alkanals formed through the oxidation of the a.-carbon of the alkyl chain are further metabolized via ~-oxidation (Van Ginkel, 2004).

15

1.4.2 Biodegradation of Natur-ally Occurring QAC by Microorganisms

1.4.2.1 Bacterial metabolism of choline

Choline is found ubiquitously in nature and the ability to degrade choline is widespread

amongst microorganisms. The aerobic degradation of choline was firstly reported in

. Achromobacter cholinophagum that metabolized choline via betaine as first oxidation

products (Shieh 1964). Subsequently, Kortstee (1970) showed that many

microorganisms of the genera Agrobacterium, Arthrobacter, Micrococcus,

Pseudomonas, Rhizobium and Streptomyces were found to grow with choline as a sole

source of carbon and nitrogen. He also found that all the tested choline-utilizing bacteria

were able to grow with betaine, N, N-dimethylglycine or sarcosine as a sole carbon and

nitrogen source.

In microorganisms, the degradation of choline proceeds by stepwise oxidation of the

ethanol-group yielding betaine aldehyde and glycine betaine (Fig. 1.6). Thereafter,

glycine betaine is degraded by subsequent splitting off the methy 1-groups, leading to the

metabolites dimethylglycine, sarcosine and finally glycine (Shieh, 1964; Kortstee, 1970;

Mori, 1981). In plants such as spinach (Rathinasabapathi et al., 1997), sugar beet and

amaranth (Russell et al., 1998), the first step reaction is catalyzed by choline

monooxygenase (EC.l.14.15. 7), a ferredoxin-dependent soluble Rieske-type protein,

whereas in animals and microorganisms, it is catalyzed by either membrane-bound

choline dehydrogenase (EC.l.l.99.1) (Nagasawa et al., 1976; Landfald and Strom, 1986;

Russell and scope, 1994; Tsuge et al., 1980) or soluble flavoprotein, choline oxidase

(EC.l.l.3.17) (lkuta et al., 1977; Yamada et al., 1977; Rozwadowski et al., 1991).

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via

betaine aldehyde as intermediate (Ikuta et al., 1977). Based on amino acid sequence

16

compansons, the enzyme can be grouped in the glucose-methanol-choline flavin

dependent (GMC) oxidoreductase enzyme superfamily, which comprises enzymes like

glucose oxidase, cholesterol oxidase, or cellobiose dehydrogenase that utilizes FAD as a

cofactor for catalysis and non-activated alcohols as a substrate. The highly GC rich

codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter

globiformis and expressed to high yield in Escherichia coli (Fan et al., 2004). The

resulting recombinant enzyme was highly purified and showed to be a dimmer of

identical subunits containing covalently bound FAD.

In the second step, the oxidation of betaine aldehyde is catalyzed by a soluble,

NAD(Pt-dependent betaine aldehyde dehydrogenase (EC.l.2.1.8) that has been

purified to homogeneity from Pseudomonas aeruginosa (Nagasawa et al., 1976), E. coli

(Falkenberg et al., 1990), Xanthomonas translucens (Mori et al., 1992), Arthrobacter

globiformis (Mori et al., 2001) and Cylindrocarpon didymum (Mori et al., 1980).

Betaine-homocysteine methyltransferase is an enzyme catalyzing methyl

transfer reaction from glycine betaine to homocysteine, forming dimethylglycine and

methinonine, respectively. Betaine-homocysteine methyltransferase (EC.l.2.1.8) has

been purified to homogeneity from rat (Lee et al., 1992), pig (Garrow, 1996), human

(Skiba et al., 1982) and cyanobacteria (Waditee and Incharoensakdi, 2001). It is a zinc

metalloenzyme consisting of a hexamer of 45 kDa subunits and is active in the livers and

kidneys of humans and pigs, but only in the livers of rats (Mckeever et al., 1991;

Millian and Garrow, 1998). Following the betaine-homocysteine methyltransferase

reaction, the last two reactions of choline oxidation convert dimethylglycine to glycine

via sarcosine as an intermediate. The oxidative demethylation of dimethylglycine to

17

form sarcosine is catalyzed by dimethylglycine oxidase (Mori et al., 1980) or membrane

bound dimethylglycine dehydrogenase (Porter et al., 1985). Subsequently, the

formation of glycine from sarcosine is catalyzed by the mitochondrial enzyme sarcosine

dehydrogenase (Porter et al., 1985) or sarcosine oxidase (Frisell, 1971; Mori et al.,

1980).

( CH3)::.t.,-~ CH2CH.::OH

Choline

1 Choline deilydrogcuos·e Cholillt' OYidL"t.st

(CH3)3"!'(-CH:;:CHO

Betaine aldehyde l Bttotnc aldeilydc deilnirogena''"

(CH3}3~-CH2COOH

Glydnebeta ine SH( CH.::)~?'\H:1CHCOOH

Homocystei11e --~"

""'-,\

J B~.'taiiiC-hoi!!Oc:1·.sr(i•w il!erin·Tn·(l'i.Sfera.sc

CH,S( CH2)2~H2CHCOOH

~'Iethionine

/ k/

(CH3)2~CH2CC)OH

Dimethylglydne

i CH.,);'CH::COOH

Snrco::,ine

l H2::-.JCH.~COOH

Glycine

Dif!J(•rfi.i·1gf1·cf.iif.' d._:·ll] ·d1·ogt11ase

Dimerli.1·1g1~n'iue o.Yfd(l ~e

Sm·cosin~.' ·drogena.s e Sarcosi;J(' o-.:fda se

Fig. 1. 6 Choline oxidation pathway (Hassan, 2008)

18

1.4.2.2 Bacterial metabolism of 4-N-trimethylamino-1-butanol

4-N-trimethylamino-1-butanol (TMA-Butanol) is an analogue of choline, in which the

amino alcohol group is lengthened by two CH2-groups, has been shown to resemble

choline structurally. In the screening for TMA-Butanol degrading microorganisms from

soil, many bacterial strains were isolated. One of these strains, Pseudomonas sp. 13CM

was found to degrade TMA-Butanol rapidly and showed high and stable NAD+

dependent alcohol dehydrogenase activity was selected as a TMA-Butanol degrading

strain (Hassan, 2008). In Pseudomonas sp. 13CM, and similar to choline, TMA-Butanol

was oxidized to 4-N-trimethylamino-1-butyraldehyde (TMABaldehyde) by a NAD+

dependent TMA-Butanol dehydrogenase (Fig. 1.7), and consequently TMABaldehyde

was oxidized to y-butyrobetaine by a NAD+ -dependent TMABaldehyde dehydrogenase

(Hassan et al., 2007 & 2008). Kinetic studies on substrates and substrate analogs of

both enzymes showed that positively charged trimethylammoniun1 or

trimethylammonium groups of the substrates have critical effect on the catalytic activity

of the enzymes (Hassan, 2008). Similaly, Gadda et al., (2004) reported that the

trimethylammonium moiety is critically important for the binding of ligands at the

active site of choline oxidase.

19

(C:!13)3~+(~!12)4()!1

.:1-N-Trimethylamino-1-butanol

~AD-

D1A-Butanol dehydrogenase

"\TADII + ti

( Cli3)3 "\T-(~li2)3Cli()

-4-N-Trimethylmninobutyralclehyde

~AD--)\1 T~\1A.Baldehyde dehydrogena'Se

:..JADli + II l (Cli3)3~(Cli~)3C()Oli

: -Butyrobetaine

Fig. 1. 7 Degradation pathway of 4-N-trimethylamino-1-butanol in Pseudomonas sp. 13CM (Hassan, 2008)

1.4.2.3 Bacterial metabolism of L-camitine

Different Pseudomonas and Agrobacterium species are able to grow aerobically using

L-camitine as a sole source of carbon and nitrogen. Under aerobic conditions, oxidation

of L-camitine is catalyzed by NAD+ -dependent L-camitine dehydrogenase (EC.

1.1.1.108) (Goulas, 1988; Mori et al., 1988, 1994). The L-camitine dehydrogenase

gene from Xanthomonas translucens was cloned and expressed in E. coli (Mori et al.,

1988). 3-Dehydrocamitine formed by the L-camitine dehydrogenase could be degraded

to glycine betaine by the addition of NAD+, ATP, and CoA (Lindstedt et al, 1967). A

NAD+ -dependent D-camitine dehydrogenase (EC.l.l.l.254) has been purified and

characterized from various Agrobacterium species, which were able to utilize D-

camitine as a sole source of carbon and nitrogen (Hanschmann et al., 1994; Setyahadi et

20

al., 1997). During growth on D-carnitine, a carnitine racemase and L-carnitine

dehydrogenase were induced in Pseudomonas sp. AK1 (Monnich et al., 1995).

Enterobacteriacea such as E. coli, Salmonella typhimurium and Proteus

vulgaris are able to convert L-carnitine via crotonobetaine to y-butyrobetaine in the

presence of carbon and nitrogen sources under anaerobic conditions (Seim et al., 1982).

A two-step pathway, including L-carnitine dehydratase (EC. 4.2.1.89) and a

crotonobetaine reductase (EC. 1.3.99), was demonstrated in E. coli (Eichler et al., 1994)

and Proteus sp. (Engemann et al., 2005). The isolation and characterization of the cai

operon encoding the structural genes of the anaerobic L-carnitine pathway in E. coli and

Proteus sp. were reported (Bernal et al., 2007; Eichler et al., 1994; Engemann et al.,

2005).

Acinetobacter calcoaceticus 69N is able to metabolize L-carnitine, L-0-

acylcarnitines and y-butyrobetaine as a sole carbon source. A. calcoaceticus is able to

split the C-N bond of L-and D-carnitine to yield trimethylamine (Englard et al., 1983)

and a monoxyenase catalyzed cleavage has been postulated (Ditullio et al., 1994).

1.5 RESEARCH OBJECTIVES

Based on the above-mentioned literature review, research on the biodegradation of

homocholine by soil microorganisms is not yet carried out and consequently, the

biodegradation pathway of this compound remains unclear. Furthermore, from the

choline-like structure, one would expect that homocholine could be degraded similarly

and at a rate comparable to that of choline. Consequently, an osmoprotectant p -alanine

betaine could be an intermediate product in the degradation pathway of homocholine.

Therefore, the study ofhomocholine degradation pathways and the enzymes involved in

this degradation pathway is of considerable interest. From an application standpoint, the

21

recent findings that many marine bacterial and plant species accumulate ~-alanine

betaine in response to salt stress or water deficit (Hanson et al., 1994; Rathinasabapathi

et al., 2000) have spurred considerable interest and investigation on ~-alanine betaine

biosynthesis, with the goal of genetically engineering water and osmotic stress

resistance in beneficial bacteria and crop plants (Rathinasabapathi et al., 2000).

Based on the aforementioned assumptions and concepts, the overall objective of

this thesis was to obtain information to help in understanding homocholine degradation

ability by soil bacteria as well as to elucidate the degradation pathway of this compound

by the isolated strains.

The specific aims of the study were:

1- Screening of microorganisms that able to grow with homocholine as a sole

source of carbon and nitrogen.

2- Isolation, characterization and identification of microorganisms capable of

degrading homocholine.

3- Postulation of the degradation pathway ofhomocholine by the isolated strains.

4- Screening for the enzymatic activities in the degradation pathway of

homocholine by the isolated strains.

22

CHAPTER2

SCREENING, ISOLATION AND IDENTIFICATION OF

HOMOCHOLINE DEGRADING MICROORGANISMS

2.1 INTRODUCTION

Homocholine (3-N-trimethylamino-1-propanol) an analogue of choline, in which the

amino alcohol group is lengthened by one CHrgroup, has been shown to resemble

choline in many aspects of cholinergic metabolisms (Boksa and Collier, 1980). It is

transported into rat brain synaptosome, and is acetylated and released as

acetylhomocholine from a superior cervical ganglion and minces of mouse forebrain by a

calcium-dependent process during depolarization (Collier et al., 1977; Carroll and

As pry, 1980). It is also effective in preventing fat infiltration both in fat and cholesterol

fatty livers (Channon el al., 1937). It is well kno·wn that choline is an essential nutrient

that is widely distributed in foods, principally in form of phosphatidylcholine. It is also

a precursor of membrane and lipoprotein phospholipids and the neurotransmitter

acetylcholine; thus it is important for the integrity of cell membranes, lipid metabolism

and cholinergic nerve function (Zeisel et al., 1994; Zeisel 200). From the simple and

choline-like structure, one would expect that homocholine is degraded in a similar way

and rate comparable to that of choline. To date, no microorganism degrading

homocholine as a sole source of carbon and nitrogen has been isolated and consequently

23

the catabolic pathway is not yet elucidated. Considering the importance of this

compound and the design and development of similar compounds, it is important to

know the microbial strategies and the biochemical pathway of its degradation.

2.2 MATERIALS AND METHODS

2.2.1 Mater·ials

3-N-dimethy lamino-1-propanol (D MA-Propanol), 3-N-dimethylaminopropionic acid

(DMA-Propionic acid) and 3-aminopropionaldehyde diethylacetal were purchased from

Tokyo Kasai (Tokyo, Japan). Yeast extract was from Nacalai Tesque (Kyoto, Japan).

Peptone was from Nihon Seiyaku (Tokyo, Japan). Gram staining kit and methyl iodide

were purchased from Wako Chemicals (Tokyo, Japan). Primers used in this work were

obtained from Sigma Genosys (Sigma, Japan). Unless otherwise specified, all other

reagents used in this study were of analytical grade.


General weight measurements were made using IB-200H electronic balance (Shimadzu

corp. Kyoto, Japan) and smaller quantities and measurements for the preparation of

standards were made using Mettler AE240 analytical balance (Mettler, Toledo, AG,

Switzerland). pH was determined using an Horiba F-22 pH meter (Horiba Ltd, Kyoto,

Japan). Cultures and extracts were centrifuged using either a medium size centrifuge

Himac CF16RX2 or big size centrifuges Himac SCR 18B and RC 20B (Hitachi Koki Co

Ltd., Tokyo, Japan). Inoculated liquid media were incubated either in at 25°C using a

NR-300 double shaker (Taitec corporation, Cupertino, CA, USA) or at 30°C and low

temperature in LTI-601SD EYALA low temperature incubator equipped with NR-30

24

double shaker (EY ALA, Tokyo, Japan). Incubation of plates occurred in Taitec M-

260F temperature controlled incubate box. Media were autoclaved at 121°C for 20

minutes using BS-235 high pressure steam sterilizer (Tomy Seiko Co., Ltd., Tokyo,

Japan). Filter sterilization of solutions was carried out using 0.22 !ill1 disposable filters

MILLEX®-GA (Millipore, Molshiem, France). Samples were vortex using a Vortex

Genie 2 (Scientific Industries, Inc., USA). Benzoylation of samples and standards was

carried out at 90°C using Eyala MG-200 hot plate (Tokyo Rikakika Co., Ltd., Japan).

PCR Thermal Cycler Dice model TP 600 of TaKaRa was used for PCR reactions

(TaKaRa Bio Inc., Shiga, Japan).


3-N-trimethylarnino-1-propanol iodide (homocholine) was prepared by N-methylation

ofDMA-Propanol following the method of Hassan et al. (2007). Briefly, Methyl iodide

(160 mmol) was added dropwise to a solution ofDMA-Propanol (100 mmol) in diethyl

ether (200 rnl) at room temperature. After stirring for 1 h, the mixture was filtered. The

precipitate was dried in a vacurnm dessiccator for overnight and afforded white powder

(85 %). The structure and purity of homocholine were confirmed according to its 1H

NMR spectrum (ECP 500 MHz, NMR spectrometer; JEOL). Approximately 5 mg

homocholine was dissolved in D20 (Merck, Co. Inc., Darmstadt, Germany) and

analyzed using 1H NMR. The strong signal of NMR spectrum at 2.9 ppm (Fig. 2.1)

reflected the presence of trimethyl group with 99% purity.

3-N-trimethylarninopropionaldehyde was prepared from 3-arninopropion

aldehyde diethylacetal by methylation with methyl iodide. Large excess amount of

25

methyl iodide (42 ml) was added to a suspension of 3-aminopropionaldehyde

diethylacetal (5.0 g, 34.0 mmol) and K2C03 (11.7 g, 35 mmol) in 50 ml ethanol at room

temperature. After stirring at 40°C for 2 h, the rrllx.ture was cooled, and evaporated in

vacuo. The residue was dissolved in 50 ml water and then extracted with CH2Cl2 (50 ml

x 3 times). Combined organic extracts were dried over sodium sulfate, filtrated and

concentrated. The residue was crystallized from methanol-ethylacetate to give colorless

solid (8.5 g, 77%) of 3-N- trimethylaminopropionaldehyde diethylacetal iodide.

Thereafter, it was hydrolyzed by 0.1 M HCl for 3 h at room temperature to produce

TMAPaldehyde iodide as a 30 mM solution. The structure of TMAPaldehyde iodide

was determined by 1H-NMR spectroscopy and capillary electrophoresis .

.. (I I'

r "' -· ......... ----·"""- .

~--.,:--:,

~,

"2:~~-~-·" -.

I; (

"N ""''./"''oH ./

lv_ Homocholine

c

I r ll ~ . 1

);...

- -- ~r ~\._ _ _.._._,___ ~---·---·______/' ·~---

.!

-----· , ...

Fig. 2. 11H NMR spectrum of chemically synthesized 3-N-trimethy lamino-1-propanol

(Homocholine)

26

2.2.4 Screening and Isolation of Homocholine Degrading Bacteria

Enrichment cultures of soil samples from different location in Tottori University and

around Tottori City, Tottori, Japan, were used to obtain homocholine degrading strains.

Approximately 1 00 mg of each soil sample was inoculated into 5 rnl basal medium

(basal-homocholine media; Table 2.1) containing 5 giL homocholine as sole source of

carbon and nitrogen; 2 giL KH2P04; 2 giL K2HP04; 0.5 giL MgS04 • 7H20; 0.5 giL

yeast extract; and 1 giL polypeptone (pH 7.0). Cultivation was done at 30°C for 2 to 7

days in reciprocal shaker at 144 strokes/min. Subsequently, 200 ~ of the culture was

transferred to fresh basal-homocholine (basal-HC) media for another 2 days incubation.

After enrichment culture, an appropriate amount (1 rnl) of culture solution was taken

and serial 10-fold dilutions were prepared with physiological saline. Then, 100-~

samples of the 107 to 109 dilutions were plated on agar medium of basal-HC (Table 2.2)

and/or meat extract (Table 2.3) and then incubated at 30°C for 2 days. After successive

transfers to new plates, individual, distinguishable colonies were selected and stroked

into slant media. Single colonies were reinoculated in basal-HC liquid media, and the cell

growth was estimated by measuring the turbidity at 660 nm with Novaspec II

spectrophotometer (Amersham Pharmacia Biotech, Uppsala, Sweden). A suitable

control was set using basal media without substrate (homocholine). The highly growth

isolates were either stroked into basal-HC slant media and stored at soc in a cold room

or transferred to 50% glycerol and stored in a refrigerator at -80°C. Highly growth

isolates were further screened for the removal of homocholine from the growth media

using TLC plates and capillary electrophoresis as described bellow.

27

Table 2. 1 Basal-homocholine (basal-HC) liquid medium

TMA-Propanol iodide (Homocholine)

K2HP04

KH2P04

MgS04.7H20

Yeast extract

Peptone

Total volume

5.0 g

2.0 g

2.0 g

0.5 g

0.5 g

1.0 g

1000 mL

Table 2. 2 Basal-homocholine (basal-HC) agar medium

TMA-Propanol iodide (Homocholine) 5.0 g

K2HP04 2.0 g

KH2P04

MgS04.7H20

Yeast extract

Peptone

Agar

Total volume

Meat extract

Peptone

NaCl

Agar

Table 2. 3 Nutrient broth (meat extract) media

2.0 g

0.5 g

0.5 g

1.0 g

15.0 g

1000 mL

5.0 g

10.0 g

5.0 g

15.0 g

Total volume 1000 mL

The pH of all media was adjusted to pH 7.0 with either 1M NaOH or 1M HCl.

2.2.5 Morphological, Biochemical and Physiological Characterization

The morphological characterization of the cells was examined using optical microscope

(Olympus BX 41; Olympus Corp., Tokyo, Japan) under light and phase contrast

conditions, and by using a transmission electron microscope (JEOL 100 CX II Tokyo,

28

Japan). For observation of cell morphology by transmission electron llllcroscopy

(TEM), cells were grown on basal-HC liquid media to both exponential and stationary

phases, collected and suspended in physiological saline solution. A small drop of the

suspension was placed on carbon-coated copper grid (Nisshin EM Co. Ltd, Tokyo,

Japan) and negatively stained with 0.1% uranyl acetate. After air-drying, the grids were

examined by the electron microscope. The Gram staining was tested using a Gram

staining kit (Wako Pure Chemical Industries, Ltd. Tokyo, Japan) according to the

manufacture's instructions, and using KOH method (Buck 1982). Gram stained bacterial

cells were observed using optical microscope under both light and phase contrast

conditions. Catalase activity was determined by bubble formation in a 3% hydrogen

peroxide (H20 2) solution. A colony was picked from the agar plate and mixed with the

solution of 3% H20 2. Frothing indicated the catalase activity. Motility was checked by

water hanging drop method. Utilization of different carbon sources and selected

metabolic activities were investigated by using API 20 NE commercially available test

kit (Biomerieux SA, Geneva, Switzerland) following the manufacture's instructions. The

API stripes tests were visually read after 24h and 48h. Utilization of selected carbon

compounds and combined carbon and nitrogen sources were tested by measuring

turbidity in liquid culture (5 ml) at pH 7.0 using basal medium supplemented with 5 gil

(w/v) of the compound of choice. The compounds used were: choline, homocholine, 4-

N-trimethylarnino-1-butanol, glycine betaine, ~-alanine betaine, y-butyrobetaine,

dimethyl glycine, dimethylarnino-1-propionic acid, sarcosine, L-carnitine, D-carnitine,

propionate, acrylate, 3-hydroxypropionate, malonate, trimethylamine, dimethylarnine

and monomethylamine. All tests were preformed with cells taken from the colonies pre-

29

grown on basal-HC medium. Cultures were incubated at 25 oc on a shaker for 48 h, and

all tests were done in triplicates.

2.2.6 Sequencing of 16S rRNA Gene

2.2.6.1 Polymerase chain reaction (PCR)

To identify the isolated bacteria, 168 rRNA gene fragments were amplified by colony

PCR with four primers sets (Table 2.4). Bacterial isolates were stroked on basal-HC

and/ or meat extract agar plates at 25°C for 48 h and single colonies were suspended in

25 fll of PCR reaction mixtures.

Table 2. 4 Primers used in PCR amplification

Primers

314 F

907R

6F

1510R

16f27

16rl488

Ps short F

Ps short R

Nucleotides sequence

5' -CCT ACGGGAGGCAGCAG-3 I

5'-CCGTCAATTCCTTTGAGTTT-3'

5'-GGAGAGTT AGA TCCTGGCTCAG-3'

5'-GTGCTGCAGGGTT ACCTTGTT ACGACT -3'

5' -AGAGTTTGA TCCTGGCTCAG-3'

5'-CGGTT ACCTTGTT AGGACTTCACC-3'

5' -C T ACGGGAGGCAGCAGTGG-3'

5'-TCGGTAACGTCAAAACAGCAAAGT-3'

Amplification using primer set 341 F and 709 R

The amplification reaction was composed of 20 pM of each respective primer (341F

and 907R), 200 uM of dNTPs, 1 Ox reaction buffer, 1.5 mM MgCl2 and 2.5 U r-Taq

DNA polymerase (Roche Applied Science) in a final volume of 25 fll. The

temperature/time profile used was an initial denaturation of 2 min at 95°C followed by

30

40 cycles of denaturation for 45 sec at 95°C, annealing for 30 sec at 50°C and elongation

for 1.45 min at 72°C and finally an extension for 5 min at 72°C.

Amplification using primer set 6 F and 1510 R

The amplification reaction was composed of 20 pM of each primer, 200 uM of dNTPs,

lOx reaction buffer, 4 ~ of GC-rich solution and 2.5 U Pwo Super Yield DNA

Polymerase (Roche Applied Science) in a final volume of 20 ~-tl. The temperature/time

profile used for primer sets was an initial denaturation of 2 min at 95°C followed by 40

cycles of denaturation for 15 sec at 95°C, annealing for 30 sec at 55°C and elongation


Amplification using primer set 16f27 and 16r1488


lOx Sp. Taq reaction buffer, 5x Tuning buffer (10%) and 2.5 U Pwo Super Yield DNA


profile used the primer sets was an initial denaturation of 2 min at 95°C followed by 35



Amplification using primer set Ps short F and Ps short R


lOx Sp. Taq reaction buffer, 5x Tuning buffer (10%) and 2.5 U Pwo Super Yield DNA


31

profile used the primer sets was an initial denaturation of 2 min at 95°C followed by 40


for 45 sec at 72°C and finally an extension for 7 min at 72°C.

2.2.6.2 Agarose gel electrophoresis of DNA and visualization

Agarose gel electrophoresis was used to detect PCR products. Gels were composed of

1% w/v agarose in 1. 0 x Tris-Acetate EDT A (T AE) buffer and run at 1 OOV for 30

minutes in the same buffer using a Mupid mini-gel electrophoresis apparatus (Advance

Corporation, Tokyo, Japan). DNA samples were run alongside Lambda DNA molecular

weight marker to estimate product size. Gels were stained with ethidium bromide. DNA

was visualized using a UV Transilluminator. Photographs of agarose gels were taken

using the Pharmacia Image master VDS system (Pharmacia Biotechnology, Uppsala,

Sweden).

2.2.6.3 PCR product purification and sequencing

The PCR products obtained were purified using NucleoSpin Extract II kit (Macherey

Nagel, Duren, Germany) according to the instruction manual, and sequenced using Big

Dye Terminator cycle sequencing kit (Applied biosystem) following the instruction

manual. The sequences were analyzed at research institute for biological sciences

(Okayama, Japan) with automated DNA sequencer ABI PRISM 3130 XL (Applied

Biosystems, USA) following the manufacture's manual.

32

2.2. 7 Phylogenetic Analysis

In order to phylogenetically identify 16S rRNA sequences, one must compare the

sequences in a database of known species. Worldwide, there are a number of publicly

available sequence databases that build massive sequence databases compiled from

submissions from individual laboratories and large batch sequencing projects. The 16S

rRNA sequence files received in ABI format from the sequencing facility were first

opened and viewed in Codon Code Aligner software. Sequence chromatograms were

examined for overlapping nucleotide sequences and clarity of resolution. Once the

sequence was determined to be of good quality, it was saved in the F ASTA format. The

sequence was then copied into BLASTN, nucleotide database, and searched in the

DDJB using both FAST A and BLASTN algorithms (Bosshard et al., 2004) for closest

relatives. Once identified, the sequence was then compared with its closest match and

phylogenetic affiliations were made based on these matches. Sequences from closest

relatives to each isolate were downloaded for use in phylogenetic tree construction.

2.2.8 Creation of Phylogenetic Trees

The Clustal W, ver. 1.83 tool within DDJB database was used to create phylogenetic

trees to produce graphical interpretations of phylogenetic affiliations and relatedness of

the positive homocholine degrading isolates. Sequences of interest and close relatives

were uploaded into the program and aligned, distance matrixes were calculated based on

a specific algorithm of calculating evolutionary differences, and phylogenetic trees were

created. Neighbor joining trees using the distance matrix algorithm for maximum

likelihood are commonly used when comparing organisms of phylogenetically diverse

taxonomic hierarchy and were used in this study. The evolutionary tree for the datasets

33

was constructed by using the neighbour-joining method of Saito and Nei (1987) and

viewed with TreeView software. The confidence of the resultant tree topologies were

evaluated by performing bootstrap analyses of the neighbour-joining method based on

1000 resamplings.

2.2.9 Analytical Methods

2.2.9.1 Thin laye1· chromatography (TLC)

This experiment was carried out to detect metabolites in the culture broth of

homocholine-cultivated microorganisms in order to check the ability of the isolates to

degrade homocholine as well as to identify the degradation pathway of homocholine in

the isolates. A bout 5 !!L of the samples and homocholine as standard (contain about

100 nmol) were applied with capillary tube onto 7cm x 10 em pre-coated cellulose F

plates (DC-Fertigplatten). The chromatography was then developed with a mixture of

n-butanol: acetic acid: water ( 4: 1:2) as mobile phase to distances of 7~ 7.5 em. The

developed spots were visualized with either iodine vapour or dragendorff for about 2~5

min. The disappearance of the dragendorff positive spots indicated complete

degradation ofhomocholine by the isolates. Moreover, the appearance of new bands and

in comparison with the provided standards, gave initial indication for the degradation

pathway ofhomocholine by the isolated strains.

2.2.9.2 Capilla11' electrophoresis (CE)

Capillary Electrophoresis has become an accepted method for the separation of

inorganic and organic ions. Usually, direct and indirect optical detection methods were

used in conventional CE and much better detection limits in the low ppb range were

34

obtained compared to other detection methods. Due to high sensitivity of CE, it was

used in this study to detect the metabolites of homocholine by the isolated strains. The

isolates were cultivated on 50 mL basal-HC liquid media at 25°C for a time ranged from

0 to 48 h. At intervals of 6 h, aliquots of 10 ml media were withdravm and centrifuged at

10,000 x g for 20 min to separate the bacterial cells from the supernatant. Since

homocholine has no absorbance at UVNis wavelength, 0.1rnl of the supernatant was

benzoylated with benzoyl chloride (100 mg mr1 in pyridine) for 15 min at 90°C and

analyzed by low pH capillary electrophoresis (Zhang et al., 2001).

Capillary electrophoresis analysis was carried out using Photal CAPI-3300

(Otsuka, Electronics. Co. Ltd, Osaka, Japan) equipped with a fused silica capillary of

75 f..llll i.d. with a total length of 80 em (effective length of 68 em). A new capillary was

conditioned with 0.1 M NaOH for 5 min followed by 3 min distilled water and 3 min

electrolyte buffer (50 mM sodium phosphate buffer, pH 3.0). Samples and relative

standards were injected hydrostatically (25 mm, 60 sec). The applied potential was 25

kV, and the peaks were monitored at 200 nm.

2.3 RESULTS AND DISCUSSION

2.3.1 Screening ofHomocholine Degrading Microorganisms

Screening of homocholine degrading microorganisms by enrichment techniques resulted

in the isolation of 142 pure colonies, which showed growth on the basal-HC medium

(Fig. 2.2). Out of them, about 30 strains showed the turbidity at 660 nm of more than

1. 0 were selected. These isolates were further screened for their metabolic ability to

degrade homocholine and to use it as a sole source of carbon and nitrogen. Following the

35

decreasing and disappearance of the Dragendorff positive spots on the TLC plates

indicated complete removal of homocholine from the culture medium by these isolates.

Moreover, the disappearance of homocholine peak from the capillary electrophoresis

chromatogram also confirmed the degradation of the compound by the isolates. Among

these isolates, 10 strains showed the highest growth and ability to metabolize

homocholine were selected for further studies. Those isolates were chosen as most

superior homocholine degraders and were identified to the specific level according to

general principles of microbial classification. For all isolated strains, Actinobacteria and

Proteobacteria comprised the two main groups of homocholine degrading bacteria. The

Actinobacteria included mainly Rhodococcus and Arthrobacter strains. Proteobacteria

included mainly y-Proteobacteria and more specifically Pseudomonas. These results

indicated, and similar to choline, that the ability to grow on and to degrade homocholine

is widespread amongst microorganisms since a large and diverse number of bacteria were

found to grow on homocholine as a sole source of carbon and nitrogen. Although there

are very few reports on the existence of homocholine in mammalian brain and/or

cytoplasm cells and no direct report on the existence of the compounds in the plants

and microorganisms, the ability to degrade homocholine is widespread. Very few reports

on the existence of homocholine motif in alkaloids isolated from some plants as well as

recent reports identified some homocholine esters in some plants (Barker et al., 2004).

These reports shed the light on the existence and importance of this compound in the

nature. The results of this study confirmed the fact that repeated exposure to

homocholine in the soil would usually increase the adaptive capabilities of the

microorganisms and though increase the rate of degradation with a new exposure to a

36

compound. This means that homocholine is naturally and widely exist in the biosphere

although that is rarely reported.

50r----------------------------------------------------------.

"' "'

40

'§ 30 0 '"' 'o "' ... "' .::::. E 20 = z

10

0

O-Q.49 0.5-Q.99 1.0-1.49

Turbidity (T """)

1.5-1.99

Fig. 2. 2 Histogram showing the cell growth of isolated microorganisms.

Cell growth was estimated by measuring the turbidity at 660 nm.

2.0-

2.3.2 Isolation and Charactelization of Homocholine Degrading Artltrobacter sp.

Strain E5

Based on the enrichment of homocholine degrading microorganisms under conditions

described in "Materials and methods" section, pure colonies with high growth (turbidity

>1) were isolated. Out of the 30 highly growth strains, one bacterium designated as

strain E5 showed a good growth and ability to utilize homocholine. This strain also

degraded homocholine at a good rate, therefore, was selected for further study.

37

2.3.2.1 Morphological and physiological characteristics of strain E5

Strain E5 is an aerobic, non-motile and ~am-positive bacterium that forms pnmary

branching and long rods of variable length when grown on homocholine agar media

during the early growth phase. Then the cells fragmented into irregular short rods and/or

cocci, thereby completing the growth cycle as the culture aged (Fig. 2.3). The colonies

are yellow in color, convex, round and entire with smooth and regular margins on both

homocholine and nutrient agar medium. The colony size ranged from 1.5 ~3.0 mm on

homocholine-agar plates at 30°C for 2 days. These morphological characteristics were

consistent with strain E5 belonging to the genus Arthrobacter. The isolated strain E5

showed catalase activity, but no oxidase activity. The bacterium was able to grow on

homocholine media at an optimum temperature of 25-30°C, but did not grow at either

4°C or 41 oc. Some other physiochemical properties of strain E5 are summarized in

Table 2.5. Phenotypic and physiochemical tests suggest that the isolated strain E5 is

belongs to the genus Arthrobacter.

Arthrobacter is a common genus of gram-positive and obligate aerobic soil bacteria

characterized by polymorphism and gram variability. Arthrobacter species are among

the most frequently isolated, indigenous, aerobic bacterial genera found in harsh

conditions for extended periods of time (Mongodin eta!., 2006). They are metabolically

versatile and therefore can grow on a wide range of substrates, including nicotine

(Kodama eta!., 1992; Ganas eta!., 2007), herbicides and pesticides (Sqjjaphan eta!.,

2004) and other contaminants such as phenol (Karigar eta!., 2006), fluorene (Casellas et

a!., 1997) or 4- chlorophenol (Nordin eta!., 2005). Due to their ubiquitous presence in

soil, their high resistance against environmental stress factor and their ability to

38

metabolize a variety of substances, bacteria of this genus are of great interest for

potential environmental and industrial applications.

A 8

1 mm 10 1Jm

Fig. 2. 3 Morphology of Arthrobacter sp. E5 as seen by light microscopy under (A) optical (B) phase contrast conditions.

The colony morphology in panel (A) and the morphological differentiation in cells shape and size after cultivation for 48 h (B) are ty pical gram-positive bacteria.

2.3.2.2 16S rRNA gene sequence analysis

The world molecular taxonomy was revolutionized, however, in the mid-1980s with the

advent of full sequence analysis of molecular chronometers such as rRNA (Rossello-

Mora and Amann, 2001). By the mid-1 990s sequencing of the small subunit (16S)

rRNA genes had become commonplace, considered as a standard tool for microbial

taxonomists not only for elucidating phylogenetic relatedness but also as a means of

bacterial identification (Kolbert and Persing, 1999; Rossello-Mora and Amann, 2001).

Phylogenetic relationships could be inferred through the alignment and cladistic

analysis of homologous nucleotide sequences of known bacteria. To investigate the

phylogenetic relationships between strain E5 and Arthrobacter species, a 16S rRNA

39

gene sequence was compared with those of representative members of the genus

Arthrobacter. The 16S rRNA data supported the results of morphological and

phenological analysis. The phylogenie tree drawn from the partial 16S rRNA sequence

(544 nt) of the isolated strain E5 (FJ595954) clearly demonstrated that this strain

belongs to theArthrobacter nicotinovorans 16S rRNA subclade (Fig. 2.4). The organism

was most closely related to the type strains of Arthrobacter nicotinovorans

(AY833098) with a homology of 99.8%, to Arthrobacter sp. HSL strain (AY714235)

with homology of 99.8%, and to Arthrobacter histidinolovorans (AF501356) with

homology of 99.8 %. The high level of similarity observed between the 16S rRNA

sequence of the isolated strain E5 and several Arthrobacter species suggest that the

strain E5 could be a strain of one of those species. However, it is accepted that 16S

rRNA sequence comparison may indicate species level identification with a probability

but are not considered to be definitive. Thus, strain E5 should be considered as

Arthrobacter species with a close phylogenetic relationship to Arthrobacter

nicotinovorans.

40

u·•~,

0,00/

~---A. n:~siphoc:~e (AJ293364)

r--------~-------- KocurrJ sp.

m A korconSIS{AY116496)

llll A. lutcolus (00486130)

A. bontolcrJns (AB288059)

lA. lliStidmo/o}'OI:IflS (AF501356)

1,11

I A. mcormovor:ms (AY833098)

,,,_ol 1A. sp HSL (AY714235)

"'"iArtiJrobacter sp. E5

A. bontofcr.1m jAB288060}

,, ... ' .A. mrroguJ)Jcolicus {AJ512504)

A. .. wrcscons (CP00047 4)

.,,,, r A. rJmOS(IS (AM039435)

A. morhylorrophus (AF235000

Fig. 2. 4 Phylogenetic tree based on 16S rRNA sequence showing the relationship between strain E5 and most closely related species of the genus Arthrobacter.

Numbers at nodes indicated level of bootstrap support 2': 50%, based on a neighbour-joining analysis of 1000 re-sampled datasets. Bar = 0.005 nucleotide substitution per nucleotide position.

2.3.2.3 Utilization of several organic compounds by strain E5

For the determination of nutritional and biochemical properties of Arthrobacter sp.

strain E5, a variety of selected organic compounds were tested (Table 2.5). The strain

was found to grow with homocholine, which was used for its isolation and the

intermediate metabolite of its degradation pathway namely f)-alanine betaine.

Dimethylaminopropanol also was utilized for growth by the isolated strain E5. Out of

the tested substrates, Cl-compounds such as trimethylamine and monomethylamine did

not support the growth, while dimethylamine did. It is remarkable that the isolated

strain beside its preference to homocholine it prefer the other C and N-containing

compounds that have dimethyl group instead of tri- and mono methyl group. Most of

41

the substrates tested as carbon sources were utilized for growth except caprate, adipate,

malonate, and 3-hydroxypropionate.

Table 2. 5 Morphological and physiological characteristics of Arthrobacter sp. strain E5

Parameter Characteristics Parameter Characteristics Morphogenic sequence R-C Assimilation of

Colony morphology Entire & convex D-glucose +

Colony color Yellow L-arabinose + Gram reaction + D-mannose + Motility Non D-mannitol + Oxidase - N-Acety 1-gluco samine + Catalase + D-maltose + Urease - Gluconate + Reduction of nitrates to nitrite - Caprate Reduction of nitrates to - Adipate nitrogen Indol production - Malate +

Glucose fermentation - Citrate +

Arginine dehydrolase - Pheny 1 acetate +

~-galactosidase + Growth with (0.5%) ~ -glucosidase + Propionate +

Hydrolysis of Acrylate +

Esculin - 3-Hydroxypropinate Gelatin + Malonate Growth at Homocholine + 4°C - ~- Alanine betaine +

37°C + Dime thy laminopropanol + 45°C - Dimethy laminopropionic acid ± pH 5.0 - Trimethylamine ± pH8.0 + Dimethy lamine +

Monomethy lamine ±

Characteristic are scored as follow;+, positive, utilized;±, weakly positive;-, negative; R-C, rod-coccus growth cycle

2.3.3 Isolation and Characterization of Homocholine Degrading Rlwdococcus sp.

Strains A2

The genus Rhodococcus is comprised of genetically and physiologically diverse bacteria,

which have been isolated from various habitats, for example soil and seawater plants

(Martinkova et al., 2009). They share with a distantly related Pseudomonas species a

capacity to degrade a large number of organic compounds including some of the most

42

difficult compounds that cannot be easily transformed by other organisms (Larkin et al.,

2005). The large genome of rhodococci, their redundant and versatile catabolic

pathways, their ability to uptake and metabolize various organic compounds such as

aliphatic and aromatic hydrocarbons, halogenated compounds, nitriles and various

herbicides, beside their ability to produce acrylarnide and acrylic acid and to persist in

adverse conditions makes them suitable industrial microorganisms for biotransformation

and the biodegradation of many environmentally important organic compounds

(Martinkova et al., 2009).

2.3.3.1 Enrichment and isolation of strain A2

Enrichment for homocholine degrading microorganisms was preformed in batch culture

at 30°C and pH 7.0 using basal medium supplemented with homocholine as a sole

source of carbon, energy and nitrogen at a concentration of 5 g/1. This enrichment culture

was subsequently tested for growth (turbidity). Initially, a mixed culture was obtained;

however; after streaking the mixed culture onto nutrient agar and homocholine agar

plates, individual colonies were isolated and, eventually, a pure culture capable of

growing in medium with homocholine as a sole source of carbon and nitrogen was

obtained. About 30 colonies with high growth (turbidity > 1) were isolated in pure

culture, and one of these colonies was analyzed in detailed for its degradation capacity

and designed as strain A2.

2.2.3.2 Morphological and physiochemical characterization of strain A2

Strain A2 is an aerobic, non-motile, non-spore forming and Gram-positive bacterium

that forms mycelium and long rods of variable length when grown on basal-HC agar

43

media during the early growth phase. Most cells are form filaments or show elementary

branching at early growth phase (Fig. 2.5) and then fragmented into irregular short rods

and/or cocci, thereby completing the growth cycle as the culture aged. The colonies are

pale pink in color, opaque, convex, round and entire with smooth and regular margins on

both basal-HC and nutrient agar media. Colonies are about 1.5-3. 0 mm diameter after 6

days when grown on nutrient agar plates. Strain A2 showed catalase activity, but no

oxidase activity. The bacterium was able to grow on basal-HC medium at a wide range

of temperatures, with an optimum at 25-30°C and grew optimally over a broad pH

range 6.0-8.0. Some other physiochemical properties of strain A2 were listed in Table

2.6. Phenotypic and physiochemical tests suggest that strain A2 is belongs to the genus

Rhodococcus.

Table 2. 6 Morphological and physiological characteristics of Rhodococcus sp. strain A2

Parameter Characteristic

Morphogenic sequence Colony morphology Colony color Colony size Gram reaction Motility Oxidase Catalase Urease Reduction of nitrates to nitrite Reduction of nitrates to nitrogen Indol production

EB-R-C Entire & convex Pale pink 1.5- 3.0 mm

+ Non

+

Parameter

Glucose fermentation Arginine dehydrolase ~-galactosidase

Hydrolysis of Esculin Gelatin

Growth at 4°C l5°C 37°C 41°C

45°C

Characteristic

+

±

+ + ±

Characteristic are scored as follow;+, positive;±, weakly positive;-, negative; EB-R-C, elementary branching-rod-coccus growth cycle.

44

Fig. 2. 5 Morphology of Rhodococcus sp. strain A2 as seen by Phase contrast microscopy (A & B), and transmission electron microscopy (C).

Morphological differentiation in cells shape and size after cultivation for 48 h (A), and 72h (B). Cells were grown on both Basal-HC medium and nutrient broth medium. (C) Presence of fimbria-like structure (arrows) on the cell surfaces, and the V shape are typical of gram-positive bacteria. Bar= 10 fill1 (A & B), and bar= 0.5 fill1 (C).

2.3.3.2 Phylogenetic analysis of strain A2

To investigate the phylogenetic relationships between strain A2 and Rhodococcus

species, the 168 rRNA gene sequence was compared with those of representative

members of the genus Rhodococcus. The 168 rRNA data supported the results of

morphological and phenological analysis. The partial 168 rRNA sequence (1374 nt) of

the isolated strain A2 (AB473943) clearly demonstrated that this strain belongs to the

Rhodococcus erythropolis 168 rRNA subclade (Fig. 2.6). The organism was most

closely related to the type strains of Rhodococcus opacus (AY027585) with an identity

of 99.1 %, Rhodococcus wratislaviensis (Z37138) with homology of 98.8% and to

45

Rhodococcus koreensis (AF 124342) with an identity of 98.5%. The high level of

identity observed between the 16S rRNA sequence of strain A2 and several

Rhodococcus species suggests that the isolate A2 is a strain of one of those species.

However, it is accepted that 16S rRNA sequence comparison might indicate species

level identification with a probability but are not considered to be definitive.

Consequently, strain A2 should be considered as Rhodococcus sp., with a close

phylogenetic relation with Rhodococcus opacus and Rhodococcus wratislaviensis.

In many studies, 16S rRNA identity values between 99.0 and 99.8 have been

reported for representatives of several species of Rhodococcus (Goodfellow et al., 2002;

Yoon et al., 2000) that share DNA-DNA relatedness well below the 70% cut off that is

recommended for the assigmnent of organism to the same genomic species (Wayne et al.,

1987). Despite the striking similarity of strain A2 and Rhodococcus opacus and

Rhodococcus wartislaviences based on 16S rRNA, they differ in substrate range and

even shape. This demonstrates that ribosomal genetic analysis is able to provide

phylogenetic relationship, indeed, but does not necessarily supply information on the

specific metabolic ability of isolates.

46

l 0.005 I .--------Rhodococcus maa.nsha.nensis (AF416566) l Rhodococcus marinonascens (X80617) L---Rhodococcus jostii (AB046357)

...---Rhodococcus percalatus (X92114) .----Rhodococcus koreensis (AF 124342)

Rhodococcus wratislaviensis (Z37138) 77 1 r Rho do= us opa.cus (A ¥027585)

5fjl-Rhodococcus sp. TMAP A2 I R. erythropolis ----Rhodococcus gioberuius (X80619) subclade

- Rhodococcus bctritolera.ns (AB288061) Rhodococcus erythreus(X79289)

Rhodococcus erythropo}is (AY168587)

~'hodo.,'"OCCus yunna.nensis (AY602219)

1 Rhodococcus r:erddiphyllus(EU325542) 90 Rhodococcus fasc;a.ns (A ¥771756)

1 I Rhodococcus kunmingensis (DQ997045) I R. equi subclade 98 · Rhodo=us eqw (AF490539)

100 L Rhodococcus kro~penstedfii(A ¥72660.5) I .. I Rhodococcus corynebacterioides (AF430066) R. rhodnn subclade

,---------Rhodo=us rhodnii(X60823) Rhodococcus .t:riatamae(AJ854056) ~-----Rhodococcus po}yvwum (AY428603)

-Rhodo=us .:wp./Ji (AF191343) Rhodococcus phenolicus (AM933579)

~----- Rhodococcus coprophilus (X80626) I R. rhodochrous Rhodococcus aetherivwans (EU004422) subclade

Rhodococcus ru.b.;>r(X80625) Rhodococcus rhodochrous (AH:-!10967)

1 L==: Rhodococcus pyridinivwans (AF459741) 98 Rhodococcus flordoniae (EU004418)

Fig. 2. 6 Phylogenetic tree based on 16S rRNA sequence showing the relationship between strain TMAP A2 and most closely related species of the genus Rhodococcus.

Numbers at nodes indicate level of bootstrap support ::::: 50%, based on a neighbour-joining analysis of 1000 re-sarnpled datasets. Bar = 0.005 nucleotide substitution per nucleotide position.

2.3.3.3 Growth of strain A2 on homocholine and other structumlly related

compounds

A typical time course of Rhodococcus sp. strain A2 cell growth and homocholine

degradation is presented in Fig. 2. 7. The bacterial cells increased exponentially after 15 h

of cultivation with homocholine as a substrate, although increase in cell density in the

control (without substrate) was negligible throughout the experiment. About 90% of the

substrate was degraded rapidly during the initial 28 h of cultivation. Thereafter, the

degradation rate of the substrate slowed and complete degradation occurred after 42 h

from cultivation. Complete disappearance of homocholine from the medium, together

47

with differential growth in media with and without the substrate, indicated that the

strain used homocholine as a source of carbon and nitrogen.

Utilization of other structurally related homocholine analogues by strain A2 was

examined. Results showed that strain A2 utilized trimethylamino-1-butanol, y

butyrobetaine, L-carnitine, ~-alanine betaine and glycine betaine for growth, but not

choline, D-camitine, dimethyl glycine, dimethylarnino-1-propanol or dimethylarnino-1-

propionic acid (Table 2. 7). The inability of strain A2 to use choline, a natural

structurally related compound to homocholine, in addition to its ability to grow on

trimethylamino-1-butanol suggested that homocholine degrading enzymes prefer a

longer chain substrate. A similar observation was reported by Hassan et al. (2007) who

found that the activity of trimethylarnino-1-butanol dehydrogenase was greater for

increased lengths of carbon chains of the quaternary ammonium compounds. Moreover,

the inability of the strain to metabolize dimethyl-containing compounds might be

explained by the fact that a positively charged trimethylammonium group on the

substrate is critically important for the activity of the quaternary ammoruum

compound-degrading enzyme (Gadda eta!., 2004; Hassan et al., 2007).

48

25 3

20 -== --2 E E 1:

-15- 0 Cl)

<0 <0

.5 c 0 0 .c -() .c 0 10- i E 0 --1 e :I: "

5

D

0 fi ===:: , r\ 'iC .........,.. • 1 ·-0 0 1 0 20 30 40 50 60

Time(h)

Fig. 2. 7 Time course study of the growth and metabolism of homocholine by Rhodococcus sp. strain A2.

Degradation of homocholine (1111) and growth of cells in basal medium supplemented with 20 mM homocholine ( • ). The control was the basal medium without homocholine (M. Each value represents an average of three independent experiments.

Table 2. 7 Utilization of homocholine and other structurally related compounds by Rhodococcus sp. strain A2

Compound

Homocholine

Choline

Growth

++

Trimethylamino-1-butanol ++ + L-camitine + + D-camitine Glycine betaine + + y- butyrobetaine + + ~- alanine betaine + Dimethyl glycine Dimethy lamino-1-propanol Dimethy larnino-1-propionic acid Control (no substrate)

Growth was determined by measuring the turbidity at 660 nm (OD66o nm). Symbols:(+++) OD660nm::: 3.0; (+ +) OD660nm::: 2.0; (+) OD660nm::: 1.0; -,no growth

Each datum represents the average of three independent experiments.

49

2.3.4 Isolation and Cha1·acterization of Homocholine Degrading Pseudomonas sp.

Strains A9 and B9b

2.3.4.1 Morphological and physiological charactelistics of the isolated strains

Quaternary ammonium compound degrading bacteria exist widely in the environment

and they are usually isolated from soil. In the present study two strains (A9 and B9b)

were isolated using homocholine as a substrate and identified as species of the genus

Pseudomonas, based on the analysis of phenotypic and genotypic characteristics.

Isolated strains are aerobic, gram-negative rods and motile by means of single

(B9b) or multiple (A9) polar and sub-polar flagella (Fig. 2.8). Cells of strain A9 formed

short rods measuring approximately 0.5-1 x 1.5-2.0 llm in size while those of B9b

formed long rods of 0.5-1 x 2.5-3.0 jlm during the early growth phase on both nutrient

broth and basal-HC media. The colonies of both strains are non-pigmented, convex,

transparent at the edges and opaque at the center and undulate with slightly irregular

margins on both basal-HC and nutrient agar medium. Colonies were about 2-3 mm in

diameter after 5 days of growth on nutrient agar plates. Metabolism was respiratory not

fermentative. The cytochrome oxidase test and catalase activity were positive. Nitrate

reduction and denitrification were negative. Strain A9 was able to grow on basal-HC

media at a wide range of temperatures ( 4 - 41 °C) whereas strain B9b was not able to

grow at either 4 or 41 °C. Both strains showed optimum growth at temperature range of

25-30 oc and pH range of 6.0-8.0. Some other physiochemical properties of strain A9

and B9b are summarized in Table 2.8. Phenotypic and physiochemical tests suggest that

both strains belong to the genus Pseudomonas.

50

A c

B D

1 J.lm 1 J.lm

Fig. 2. 8 Morphology of Pseudomonas sp. strain A9 and B9b as seen by phase contrast microscopy (A & C) and transmission electron microscopy (B & D), respectively.

The morphological differences in the cells size and the number of flagella.

2.3.4.2 16S rRNA gene sequence analysis of strains A9 and B9b

Phylogenetic relationships could be inferred through the alignment and cladistic analysis

of homologous nucleotide sequences of known bacteria. To investigate the phylogenetic

relationships between strains A9 and B9b and Pseudomonas species, 16S rRNA gene

sequences were compared with those of representative members of the genus

Pseudomonas. The 16S rRNA data supported the results of morphological and

phenological analysis. The phylogenetic relationship drawn from the partial 16S rRNA

sequence (1408 nt) of the isolated strain A9 (FJ 605430) clearly demonstrated that this

strain belongs to the Pseudomonas putida 16S rRNA subclade (Fig. 2.9). The organism

51

was most closely clustered with the type strains of Pseudomonas putida (AY 973266)

with a homology of 99.4 % and Pseudomonas umsongensis (AF 460450) with a

homology of 99.3 %. Whereas, the partial (1421 nt) 16S rRNA gene sequence of strain

B9b (FJ 605432) demonstrated its phylogenetic position in the Pseudomonas fulva

subclade. The strain is closely clustered with two types of strain, Pseudomonas

plecoglossicida (EU 594553) and Pseudomonas fulva (DQ 141541) having sequence

similarities of 99.8 % and 99.7 %, respectively. Our 16S rRNA sequence comparison

confirmed that strains A9 and B9b belong to the genus Pseudomonas.

The high level of similarity observed between the 16S rRNA sequence of these

strains and several Pseudomonas species suggest that the isolated strains could be strain

of one of those species. However, it is accepted that 16S rRNA sequence comparison

may indicate species level identification with probability but are not considered to be

definitive. In many studies, 16S rRNA similarity values more than 99.0 have been

reported for representatives of several species of Pseudomonas (Behrendt et al., 2003;

Hauser et al., 2004; Uchino et al., 2001) that share DNA-DNA relatedness well below

the 70 % cut off recommended for the assignment of organism to the same genomic

species (Wayne et al., 1987). In contrast, it was pointed out that strains with lower

similarity in the 16S rRNA gene sequence would re-associate with rates above the

species limit (Tvrzova eta!., 2006). Combined genotypic and phenotypic data suggest

that strains A9 and B9b merits recognition as new species within the genus

Pseudomonas, we tentatively named them as Pseudomonas sp. strain A9 and

Pseudomonas sp. strain B9b.

52

100

Pseudomonas sp. B9b Psevcfornonas pleco_qlossicicla (EU594553_1)

Pseudomonas fvllta (DQ1 4154·1_"1) Psevdomonas mossefii (ft.M184215_·1)

,-----Pseudomonas entomophila (CT573326_6) Pseudomonas montei.lii (EU668000_1)

Psevdon?ona.s tai•Nanen.sis (EU·t 03629_·1) ,-------- P.sevdomonas raoraviensis (.A.Y970952_1)

Pseudomonas koreen.sis (AF 468452_1) Pseudornonas pa·.ronaceae (D84Jl1 9_'1)

'------Pseudomonas teessiciea (/I.M4 ·1 9154 _1)

Pseudomonas ~'ancou·...-eren.sis (iU01'1507 _1) Pseudornonas moorei (AM293566_1)

,-----------Pseudomonas migulae{ft.F074383 1'• .-----Pseudomonas sp. A9 ' - '

Pseudomonas umsongensis (.A.F468450_1) Pseudomona.s putid8 (./I.Y97326f3_·1)

.---------Pseudomonas corrugata (.A.Y050495_1) Pseudomonas f!ovrescens (ft.Y5126'14_1)

'----------Pseudomonas gingeri (.I\F3325.11_1) 1 I .c·seudomonas _gramini.s ({11.150_.1)

9& Pseudomonas lute.:~ (EU184082_1) I 0.002 I

Fig. 2. 9 Phylogenetic tree based on 16S rRNA sequence showing the relationship between the isolated strains A9 (FJ605430) and B9b (FJ605432), and most of the closely related Pseudomonas species.

Numbers at nodes indicate level of bootstrap support 2:: 50 %, based on a neighbour-joining analysis of 1000 re-sampled datasets. Bar = 0.002 nucleotide substitution per nucleotide position.

Diverse ranges of microbial taxa capable of aerobic degradation of quaternary

ammonium compounds have been reported (Seim et a!., 1982; Miura-Fraboni and

Englard, 1983; Mori eta!., 1988; Van Ginkel et al., 1992; Keach eta!., 2005). Among

them, Pseudomonas species are predominant, due to their versatility and capability of

degrading a number of different quaternary ammonium compounds (Van Ginkel, 1996).

The genus Pseudomonas is present in ecologically diverse niches, playing important

roles in the biogeochemical cycles. Bacteria belonging to this genus are engaged in

important metabolic activities in the environment, including element cycling and the

53

degradation of biogenic and xenobiotic pollutants (Timmis, 2002). They play a key role

in the metabolisms of quaternary ammonium compounds, e.g., choline (Nagasawa et al.,

1976), y-butyrobetaine (Miura-Fraboni and Englard, 1983), 3-N-trimethylamino-1-

butanol (Hassan et al., 2007), 2,3-dihydroxy propyl-trimethyl ammonium (Keach et al.,

2005) and many others long chain quaternary ammoni urn compounds (Van Ginkel et al.,

1992; Nishihara et al., 2000).

Additionally we also reported here the isolation of two Pseudomonas strains able

to degrade homocholine and to use it as a sole carbon and nitrogen source. Despite the

striking similarity of strain A9 and B9b in substrate range, they differ in 16S rRNA gene

sequences and even shape. This demonstrates that the metabolic ability of the isolates

of the same genus might be similar although they are differing in the species level.

2.3.4.3 Utilization of several C.N. sources by strains A9 and B9b

For the determination of nutritional and biochemical properties of the isolated strains

A9 and B9b, a variety of selected organic compounds were tested (Table 2.8). Both

strains were able to grow on homocholine, which was used for their isolation and

weakly on P-alanine betaine. Choline and its degradation pathway metabolites namely

glycine betaine, dimethylglycine, and sarcosine were used as substrates for both strains.

Trimethylamino-1-butanol, an analogue of homocholine in which the amino alcohol

chain lengthened by one CH2 group, and an intermediate metabolite of its degradation

pathway namely y-butyrobetaine were not utilized for growth by both strains. L

carnitine, the naturally widely occurring quaternary ammonium compound was also

supported the growth of both strains, whereas D-carnitine did not. It is clear that the

preference of quaternary ammonium compounds as substrates for both strains is quite

54

similar. The only exception is that strain B9b showed positive growth with

dimethylamino-1-propanol whereas that of A9 was negative. Out of the additionally

substrates tested as carbon sources D-maltose, adipate, and acrylate did not support the

growth of both strains. In addition to these substrates strain B9b did not also utilized

both D-mannitol and N-acetyl-glucosamine. Moreover, the cell growth of both strains

under the controls (basal media without the substrate) was negligible throughout the

experiment.

Table 2. 8 Phenotypic characteristics of homocholine-degrading Pseudomonas sp. strains A9 and B9b isolated from soil.

Characteristic A9 B9b Characteristic A9 B9b

Flagellation Multiple Single, Assimilation of polar

Shape Short rod Long rod D-maltose Motility + + Gluconate + + Oxidase + + Caprate + + Catalase + + Adipate Urease + - Malate + + Nitrate reduction - - Citrate + + Indol production - - Phenyl acetate + + Glucose fermentation - - Propionate + + Arginine dehydrolase + + Acrylate ~-galactosidase - - 3 -hydroxypropionate + + Hydrolysis of Malonate +

Esculin - - Homocholine + + Gelatin + - Choline + + Growth at: Trimethy laminobutanol

4 oc + - L-carnitine + + 41°C + - D-camitine pH5.0 + + Glycine betaine + + pH8.0 + + y- butyrobetaine NaCl (4 %) + - ~- alaninebetaine ± ±

Assimilation of Dimethy 1 glycine + +

D-glucose + + Dimethy larninopropanol - + L-arabinose + + Dimethy laminopropionic acid D-mannose + + Sarcosine + + D-mannitol + - Propanol N-acetyl- + - Trimethylamine glucosamine Characteristics were scored as follow:+, positive;-, negative;±, weakly positive Each datum represents the average of three independent experiments

55

2.4 CONCLUSIONS

The present study is an attempt to investigate the ability of soil microorganisms to

degrade homocholine. As expected, the ability to degrade homocholine is widespread

among aerobic microorganisms since representatives of the genus Arthrobacter,

R.hodococcus and Pseudomonas, were found to grow with homocholine as a sole source

of carbon and nitrogen. All these microorganisms are widely distributed in the

biosphere, and particularly in the soil, they probably play an important role in the

degradation of homocholine in the nature. Although there are very few reports on the

presence of homocholine in mammalian brains and there is no direct evidence on the

presence of such compound in plants. The widespread utilization of homocholine by

soil microorganisms provides indirect evidence that the compound is widely exists in the

nature and may be in the plant kingdom. In this study we reported the isolation of

homocholine degrading bacteria from soil as a first study. Strains belongs to the genus

Arthrobacter, R.hodococcus and Pseudomonas were isolated, identified and characterized

for their ability to grow with homocholine and its related analogues. With few

exceptions, most of the bacteria isolated and identified so far that degrade quaternary

ammonium compounds were from the genus Pseudomonas and Arthrobacter (Van

Ginkel et al., 1992; Van Ginkel, 1996; Hassan et al., 2007). However, R.hodococcus

species capable of degrading quaternary ammonium compounds have not yet been

reported despite their versatility and ability to degrade numerous organic compounds,

including some difficult and toxic compounds. In the present study we are able to isolate

R.hodococcus strains that metabolized homocholine as a sole source of carbon and

nitrogen.

56

CHAPTER3

DEGRADATION OF HOMOCHOLINE BY RESTING

CELL SUSPENSIONS AND GROWING CELL CULTURES

OF THE ISOLATED STRAINS

3.1 INTRODUCTION

Traditionally, organisms for bioremediation purposes have been sourced from

environments exposed to the target compounds on the assumption that there is a better

likelihood for these organisms to have potential to degrade these compounds. This has

been undertaken in numerous studies on a variety of quaternary ammonium compounds

including: cetyl trimethyl ammonium bromide (CTAB), dodecyl benzyl dimethyl

ammonium chloride and didecyl dimethyl ammonium chloride (Gerike, 1982; Van Ginkel

et al., 1992; Takenaka et al., 2007), choline (Shieh, 1964; Kortstee, 1970; Mori, 1981)

and L-carnitine (Goulas, 1988; Mori et al., 1988, Mori et al., 1994) among others. It is

thought that the soil environment exerts a selective pressure that selects for organisms

with the metabolic capability to degrade the target compound as a carbon and energy

source and that the likelihood of isolating an organism with degradative capabilities from

these sources is greater (Margesin et al., 2003).

Quaternary ammonium compounds such as choline, glycine betaine and carnitine

are widely distributed components in many organisms. These naturally occurring

57

quaternary ammonium compounds as well as synthetic quaternary ammomum

compounds are metabolized in the nature by soil microorganisms. Many

microorganisms used choline as a sole source of carbon and nitrogen has been isolated

from soil and the degradation pathway of choline of these isolates was studied (Ikuta et

al., 1977; Mori et al., 1980, 1992, 2002; Nagasawa eta!., 1976). In microorganisms

choline is oxidized by either choline oxidase (EC 1.1.3.17) or membrane bound choline

dehydrogenase (EC.l.l.99.1) to betaine aldehyde, which in tum is oxidized to glycine

betaine by a soluble betaine aldehyde dehydrogenase (EC 1.2.1.8) (Andersen et a!.,

1988; Mori eta!., 1980; Nagasawa et al., 1976; Tani et al., 1977).

Despite its similarity to choline in many aspects, very few microorganisms

degrading homocholine as only source of carbon and nitrogen has been isolated (Chapter

2) and consequently, the catabolic pathway remains unclear. From the choline-like

structure, one would expect that homocholine could be degraded in a similarly and at a

rate comparable to that of choline. Thus, an osmoprotectant B -alanine betaine could be

an intermediate product in the degradation pathway of homocholine. Therefore, the

study of homocholine degradation pathway and the enzymes involve in is of

considerable interest. From an applied standpoint, the recent findings that many marine

bacteria and plant species accumulate B-alanine betaine in response to salt stress or

water deficit (Hanson et al., 1994; Rathinasabapathi et a!., 2000) have prompted

considerable interest in research on B-alanine betaine biosynthesis, with the goal of

genetically engineering water/osmotic stress resistance in beneficial bacteria and crop

plants (Rathinasabapathi et al., 2000). With this in mind, an investigation was carried

out to see which mechanism is followed in the degradation of homocholine by soil

58

microorganisms. This beside, whether B-alanine betaine is accumulate as an intermediate

metabolite or not. Therefore, this study was carried out to identify and quantify the

metabolites released from homocholine by the isolated strains that utilize homocholine

as a carbon and nitrogen source, as well as to elucidate the degradation pathway of

homocholine by these isolates. In this chapter, we have isolated and identified

metabolites librated from homocholine by the isolated strains. Furthermore, the partial

pathway for homocholine degradation was also postulated.


3.2.1 Materials

3-N-Dimethylaminopropionic acid (DMA-Propionic acid), and p-bromophenacyl

bromide were purchased from Tokyo Kasai (Tokyo, Japan). Benzoyl chloride,

dehydrated pyridine and methyl iodide were from Wako Pure Chemicals Co. Ltd.

(Tokyo, Japan). 0-(4-Nitrobenzyl)-hydroxyarnine was obtained from Fluka Chemicals

(GmbH, Switzerland). Unless otherwise specified, all other reagents were of analytical

grade from either Wako (Tokyo, Japan) or Sigma (St. Louis, MO, USA).

3-N-Trimethylarnino-1-propanol iodide (homocholine) was prepared from 3-N

dimethylarnino-1-propanol as described in Chapter 2. 3-N-Trimethylaminopro

pionaldehyde was also prepared from 3-arninopropion-aldehyde diethylacetal as

described in Chapter 2.


General weight measurements were made using IB-200H electronic balance (Shimadzu

corp. Kyoto, Japan) and smaller quantities and measurements for the preparation of

59

standards were made using Mettler AE240 analytical balance (Mettler, Toledo, AG,

Switzerland). pH was determined using Horiba F-22 pH meter (Horiba Ltd, Kyoto,

Japan). Cultures and extracts were centrifuged using either a medium size centrifuge

Himac CF16RX2 or big size centrifuges Himac SCR 18B and RC 20B (Hitachi Koki Co

Ltd., Tokyo, Japan). Media were autoclaved at 121 oc for 20 min using BS-235 High

Pressure Steam Sterilizer (Tomy Seiko Co., Ltd., Tokyo, Japan). Filter sterilization of

solutions was carried out using 0.22 !!Ill disposable filters MILLEX®-GA (Millipore,

Molshiem, France). Samples were vortex using a Vortex Genie 2 (Scientific Industries,

Inc., USA). Benzoylation, esterification and hydroxylation of samples and standards

were carried out at 90°C using Eyala MG-200 hot plate (Tokyo Rikakika Co., Ltd.,

Japan).


3-N-Trimethylarninopropionic acid (~-alanine betaine) was synthesized by N

methylation of dimethylarninopropionic acid (Tokyo Kasei Kogyo Co. Ltd., Tokyo,

Japan) with methyl iodide. Methyl iodide (4 rnl) was added to a suspension of

dimethylarninopropionic acid (1 g, 6.5 mmol) and KHC03 (1.3 g, 13 mmol) in 20 rnl of

methanol. Then the mixture was stirred overnight at room temperature. The mixture was

decanted. Subsequently, the liquid phase was concentrated and the residue was extracted

using a mixed solvent (acetonitrile: methanol =10:1, v/v, 15 rnl x 3). The combined

extracts were dried under a nitrogen stream to give ~-alanine betaine as a colourless

powder (1.2 g, 63.2%). The structure and purity of ~-alanine betaine were confirmed

using 1H NMR (Fig. 3.1) and capillary electrophoresis.

60

H

!i"Ahmm"

Fig. 3. 1 1 H NMR spectrum of chemically synthesized 3-N-trimethylamino-1-propionic acid (~-alanine betaine)

3.2.4 Growth and Homocholine Degradation by the Isolated Strains

The isolated strain was cultivated at 25°C on 75 ml of basal-HC liquid media containing

20 mM homocholine as a sole source of carbon, nitrogen and energy. At intervals of 9,

12, 15, 18 and 24h about 10 m1 culture was withdrawn by pasteurized pipette and

centrifuged at 10,000 x g for 20 min at 4°C. The supernatant was collected and

preserved at -20°C until used for detection of residual homocholine and the metabolites

of its degradation as described below.

61

3.2.5 Homocholine Degradation by Resting Cells of the Isolated Strains

The isolated strain was cultivated (24 h at 25°C) on 75 ml of basal-HC liquid media

containing 20 mM homocholine as a sole source of carbon, nitrogen and energy. The

cells were harvested at the exponential phase by centrifugation at 10,000 x g for 20 min

at 4°C, washed three times with saline solution (8.5 giL KCl) and re-suspended in 50

mM potassium phosphate buffer (pH 7.5). The resting cell reaction was started by the

addition ofhomocholine (20 mM) to the cell suspension. The suspension was incubated

on a shaker at 120 rpm and 30°C. At appropriate time intervals (30 min, 1, 2, 3 and 6 h),

aliquots of the cell suspension were withdrawn and boiled for 3-5 min to stop the

reaction. After centrifugation, the supernatants were preserved at -20°C and used for

metabolites detection as described below.

3.2.6 Isolation and Quantification of the Metabolites of Homocholine

3.2.6.1 Homocholine

Since quaternary ammonium compounds lack an absorbance in UV NIS light range,

derivetization is most important. The decreasing amount of homocholine was detected

during the degradation of the compound by the isolated strains according to the method

of Zhang et al. (200 1 ). In this method, 0.1 ml of the supernatants of either culture

filtrate or intact cell reaction mixtures and an authentic standard ofhomocholine (10 mM

in 2-propanol containing 3% water) were mixed with 0.3 ml of benzoyl chloride (10% in

pyridine). The tube was caped and heated at 80°C for 15 min. After cooling to room

temperature, 1 ml of chloroform and 0.5 ml of water were added and the mixture was

centrifuged at 10,000 x g for 20 min. The supernatant contained benzoylated

homocholine was applied directly to CE at hydrostatic mode (25 em height for 30 sec)

62

and electrophoresis was carried out at 20 kV for 20 min. Standards homocholine was

treated in the same manner. Each experiment was carried out in triplicate.

3.2.6.2 T1imethylaminopropionaldehyde (TMAPaldehyde)

The supernatants of either culture filtrate or intact cell reaction mixtures (0.1 rnl) were

treated with 0.3 rnl of 0-(4-nitrobenzyl) hydroxylamine in methanol (5 mg/ rnl) and

heated at 80°C for 15 min. After cooling to room temperature, 0.1 rnl of 50 mM sodium

phosphate buffer (pH 3.0) was added to the reaction mixture and the existance of the

metabolite trimethy larninopropionaldehyde-oxime was detected using low pH capillary

electrophoresis (Zhang et al., 1997).

3.2.6.3 P-alanine betaine and trimethylamine (TMA)

Both ~-alanine betaine and TMA were detected by low pH capillary electrophoresis

after esterification with p-bromophenacyl bromide following the methods of Nishimura

et al. (2001) with slight modifications. In micro tube, 0.1 ml of the collected

supernatants of either culture filtrate or intact cell reaction mixture and authentic

standards (~-alanine betaine and TMA) were placed and mixed with 0.05 rnl of a buffer

solution (1 00 mM KH2P04: distilled water: acetonitrile= 1: 1:4). To the mixture, 0.3 ml

of p-bromophenacyl bromide (20 mg/ml in acetonitrile) was added. The tube was

capped and heated at 90°C for 90 min. The reaction mixture was evaporated to dryness

with a centrifugal evaporator. Three hundreds microliters of 50 mM sodium phosphate

buffer (pH 3.0) was added, mixed well and centrifuged at 10,000 x g for 20 min at 4°C.

The supernatants, which contained esters of the metabolites ~-alanine betaine and TMA,

were analyzed using capillary electrophoresis.

63

3.2.6.4 Tiimethylamine (TMA)

In glass tubes, the supernatants of either culture filtrate or intact cell reaction mixtures

(1 ml) were placed and then 0.5 ml toluene and 0.5 ml of 8N NaOH were added and

mixed by vortex for 15 sec. The mixtures were stand at room temperature for about 5

min, and then vortex mixed again for 15 sec, and thereafter stands at room temperature

for another 5 min. The organic layers were transferred to new glass tube and tightly

close. The upper layer contained the metabolite TMA, which was identified by GC

MS as described below. The authentic standard ofTMA was treated in the similar way.

3.2. 7 Analytical Methods

Degradation of homocholine and the production of metabolites by intact cells of the

isolated strains were detected using capillary electrophoresis, GC-MS and F AB-MS.

3.2.7.1 Capillary electrophoresis (CE)

Capillary electrophoresis analysis was conducted with a device (Photal CAPI-3300;

Otsuka Electronics. Co. Ltd., Osaka, Japan) equipped with a fused silica capillary of 75

J..Ull i.d. with a total length of 80 em (effective length of 68 em). A new capillary was

conditioned with 0.1M NaOH for 5 min followed by 3 min distilled water and 3 min

electrolyte buffer (50 mM sodium phosphate buffer, pH 3.0). Samples and relative

standards were injected hydrostatically (25 mm, 60 s). The applied potential was 20

kV, and the peaks of benzoyl-homocholine, trimethylarninopropionaldehyde-oxime, and

TMA- and ~-alanine betaine-esters were monitored, respectively, at 200, 270 and 254

nm.

64

3.2.7.2 Gas chromatography-mass spectrometry (GC-MS)

Gas chromatography-mass spectrometry analysis was conducted usmg a mass

spectrometer (JEOL AX505 HA; JEOL, Tokyo, Japan) with electron-impact ionization

(70 eV) coupled with a gas chromatograph (5890 series II; Hewlett-Packard Co.,

Wilmington, DE, USA). A fused silica capillary column (0.25 mm i.d., 30 m long)

packed with DB1 (J&W Scientific Inc., Folsom, CA, USA) was used. Helium was used

as the carrier gas at a flow rate of 15 ml min-1. The column temperature was maintained

at 50°C and samples (1 ~) were injected onto the GC at an injection port temperature

of250°C.

3.2.7.3 Fast atom bombardment- mass spectrometry (FAB-MS)

Fast atom bombardment - mass spectrometry analysis was used to detect ~-alanine

betaine and 3-N-trimethylarninopropionaldehyde following the method described by

Rhodes et al. (1987). The supernatants of resting-cell reaction mixtures and the culture

filtrate, alongside with the authentic standards, were applied directly to the FAB-MS

spectrometer (JEOL AX505 HA; JEOL, Tokyo, Japan). Aliquots of 1 ~ sample and

authentic standard were mixed with glycerol and applied to the F AB probe and

irradiated with Xenon (3 kV, 50°C).


3.3.1 Identification of Homocholine Degrading Metabolites

Degradation of quaternary ammonium compounds, such as choline, L-camitine and

trimethylamino-1-butanol, has been reported for strains of the genera Arthrobacter,

Xanthomonas, Agrobacterium and Pseudomonas among others. Betaine aldehyde,

65

glycine betaine, dimethyl glycine, sarcosine, 3-dehydrocamitine, trimethylamino-1-

butyraldehyde and gamma-butyrobetaine were identified as intermediate metabolites

(Ikuta et al., 1976; Mori et al., 1988; Hanschmann et al., 1996; Hassan, 2008). They all

seem to adopt the oxidation pathway by either a dehydrogenase or oxidase enzymes.

In the previous chapter, strains belong to the genera Arthrobacter, Rhodococcus,

and Pseudomonas were isolated from soil using homocholine as a sole source of carbon,

energy and nitrogen.

In this chapter, the degradation of homocholine by the resting cell suspensions and

growing cell cultures of isolated strains and the detection of formed metabolites were

tested by capillary electrophoresis, GC-MS and F AB-MS methods. During the

consumption of homocholine by the resting cell suspensions and the growing cell

cultures of the isolated strains (Arthrobacter sp. strain E5, Rhodococcus sp. strain A2,

and Pseudomonas sp. strain A9 and B9b), there were a concurrent formation and

accumulation of some soluble metabolites as detected by capillary electrophoresis

analysis (Fig. 3.2). These metabolites were found to be trimethylamine (peak 1, TMA)

and ~-alanine betaine (peak 2, ~-AB) as compared to the authentic standards of TMA

and ~-AB.

66

<II u c ~ 6 .! <(

0.06 < ~

Standard

0.04

Pseudomonas sp. Strain A9

Arthrobacter sp. Strain E5

0.00 i ·~. •• . •• ~- .

10 12 14 16 18 20 Retention time (min)

Fig. 3. 2 Capillary electrophoresis chromatogram of the intermediate metabolites of homocholine biodegradation.

The chromatogram showed the generated metabolite peaks from the degraded homocholine by the isolated strains alongside with authentic standards peaks of trimethylamine (TMA) and ~-alanine betaine (~-AB).

The first evidence for the accumulation ofTMA was the remarkable fishy-odor of

the culture filtrate of homocholine growing cells. Further analysis of the culture filtrate

and the intact cell reaction mixtures of the isolated strains by GC-MS confirmed the

accumulation ofTMA (Fig. 3.3). The mass spectra (M+, 59) and the retention time (1.5

min) of the observed metabolite, agreed with those of authentic standard of TMA

treated and extracted in the same way. These results demonstrated that TMA is a major

metabolite of the degraded homocholine by the isolated strains.

67

b ";) c: $ .5% j

80

1i 40

I.

57

56

55 56 57

+ M 59

Time (min)

53 59 6 61 62 mJz

Fig. 3. 3 GC-MS spectra of the metabolite (TMA) formed during the degradation of homocholine by the isolated strains.

Furthermore, analysis of the culture filtrate and the intact cell reaction products

by F AB-MS demonstrated the accumulation of ~-alanine betaine and 3-N-

trimethylaminopropionaldehyde (TMAPaldehyde ). The culture filtrate and the intact

cell reaction products spectrum of the isolated strains (Fig. 3.4) showed signals at m/z

118 [M + Ht of homocholine, m/z 131 [M+] of ~-alanine betaine and m/z 115 [M+] of

TMAPaldehyde. Those signals agreed with the signals and the theoretical masses of the

authentic standards of homocholine, ~-alanine betaine and TMAPaldehyde, respectively.

As expected, the results demonstrated that all the isolated strains oxidized homocholine

to TMAPaldehyde and ~-alanine betaine.

68

~ ·;; c s c ·;

0/o .c: -m

~

20 +

:\1 115

+ (M+H)

m/z

+ 2\1 131

135

Fig. 3. 4 F AB-MS spectra of metabolites in the degradation pathway of homocholine by the isolated strains.

Metabolites with M+ of 115 and M+ of 131 were identified respectively as TMAPaldehyde and ~-alanine betaine.

3.3.2 Degradation of Homocholine by Resting Cells of the Isolated Strains

During the degradation of homocholine by resting cells of the isolated strains, the

amount of homocholine decreased concomitantly with the increase of metabolites,

identified as ~-alanine betaine and trimethylamine. The time course degradation of

homocholine by resting cells of the isolated strains is depicted in Fig. 3.5. Resting cells

of the isolated strains almost completely degraded homocholine within 6 h, and the

metabolites TMAPaldehyde and ~-alanine betaine formed and metabolized when the

reaction elevated. The transient accumulation of ~-alanine betaine in the medium

indicated that it was an intermediate metabolite during the degradation of homocholine

and not a dead-end product. These results indicated the sequential oxidation of

69

homocholine to trimethylaminopropionaldehyde and ~-alanine betaine. In agreement

with our results, consequent oxidation of alcohol groups of the quaternary ammonium

compound to aldehyde and acid was reported (Nagasawa et al., 1976; Mori et al., 1980,

1992, 2002; Hassan et al., 2007).

It is particularly notable that the detection of ~-alanine betaine as an intermediate

in the homocholine degradation pathway by the isolated strains is important from a

biotechnological standpoint. We assume that the enzymes responsible for the formation

of ~-alanine betaine might be useful in biotechnology for the engineering of osmotic

stress tolerant crop plants. It was proposed that genetic engineering of osmotolerance in

plants could be achieved by producing betaine in non-accumulators (McCue and Hanson,

1990). This has been demonstrated in several reports where transgenic plants

accumulating glycine betaine exhibit tolerance to salt, cold and heat stresses (Hayashi et

al., 1997; Alia et al., 1998a, b).

70

A 25 0.5

20 0.4 ~ :E § g <( "' ~ 15

c: 0.3 ,.

«l (; .0

"' "' .!: c: 0 ·r: 13 0.2 .:: 0 l. "' E '" 0 I

~-~--~-----"--! ii)

:c ro

r0.1

Iii I'

0 8 25

I 0.4 ~ ~ 20·~ :E ..

E \ -"- ---~~- ,~.~~'>~- g - \ "' <( \ r;: :E Iii ~ 15 \ 0.3 -;::; \ .,

Iii .0

"'

~02 "' £ ~---,_ c:

0 ·r: J:: "' g 10 iii E ~ 0 :c \ ro

0.1

c

:? ::: E \ g <(

Ill c:

:::;: 15 0.3 ·c; 1- c;

.0

"' - - "' £ c ·r: :g 10 0.2 "' () iii 0 ,;, E I ~ 0 ro :c

Time (h)

Fig. 3. 5 Degradation of homocholine by resting cells of the isolated strains.

Time course degradation ofhomocholine (1111) and the generation of the metabolites, TMA ( o) and beta-alanine betaine (1:::.) by strains: (A)Arthrobacter sp. strain E5, (B)

Rhodococcus sp. strain A2 and (C) Pseudomonas sp. strains A9.

71

Moreover, the degradation ofhomocholine by the isolated strain was accompanied

by a stoichiometric formation of trimethylamine and degradation of the carbon skeleton.

This might be a result of the cleavage of a C-N bond of B-alanine betaine by a

monooxygenase enzyme. This was further confirmed by the detection of TMA in the

culture filtrate of the isolated strains grown on B-alanine betaine as a sole source of

carbon and nitrogen (data not shown). In addition, the intact cell reaction of these cells

with B-alanine betaine again showed an accumulation of TMA as major metabolites.

Similarly, utilization of choline, y-butyrobetaine, DL-camitine by Candida tropicalis,

Acinetobacter calcoaceticus, Pseudomonas putida and Proteus vulgaris are correlated

respectively with the stoichiometric formation ofTMA and completedegradation of the

carbon skeleton (Seim et al., 1982; Miura-Fraboni and Englard, 1983; Mori et al., 1988).

Additionally, the initial microbial degradation of methylamines, including long chain

quaternary ammonium compounds, tetramethylammonium chloride, and nitrilotriacetic

acid, always involves the breakage of C-N linkages (Tiedje et al., 1973; Hampton and

Zatman, 1983; Van Ginkel et al., 1992). Initial cleavage of the C-N bond was proposed

as a general strategy of microorganisms to gain access to the alkyl chains of quaternary

ammonium compounds (Van Ginkel, 1996). Furthermore, the pathways in bacteria that

have been suggested for TMA formation from choline and camitine are based

exclusively on reductive cleavage of C-N bond of these compounds, and thus yielded

ethylene glycol and B-hydroxybutyrate, respectively (Mori et al., 1988: Kleber et al.,

1978). In accordance with the above-mentioned pathways, we proposed that cleavage of

C-N bond of B-alanine betaine accompanied with the formation of TMA and 3-

72

hydroxypropionate. Trimethylamine is further utilized as a nitrogen source, whereas 3-

hydroxypropionate is used as carbon and energy source.

Although our study showed the cleavage of C-N bond as in the aforementioned

studies, but this was not the initial step in the degradation of homocholine by the

isolated strains. This demonstrated the novel degradation pathway of homocholine by

the isolated strain that is quite different from the above-mentioned studies. In this

study, we demonstrated that the alcohol group ( -OH) of homocholine was consequently

oxidized to aldehyde (-CHO) and carboxyl (-COOH) group, with cleavage of C-N bond

providing TMA and alkyl chain (C-3 moiety) (Fig. 3.6).

Although the possibility of the demethylation pathway, from ~-alanine betaine to

dimethylaminopropionic acid, particularly in Arthrobacter sp. strain E5, is still

considered since the amount of the formed TMA is quite low compared to the degraded

substrate (homocholine). This beside that the substrate dimethylaminopropionic acid

was also utilized for growth by the isolated strain.

73

"+ /~~OH 3-:\- t rim(•th~·lamino-1-propanol

rhomorholinl·l

---!!!>-

0

""'+~ /N H I

J-:\- trimetln lamino-1-propionaldchydr !T\1:\PALI

1 0 C0 2

+ ~...- ...--""- + II

C3-carbon N~OH chain / 1

H2 0

+

biomass ...-~~

/..s ·ldmeth.' lom;u<>·l·pmp;onk "dd N 1 ~-alanint helaine)

I Trimdh~ laminl'

Fig. 3. 6 Proposed pathway for the aerobic biodegradation ofhomocholine by the isolated strains

3.4 CONCLUSIONS

This study was attempted to characterize and identify the metabolites of homocholine

degradation by the isolated strains as well as to elucidate the degradation pathway of the

compound by these isolates. As expected, the metabolites TMAPaldehyde, ~-alanine

betaine and trimethylamine were detected in the culture filtrate and intact cell reaction

mixtures of the isolated strains.

The degradation pathway of homocholine was found to be through consequent

oxidation of alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH), and thereafter

cleavage of C-N bond of ~-alanine betaine to give trimethylamine and alkyl chain (C3-

moiety).

74

It is particularly notable that the detection of P-alanine betaine as an intermediate

during homocholine degradation pathway by the isolated strains is important from a


of P-alanine betaine might be useful in biotechnology for the engineering of osmotic

stress tolerant crop plants. Therefore, our further research will be focused on isolation,

characterization and possible application of homocholine degrading enzymes. This

information will be important to understand the mechanisms involved in the degradation

pathway of this compound.

75

CHAPTER4

ENZYMATIC ACTIVITY IN THE DEGRADATION

PATHWAY OF HOMOCHOLINE BY THE ISOLATED

STRAINS

4.1 INTRODUCTION

Enzymes are remarkable catalysts capable of accepting a wide array of complex

molecules as substrates and catalyzing reactions with unparalleled chiral (enantio-) and

positional (regio-) selectivities (Schmid et al., 2001). Such high selectivity also affords

efficient reactions with few by-products, thereby making enzymes an environmentally

friendly alternative to conventional chemical catalysts (Schmid et al., 2001). Among

various classes of enzymes, oxidoreductases represent a highly versatile class of

biocatalysts for specific reduction and oxidation reactions, and currently used for the

production of a wide variety of chemical and pharmaceutical products (Johannes et al.,

2006). To this decade, more than 1,000 oxidoreductases are known from which 80% use

NADH as cofactor, 10% use the corresponding phosphates. Flavins and

pyrroloquinoline quinone are involved more rarely (Peters, 2000). The glucose

methanol-choline (GMC) oxidoreductase enzyme superfamily is composed of a group

of enzymes that are able to catalyze the conversion of alcohols to the corresponding

aldehydes or ketones. This family includes alcohol dehydrogenase, glucose oxidase,

77

cholesterol oxidase, cellobiose dehydrogenase, pyranose 2-oxidase, choline

dehydrogenase, choline oxidase and betaine aldehyde dehydrogenase (Cavener, 1992).

The later three enzymes play an important role in the metabolism of choline in both

mammalian and microorganisms.

In choline degradation pathway, choline is oxidized to glycine betaine by two

reactions: (i) choline to betaine aldehyde by either choline oxidase or choline

dehydrogenase (Ikuta et al., 1977; Landfald et al., 1986; Nagasawa et al., 1976;

Rathinasabapathi et al., 1997; Rozwadowski et al., 1991; Russell et al., 1994; Russell et

al., 1998; Tsuge et al., 1980; Yamada et al., 1977) and (ii) betaine aldehyde to glycine

betaine by a NAD+ -dependent betaine aldehyde dehydrogenase (Arakawa et al., 1987;

Falkenberg et al., 1990; Nagasawa et al., 1976; Mori et al., 1992; Mori et al., 1980; Mori

et al., 2001; Valenzuela-Soto et al., 1994).

TMA-Butanol, a choline analogue, had a similar pathway as choline. In oxidative

reaction, TMA-Butanol is converted to TMABaldehyde by a NAD+ -dependent TMA

butanol dehydrogenase. Consequently, TMABaldehyde is oxidized to y-butyrobetaine

by a NAD+ -dependent TMABaldehyde dehydrogenase. Both enzyme have been

isolated and purified from Pseudomonas sp. 13CM (Hassan, 2008). On the other hand,

enzymatic studies on the degradation pathway of homocholine, an analogue of choline

and TMA-Butanol, by soil microorganisms were not yet carried out.

In the previous chapters, the author postulated the degradation pathway of

homocholine by strains belongs to the genera Arthrobacter, Rhodococcus and

Pseudomonas. Similar to choline and TMA-Butanol, homocholine was consequently

oxidized to TMAPaldehyde and ~-alanine betaine. Thereafter, cleavages of ~-alanine

78

betaine C-N bond yielded trimethylamine and alkyl chain. In this chapter, screening and

preliminary studies on the oxidative enzymes in the degradation pathway of

homocholine by Pseudomonas sp. strain A9 will be described.


4.2.1 Materials

3-Dimethylamino-1-propanol (DMA-Propanol), 4-dimethylamino-1-butanol (DMA-

Butanol), and 4-aminobutanol were purchased from Tokyo Kassei (Tokyo, Japan).

Homocholine iodide was prepared from 3-dimethylamino-1-propanol according to the

method described in Chapter 1. TMAPaldehyde iodide was prepared from 3-

aminopropoinaldehyde dimethylacetal according to the method described previously in

Chapter 1. Protease inhibitor cocktail Set II was from Calbiochem (Darmstadt,

Germany; Table 4.1). TSK-gel G3000SW was from Tosoh (Tokyo, Japan). Standard

proteins for gel filtration were from BIO-RAD (Hercules, USA). All other reagents

were commercial products of analytical grade from Wako Pure Chemicals Co. Ltd.

(Tokyo, Japan).

Protease inhibitor

AEBSF hydrochloride

Bestatin

E-64, protease inhibitor

EDT A, disodium

Pepstatin A

Table 4. 1 Protease inhibitor cocktail

Target protease

Serine protease

Aminopeptidase B, leucine aminopeptidase

Cysteine protease

Metallo protease

Aspartic protease

79


Novaspec II from Amersham Pharmachia Biotech was used to check the turbidity.

HITACHI HIMAC SCR 18B (rotor: RPR9-2) was used for centrifugation. For

measurement of enzyme activity, Shimadzu UV-2100S was used. For HPLC system,

Gilson HPLC System was used. Unipoint TM HPLC system control software was

used for data analysis. AE-6400 Rapidus-NirenMinisurabu Electrophoresis· Apparatus

from ATTO Corporation was used for polyacrylamide gel electrophoresis.

4.2.3 Screening for Homocholine degrading Enzymatic Activities

4.2.3.1 Plate replica staining method

An additional screening step was setup in order to narrow the selection range and to see

the metabolic ability of the isolated strains. In this step, the isolated strains (30 highly

growth strains) were screened for homocholine oxidation activities using plate replica

activity staining methods. In this method, the isolated strains were transfer individually

using sterilized tooth stick to a single basal-HC agar plate medium. The plate was

incubated at 30°C for either 24 or 48 h. The grown colonies were replicated onto a filter

paper, treated with 1 ml of lysing solution (0.5% lysozyme and 10 mM EDTA in 100

mM potassium phosphate buffer, pH 8.0) and incubated at 37°C for 30 min. After

lysis of the cells, the filter paper was sunk in a solution containing inhibitors of energy

generating system (10 mM of NaN3, 10 mM of NaF, 1 mM of Na2As04) for 5 min.

Then, the filter paper was frozen and thawed three times to inactivate the energy

generating system. After drying, the dried filter paper was sunk in 2 ml of activity

staining solution containing 100 mM of glycine-NaOH buffer (pH 9.5), 64 ~M of 1-

methoxy phenazine methosulfate, 0.24 mM of nitroblue tetrazolium (NTB), 1 mM of

80

NAD+ and either 2 mM homocholine or 1 mM ofTMAPal iodide. The reduction of the

NTB lead to the formation of the purple color is an indictor of the enzymatic activity.

Strains showed positive result with replica staining were selected and used for

preparation of cell free extract to confirm the enzyme activity by the

s pectrophotometeric methods.

4.2.3.2 Cell free extract preparation

To check the enzyme activities of the isolated strains, the strains were inoculated into

75 ml of basal-HC liquid medium and incubated at 25°C for 2 days in reciprocal shaker.

At the end of cultivation period, bacterial cells were harvested by centrifugation at

10,000 x g for 20 min and washed with 0.85 % KCl. The harvested bacterial cells were

suspended in 30 ml of 0.2 M potassium phosphate buffer pH 7.5 containing 1mM

dithiothreitol (DTT) and disrupted by either sonication at 4~ 11 oc for 15 min with 1.5

min run intervals or by glass bead (0.1 mm) on dzy ice or in a cold room for 10 cycles

with 30 sec for each cycle. Cell free extract was obtained by centrifugation at 10,000 x g

for 20 min as supernatant and assayed for dehydrogenase and oxidase activities.

4.2.3.3 NAD+ -dependent homocholine dehydrogenase

Homocholine dehydrogenase activity was measured as an increase in the absorbance at

340 nm at 300C. The standard reaction mixture (1.5 ml) contained 225 !J.mol of Tris

HCl buffer (pH 7.5), 33.3 !J.mol of homocholine iodide, 3 !J.mol of NAD+ and an

appropriate amount of the enzyme. The reaction was started by the addition of NAD+

and the enzyme activity was calculated using an extinction coefficient of 6,200 M- 1·cm-1

of NADH at 340 nm. One unit of enzyme activity was defined as the amount of

81

enzyme that catalyzes the formation of 1 11mol of NADH per min under the assay

conditions. Specific activity was defined as units of enzyme activity per mg protein.

4.2.3.4 NAD+ -dependent TMAPaldehyde dehydrogenase

The standard reaction mixture (1.5 rnl) contained 225 11mol of potassium phosphate

buffer (pH 8.0), 15 11mol of TMAPaldehyde iodide, 3 11mol of NAD +, and an

appropriate amount of the enzyme. The reaction was started by the addition of NAD+

and the enzyme activity was calculated using an extinction coefficient of 6,200 M- 1·cm-1

of NADH at 340 nm. One unit of enzyme activity was defined as the amount of

enzyme that catalyzes the formation of 1 11mol of NADH per min under the assay

conditions. Specific activity was defined as units of enzyme activity per mg protein.

4.2.3.5 Membrane-bound homocholine dehydrogenase

Membrane-bound homocholine dehydrogenase activity was measured as a decrease in

the absorbance at 600 nm at 30°C using phenazine methosulfate (PMS)-2,6-

dichlorophenol-indophenol (DCIP) assay system, according to the method of Nagasawa

et al. (1976). The assay mixture (1.5 rnl) contained 67.5 11mol of potassium phosphate

buffer (pH 8.0), 1.5 11mol of potassium cyanide, 0.375 11mol of 1-methoxy PMS, 0.15

11mol ofDCIP, 22.5 11mol ofhomocholine iodide and an appropriate amount of enzyme.

The assay was started by the addition of homocholine iodide. The activity was

calculated using an extinction coefficient of 21,500 M- 1·cm-1 for oxidized DCIP at 600

nm. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the

reduction of 1 11mole of DCIP per min under the standard assay conditions.

82

4.2.3.6 Homocholine oxidase

Homocholine oxidase activity was measured by the modification of the procedure

described by Tani et al. (1977). The reaction mixture contained 135 11mol of potassium

phosphate buffer (pH 8.0), 45 11mol of homocholine iodide and a suitable amount of

enzyme in a total volume of 3.0 rnl. The reaction was carried out with shaking at 3CfC

for 10 min. One rnl of the reaction mixture was mixed with 1 rnl of 0.1% solution of

2,4-dinitrophenyhydrazine in 1 M HCl and heated in a boiling water bath for 7 min.

The mixture was cooled to room temperature and then added to 6.4 rnl of distilled water.

After the addition of 1.6 rnl of 2 M NaOH, the absorbance at 440 nm was measured.

TMAPaldehyde was used to create the standard curve to calculate the enzyme activity.

One unit of the enzyme activity was defined as the amount of the enzyme that

catalyses the formation of 111mol of TMAPaldehyde per min under the above assay

conditions.

4.2.3. 7 NAD+ -dependent 3-hydroxypropionate dehydrogenase

3-Hydroxypropionate dehydrogenase activity was measured as an increase in the

absorbance at 340 nm and at 30°C. The standard reaction mixture (1.5 rnl) contained

225 11mol of Tris-HCl buffer (pH 7.5), 33.3 11mol of 3-hydroxypropionate, 3 11mol of

NAD+ and an appropriate amount of the enzyme. The reaction was started by the

addition ofNAD+ and the enzyme activity was calculated using an extinction coefficient

of 6,200 M-1-cm-1 of NADH at 340 nrn. One unit of enzyme activity was defmed as

the amount of enzyme that catalyzes the formation of 1 11mol ofNADH per min under

the assay conditions. Specific activity was defined as units of enzyme activity per mg

protein.

83

4.2.4 Time Course of the Growth and Enzyme Formation of Pseudomonas sp.

stmins A9

In order to determine the cultivation time needed for high enzyme activity formation,

time course experiment was preformed. Seed culture was prepared by cultivation of

Pseudomonas sp. strain A9 in 5 ml basal-HC medium at 25°C for 24h with reciprocal

shaking at 122 strokes/min. Then the turbidity of the seed culture was determined by

measuring the optical density at 660 nm. At the growth of 2.0 (T660 =2.0), 200 ~of

the seed culture broth was inoculated in 300 ml of basal-HC medium. Cultivation was

carried out at 25°C on a reciprocal shaker. At intervals of 12,15, 18, 22, and 26h the

growth was estimated and the bacterial cells were collected by centrifugation at 6, 000 x

g (HITACHI HIMAC SCR 18B rotor: RPR9-2) for 30 min at 4°C and washed three

times with 0.85% KCl. The harvested bacterial cells were suspended in 10 ml of 0.1 M

potassium phosphate buffer, pH 7.5, containing 1mM dithiothreitol (DTT) and 1 mM

EDT A, and disrupted by glass bead (0.1 mm) on dry ice for 5 min. Cell free extract was

obtained by centrifugation at 10,000 x g for 30 min as a supernatant and assayed for

homocholine dehydrogenase activity.

4.2.5 Stability of Homocholine Dehydrogenase Activity of Strain A9

In order to select hornocholine' degrading strain, stability of the homocholine

dehydrogenase was evaluated. Cell free extract of each selected strain was dialyzed

against potassium phosphate buffer, tris-HCl buffer and triethanolamine buffer (50 mM,

pH 7.5) containing 1 mM of DTT and 1mM EDTA for overnight at 4°C. The dialyzed

extracts were stored at 4°C for 0 tolO days and the remaining enzyme activity was

estimated. The stability of the enzyme against storage was also evaluated. The cell free

84

extracts were stored at 4°C for 0 to 10 days and the remaining activity was measured.

The stability of homocholine dehydrogenase was also examined in the presence of SH

protecting reagents such as DTT, 2-mertcaptoethanol and glutathione, as well as in the

presence of stabilizing reagents such as ethanol, glycerol and ethylene glycol.

4.2.6 Formation of Homocholine Dehydrogenase on Various Media

Pseudomonas sp. strain A9 was grown on basal medium supplemented with 1% of

quaternary ammonium compounds such as homocholine, choline, TMA-butanol and L

carnitine as a sole source of carbon and nitrogen. The strain was also cultivated on basal

homocholine medium supplemented with either glucose (1 %) or glycerol (1 %) as a

carbon source or ammonium sulfate (0.5%) as a nitrogen source. Cultivation was carried

out in 300 rnl medium/1L cultural flask on a reciprocal shaker (120 strokes/min) at 25°C

for 24h.

4.2. 7 Measurement of Protein Content

Protein was measured by Lowry method (Lowry et al., 1951) using bovine serum

albumin as a standard protein (Fig. 4.1), or by the absorbance at 280 nm.

85

lj b

I) 1 BSA concentra1ion,

Fig. 4. 1 Bovine serum albumin standard curve as determined by Lowry method

4.2.8 Measurement of Molecular Mass

The molecular mass of the enzyme was estimated by gel filtration on TSK-gel

G3000SW column (0.8 x 30 em) equilibrated with 0.1 M of potassium phosphate buffer

(pH 7.5) containing 1mM DTT, 1 mM EDTA and 5% ethanol. The standard proteins

used were thyroglobulin, y-globulin, ovalbumin, myoglobin and vitamin B12.

4.2.9 Polyacrylamide Gel Electrophoresis

Polyacryamide gel electrophoresis (native-PAGE) was done using either 10% or 7.5%

gels according to the methods of Laemmli (1970). Protein was stained by commasie

brilliant blue (CBB) R-250 or was checked for enzyme activity. The reaction mixture

contained 150 mM oftris-HCl buffer (pH 7.5), 64 11M of 1-methoxy PMS, 0.24 mM of

nitro-tetrazolium blue, 3 II].M of homocholine iodide and 1 mM of NAD+ (Table 4.2).

86

Table 4. 2 Reaction mixture for activity staining on native-PAGE gels

Tris-HCl buffer (pH 7.5, 450 mM) 6.70 ml

Homochline chloride (500 mM) 0.67 ml

~-NAD+ (60 mM) 0.67 ml

1-Methoxy PMS (6.4 mM) 0.20 ml

Nitro tetrazolium blue (2.4 mM) 2.00 ml

Total volume 20.00 ml

4.2.10 Intact and Dry Cell Reaction of Pseudomonas sp. Stmin A9

To further confirm the metabolic route of homocholine in Pseudomonas sp. strain A9

both intact and dry cell reaction experiments were carried out Intact cell reaction was

carried out as described in chapter 3. The only exception is that at intervals of 0, 30, 60,

90, 120, 150 and 180 min, aliquots of the cell suspension were withdrawn and boiled to

stop the reaction.

To prepare dried cells, cells suspension was spread on glass Petri dish and dried at

room temperature with electric fan. Then the collected cells were further dried in a

vacuum discator and finely grounded using mortar. The dried cells (75 mg) were

suspended in18 ml of 50 mM potassium phosphate buffer (pH 7.5). The dried cell

reaction was started by the addition of homocholine (20 mM) with or without NAD+ (5

mM) to the cell suspension. The suspension was incubated on a shaker at 120 rpm and

30°C. At appropriate time intervals (0, 30, 90, 150, 210, and 300 min), aliquots of the

cell suspension were withdrawn and boiled for 3-5 min to stop the reaction. After

centrifugation, the supernatant was preserved at -20°C and used for metabolites

detection as described in chapter 3. An exception is that TMAPaldehyde concentration

in the reaction mixture was quantified by DNPH methods as described elsewhere (Tani

87

et al., 1977). The amount of TMAPaldehyde in the reaction mixture was calculated from

the standard curve that generated using different concentration ofTMAPaldehyde.

4.2.11 Detection of 3-Hydroxypropionate by LC/MS/MS

Analyses of the intact cell reaction mixtures of Pseudomonas sp. strain A9 and the

authentic standard of 3-hydroxypropionate were carried out using a Waters LC/MS

instrument consisting of a Waters 2695 liquid chromatograph coupled with a Waters

Quatromicro API mass spectrometer. LC separations were made using a 4.6 x 150 mm

Waters Symmetry C18 column at room temperature and at flow rate of 0.2 ml/min.

Solvent A was 0.1% formic acid in acitonitrile, and solvent B was 0.1% formic acid in

distilled water. The following gradient ofB was applied: 0 min, 100%; 5 min, 100%; 15

min, 0%; 25 min, 0%: 27 min, 100%. MS analysis was done in the negative ESI

MS/MS mode.

88


4.3.1 Screening for Homocholine degrading Activity

Screening of the homocholine oxidation activity in the isolated strains by replica staining

and spectrophotometer assays demonstrated that NAD+-dependent dehydrogenase

enzymes are predominant in homocholine degrader. Another oxidation activities such as

oxidase and membrane-bound dehydrogenase were not detected in all isolates. In replica

staining screening test, about 10 strains showed positive results (formation of purple

color), which indicated the presence ofNAD+-dehydrogenase activities in these isolates

(Fig. 4.2a). Cell free extracts of these isolates again confirmed the presence of both

NAD+-dependent alcohol and aldehyde dehydrogenase activities (Fig. 4.2b). Those

isolates were chosen as the most superior homocholine degraders and were identified to

the specific level (as described in chapter 2) according to general principles of microbial

classification. From those isolates, Pseudomonas sp. strain A9 was selected as a

preferred homocholine degrader, since it showed quite higher activity of both NAD+

dependent homocholine and TMAPaldehyde dehydrogenases. In the preliminary

experiment, these enzymes were found to be unstable during the cell free preparation

and against dialysis. Although Rhodococcus and Arthrobacter strains were also showed

both activities but these strains were excluded because cell free extract preparation is

quite difficult and need more extraction time that significantly affect the enzyme

stability. Moreover, Rhodococcus andArthrobacter strains require more cultivation time

( 48h) compare to Pseudomonas strains (24h).

89

B 0.014 .--------------------------------------..,

-eo s

0.012

0.010

2 0.008

.E' ~ u ..

.;:: 0.006 ·;:; " Q.

"' 0.004

0.002

i\2 A4 A9

Rlwdocaccus sp.

Honwcholinc·DH • TM:\PAL~DH

Bl 87 88 89 1'1 ES E6

p,·eudomonas sp. Al·thrnbat'fer sp.

Fig. 4. 2 Screening of NAD+ -dependent dehydrogenase activity in the isolated strains by (A) replica staining and (B) spectrophotometric assays

4.3.2 Time Course of the Gr·owth and Enzyme Formation of Pseudollwllas sp.

Strains A9

The time course of the formation of homocholine dehydrogenase and cell growth of

Pseudomonas sp. strain A9 was examined in basal-HC medium (Fig. 4.3). The

90

production of homocholine dehydrogenase activity was significantly increased with the

increase in the cell growth (T 660 nm). The maximum enzyme activity formation was

observed in late exponential phase at about 24h. Thereafter, the activity was rapidly

decreased after 24h cultivation. Hassan (2008) also found that the formation of TMA

Butanol dehydrogenase activity by Pseudomonas sp. 13CM was rapidly decreased

after 6 h cultivation. This phenomenon looks similar in quaternary ammonium alcohol

degrader. To this point we do not know the actual reasons for the rapid decrease in the

enzyme activity after maximum formation. It might be resulted from the inhibition of

the cellular enzymes by low molecular weight metabolites that accumulated in high

concentration in the culture medium during the degradation of the substrate and then

penetrate into the cellular components. These metabolites might be homocholine

analogue such as trimethylamine. During the growth of strain A9 on homocholine basal

medium, the pH of the medium was decreased gradually from 7.0 to 6.3.

91

::1: c.

7.5---.---------------------.

7

6.5

6~--------~--------~--------~

0 10 20 30

2.5 2.5

21 I 12 -f

1.5 + E 1.5 c

:::l 0 -CD l;' CD .... ·; -.c 1 1 :;::;

~ (.)

<( 0 l..

G

0.5-i / sf. 1-0.5

0 0 0 10 20 30

Cultivation time (h)

Fig. 4. 3 Time course of the growth and homocholine dehydrogenase activity formation of Pseudomonas sp. strain A9.

Cell-free extract was prepared from cells that grown on 300 ml of culture broth.

92

4.3.3 Formation of Homocholine Dehydrogenase on Various Media

To assess the expression ofhomocholine dehydrogenase by Pseudomonas sp. strain A9,

the bacterium was cultivated on basal media of various homocholine analogues as C and

N source, as well as on basal-homocholine medium supplemented with additional C or

N sources. The cell free extracts of the above media was prepared and assayed for

NAD+ -dependent homocholine dehydrogenase activity. The results (Table 4.3) showed

that homocholine dehydrogenase activity could only be observed in the cell-free extract

of cultures grown on homocholine. In assays with cell extracts from cultures grown on

choline, 4-N-trimethylamino-1-butanol (TMA-Butanol) and glucose, homocholine

dehydrogenase activity could not be observed. It is also notable that glucose exerted a

total repression of homocholine dehydrogenase activity induction, since no activity was

detected on homocholine-glucose growing cells. Similar observation was reported for

betaine aldehyde dehydrogenase from Pseudomonas aeruginosa (Nagasawa et al.,

1976; Velasco-Garcia et al., 2006). Moreover, the intact cell reaction of strain A9

grown on homocholine and glucose was preformed. The metabolites such as

TMAPaldehyde, P-alanine betaine and TMA were only detected in the reaction mixture

of homocholine growing cells, whereas they were not detected in the reaction mixture

of glucose growing cells. These observations demonstrated that the enzymes responsible

for degradation of homocholine were induced during the growth on homocholine. The

induction of quaternary ammonium compounds degrading activities was also observed

in many reports (Nagasawa et al., 1976; Velasco-Garcia et al., 2006; Setyahadi, 1998)

among others.

93

Table 4. 3 Formation ofHomocholine dehydrogenase on various media

Medium Growth Enzyme activity Specific activity

(1.0%) (r660nrn) (mU/ml broth) (mU/mg)

Homocholine 2.57 6.21 30.0

Choline 2.65 0.00 0.0

TMA-Butanol 0.58 0.00 0.0

Glucose & 0.5% (NH4)2 S04 3.09 0.00 0.0

Supplemented with carbon source (0.5%)

Glucose 1.63 0.00 0.0

Glycerol 2.85 3.98 22.2

Supplemented with nitrogen source (0.1 %)

(NH4h so4 3.05 9.34 21.5

Basal medium: 1% substrate, 0.2% K2HP04, 0.2% KH2P04, 0.05% MgS04. 7H20, 0.05% yeast extract, and 0.1% polypeptone.

4.3.4 Stability of Homocholine Dehydrogenase of Pseudomonas sp. Strain A9

In a preliminary experiment, the stability of homocholine dehydrogenase was evaluated

under several conditions of storage, pH, stabilizers and SH-group protecting reagents in

order to optimize the cell free extract preparation and enzyme assay conditions. The

overall observation is that the enzyme is unstable and liable to degradation by other

enzymes. Addition of protease inhibitor cocktail besides the dithiothreitol (lmM)

stabilized the enzyme to some extent, and after dialysis the activity was significantly

decreased. Replacement of protease inhibitor cocktail by EDT A (1 mM) in the buffer

gave similar results (data not shovvn), which indicated that metallo-protease might

degrade homocholine dehydrogenase. Moreover, the effect of some stabilizers such as

dithiothreitol, glutathione (lmM), ethanol (5%) and ethylene glycol (10%) were also

tested. The results (Fig. 4.4) showed that ethanol and ethylene glycol are effective

stabilizers of homocholine dehydrogenase activity. About 70% of the enzyme activity

was retained after 7 days at 4°C in 50 mM potassium phosphate buffer, pH 7.5,

94

containing either 5% ethanol or 10 % ethylene glycol. Furthermore, different dialysis

buffers were examined, and 50 mM potassium phosphate buffer gave better results

compared to others buffers. On the other hand, the cell free extract was preserved at

4°C without dialysis. The results (Fig. 4.5) showed that homocholine dehydrogenase

retained about 98% of its activity after 3 days. However, after 7 days only 40% of the

enzyme activity was retained. This instability ofhomocholine dehydrogenase makes the

purification and characterization of this enzyme is a challenge. In choline degradation

pathway, choline dehydrogenase (E. C. 1.1. 99.1) is an inner mitochondrial membrane

protein that catalyzes the four-electron oxidation of choline to glycine betaine via a

betaine aldehyde intermediate and requires an electron acceptor other than oxygen. To

date, no in depth biochemical or kinetic characterization has been performed on choline

dehydrogenase, mainly due to the difficulty in its purification because of the instability

of the enzyme in vitro (Ghanem, 2006). Recently, Gadda and McAllister- Wilkens

(2003) reported the first recombinant choline dehydrogenase. This recombinant form

choline dehydrogenase was highly unstable in vitro, which hindered any further

biochemical and mechanistic investigations of the enzyme (Gadda and McAllister

Wilkens, 2003).

95

~ ._. ~ :; ts ro t:n c: '2 'iii E (J)

a::

120

100

80

60

40

20

2 4

Time (days)

6 8

Fig. 4. 4 Effect of stabilizers on the stability of homocholine dehydrogenase of Pseudomonas sp. strain A9.

Cell free extract from homocholine-growing cells was prepared by glass bead in 100 mM potassium phosphate buffer, pH 7.5, containing 1mM DTT and 1 mM EDT A. About 1 ml of this extract was dialyzed overnight against 50 mM potassium

phosphate buffer containing either 1mM DTT (•), 1mM glutathione (•), 5% ethanol ( .i.. ), or 10% ethylene glycol ( +) and stored at 4 oc for 1 week

120~----------------------------~

100. 1111

.... c 80 >-~ ts rn 60 C) c c Iii E 40 (J)

a::

20

I 0 4 0 8

Storage time (days) 12

Fig. 4. 5 Evaluation ofthe stability ofhomocholine dehydrogenase during storage at 4°C

96

4.3.5 Intact and Dry Cell Reaction of Pseudomonas sp. Strain A9

During the degradation of homocholine by resting cells of Pseudomonas sp. strain A9,

the amount of homocholine decreased concomitantly with the increase of metabolites,

identified as TMAPaldehyde, J3-alanine betaine and TMA (Fig. 4.6). The results

confirmed the sequential oxidation of homocholine to TMAPaldehyde and J3-alanine

betaine. Thereafter, cleavage of C-N bond of B-alanine betaine provided TMA and alkyl

chain.

25 1

:? s ._.

20

~ 0.8 ~

.-. >-~ J:;

(!)

..§. '0 iii <t

0.6 ~ ~ 15 ~

"'S 1-Ill ell .5 Ill

:g 10 I c

0.4 'iii tl Qj 0 .D E Ill 0 c :::r: '2

5 ~~ 0.2 Ill a;

I Cll Qj O'l

0 ~·

0 I

0 30 60 90 120 150 180

Reaction time (min}

Fig. 4. 6 Degradation ofhomocholine by resting cells of Pseudomonas sp. strain A9.

Time course degradation ofhomocholine (1111) and the generation of the metabolites, TMAPaldehyde (L::.) beta-alanine betaine (.A) and TMA (•) by intact cells of Pseudomonas sp. strains A9.

Dry cell reaction was carried out using homocholine (20 mM) as a substrate with

and without NAD+. The results (Fig. 4.7a) showed that addition of NAD+ to the

reaction mixture significantly increased the degradation rate of homocholine, as well as

the production rate of the metabolites TMA and B-alanine betaine. Whereas in the

97

reaction mixture without NAD+, the degradation rate of homocholine; as well as the

metabolites formation was very slow (Fig.4. 7b ). The slight degradation of homocholine

in the absence of added NAD+ might be resulted from the remained NAD+ with the

dried cells. The results demonstrated that the enzymes responsible for the metabolism

of homocholine are alcohol and aldehyde dehydrogenases that require NAD+ as electron

acceptor.

A 25 1.5

~ i' 20,,\ l ! ~I ~ 1< < ::E 15 1-~

jl :g 10 u 0 E 0

::r:: 5

e ell r:::

'" o; .c Ql c::

0.5 ~ 'iii cls

-~~--...____ I ~ ----~-----,-------,------.-------,------,------+0

50 100 150 200 250 300

B Reaction time <min) 25 0.025

i' 20 ------• ...____ /~ E ---- -_ ¢ ~-- ~

0.02 i' E m"

~ 15 7------elj -~--.

Ql

~10 ~ g / E I ~ I ____________ _

5 .-----

1 --------

_______ ...

"f. o.o1s e

0.01

~

'" o; .c

~ c <0

% o;

0.005 m

0 -~---- 0

0 50 100 150 200 250 300

Reaction time <min}

Fig. 4. 7 Degradation ofhomocholine by dried cells of Pseudomonas sp. strain A9.

Time course degradation of homocholine ( 111) and the generation of the metabolites beta-alanine betaine (.A) and TMA (•) by dried cells of Pseudomonas sp. strains A9. Dry cell reaction was preformed with (A) and without (B) NAD+.

98

4.3.6 Substrate Specificity ofHomocholine Dehydrogenase

An attempt was made to detennine the substrate spectrum of homocholine

dehydrogenase in the crude preparation, because the enzyme is unstable and lost its

activity during purification processes. The substrate specificity of homocholine

dehydrogenase was detennined at 30°C and pH 7.5 using various alcohols and

quaternary ammonium compounds in the presence of NAD+ as a cofactor. Among

alcohols tested, DMA-butanol appears to be the most favorable substrate for

homocholine dehydrogenase followed by TMA-Butanol, homocholine (TMA

Propanol), 1-butanol, 4-arnino-1-butanol and dimethylarnino-1-propanol (DMA

Propanol) (Table 4.4). It is particularly interesting that no detectable activity was found

for choline, an analogue of homocholine that is found ubiquitously in nature and the

ability to degrade choline are widespread amongst microorganisms. Thus, the ability to

degrade homocholine does not go alongside with the ability to catabolize choline.

Furthermore, the inability of homocholine dehydrogenase to oxidize choline, in

accordance with the induction of the enzyme activity with homocholine, ruled out the

enzyme to be a choline-oxidizing enzyme. It was also interesting that homocholine

dehydrogenase of strain A9 showed high preference to DMA-Butanol and TMA

Butanol, although this bacterium was unable to grow on these substrates. The inability

of the bacterium to grow on these substrates as well as the induction of the enzyme

only by homocholine also excluded the enzyme to be a TMA-Butanol-oxidizing

enzyme. Recently, a NAD+-dependent TMA-Butanol dehydrogenase was purified and

characterized from Pseudomonas sp. 13CM. This enzyme did not react with TMA

Propanol, DMA-Propanol and choline (Hassan, 2008). Thus, it could be assumed that

99

homocholine dehydrogenase is a new enzyme and is not a choline or TMA-Butanol

oxidizing enzyme.

Table 4. 4 Substrate specificity ofhomocholine dehydrogenase from strain A9

Substrate (33 .3 mM) Activity Relative (mU/ml} activi!Y (%)

Homocoline (CH3)3N+(CH2h CH20H 649 100

TMA-Butanol (CH3hN\CH2)3 CH20H 902 139

TMA-Pentanol (CH3hN\CH2)4 CH20H 0 0

TMA-Hexanol (CH3)3N+(CH2)s CH20H 0 0

Choline (CH3)3N+CH2CH20H 0 0

P-methyl choline (CH3)3N+CH(CH2)0H 0 0

DMA-Propanol (CH3)zN(CH2h CH20H 141 22

DMA-Butanol (CH3)zN(CH2)3 CH20H 1020 157

DMA-Hexanol (CH3)zN(CH2)s CH20H 0 0

4-Amino-1-butanol H2W(CH2)3 CH20H 215 33

1-Butanol CH3 (CH2h CH20H 236 36

1-Propanol CH3 CH2 CH20H 43 7

2-Propanol CH3 CH (OH) CH3 0 0

Ethanol CH3CH20H 0 0

Methanol CH30H 0 0

L-Carnitine (CH3)3N+CH2CH(OH)CH2COOH 0 0

D-Carnitine (CH3hN+CH2CH(OH)CH2COOH 0 0

To ascertain if the activity detected in the cell free extract of homocholine growing

cells with different substrate was catalyzed by the same enzyme, crude cell free extract

was analyzed by native-PAGE, and then, NAD+ dependent activity was located in gels

using different substrates. The result showed that only single band of activity was

detected (Fig. 4.8). The activity band with DMA-buanol as a substrate was much more

intense than that with homocholine as a substrate. This result is in good agreement with

the activity measured in vitro for both substrate (Table 4.4). The results obtained

demonstrated that homocholine dehydrogenase has a broad substrate range. Broad

100

substrate specificities were also reported for many primary and secondary alcohol and

aldehyde dehydrogenases (Vangnai and Arp, 2001; Schenkels and Duine, 2000; Tani et

al. , 2000; Jaureguibeitia et al., 2007; Jo et al. , 2008).

1 2 3 4 5 6 7

Fig. 4. 8 Native-PAGE analysis of crude homocholine dehydrogenase.

Native gels (10%), after PAGE, were stained for NAD+ dependent dehydrogenase activity for 5 min without substrate (lane 1) and with the substrates; homocholine (lane 2), trimethylamino-1-butanol (lane 3) dimethylamino-1-propanol (lane 4), dimethylamino-1-butanol (lane 5), 4-amino-1-butanol (lane 6) and 1-buanol (lane 7)

4.3. 7 Measurement of the Native Molecular Mass of Homocholine Dehydrogenase

The native molecular mass of the enzyme was estimated by size exclusion

chromatography on TSK-gel. The result showed that the enzyme has a molecular mass

of 160 kDa (Fig. 4.9). Such high molecular masses (150~ 170 kDa) were also reported for

medium-chain alcohol dehydrogenase from Acinetobacter sp. strain M-1 (Tani et al. ,

2000), a nicotinoprotein alcohol dehydrogenase from Rhodococcus erythropolis DSM

1069 (Schenkels and Duine, 2000) and an aldehyde dehydrogenase from Rhodococcus

erythropolis UPV -1 (Jaureguibeitia et al., 2007).

101

10000~-------,.-------------------------~

-;- 1000 c .lll:: -.s:: C'l

'Q)

1001 Homocholine == ,_

dehydrogenase ~ ::l

(160 kDa) () Q)

0 :E 10

1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I,., 0 2 4 6 8 1 0 12 14 16 18 20

Retention time (min)

Fig. 4. 9 Estimation of the native molecular mass of homocholine dehydrogenase by size exclusion chromatography on TSK-gel G3000 SW column

4.3.8 Detection of 3-hydroxypropionate Dehydrogenase Activity

As described in chapter 3, j3-alanine betaine degraded to TMA and C-3 moiety such as

3-hydroxypropionate. A NAD+ -dependent 3-hydroxypropionate-dehydrogenase activity

was also detected in the cell free extract of Pseudomonas sp. strain A9. This activity

was only detected in the cell free extract of homocholine-growing cells and no activity

was detected on both choline- and glucose-growing cells (Table 4.5). Furthermore, this

enzyme activity was also detected on native-PAGE only in the cell free extract of

homocholine growing cells (Fig. 4.1 0). These results demonstrated the induction of 3-

hydroxypropionate dehydrogenase activity by homocholine and indicated the presence

of 3-hydroxypropionate as an intermediate metabolite in the degradation pathway of

homocholine by Pseudomonas sp. strain A9. The upper band was assumed to be for

homocholine dehydrogenase activity because it appeared in the same position of

102

homocholine dehydrogenase activity band.

Table 4. 5 NAD+ -dependent 3-hydroxypropionate dehydrogenase activity in the cell extracts of homocholine-, choline- and glucose-growing cells

Medium Substrate Activity (1.0%) (mU/ml)

Homocholine 125

Choline 0

Glucose 0

3-HPDH_.

Fig. 4. 10 Native-PAGE analysis of crude 3-hydroxypropionate dehydrogenase.

Non-denaturing gel (7.5% gel) stained for NAD+-dependent 3-hydroxypropionate dehydrogenase activity. Lane 1; homocholine-growing cells, lane 2; choline-growing cells, and lane 3; glucose-growing cells.

Moreover, to confirm the presence of 3-hydrpxypropionate as an intermediate

metabolite, the intact cell reaction mixtures of strain A9 were analyzed by LC-MS. The

results demonstrated the accumulation of 3-hydroxypropionate as an intermediate

103

metabolite (Fig. 4.11). The mass spectrum (~ 90.08) and the retention time (6.7 min)

of the observed metabolite, agreed with those of authentic standard of 3-

hydroxypropionate.

A

Titne (min)

Fig. 4. 11 LC/MS/MS analysis of 3-hydroxypropionate in the degradation pathway of homocholine by the intacted cells of Pseudomonas sp. strain A9.

(A) Intact cell reaction mixture of strain A9 and (B) authentic standard of 3-hydroxypropionate.

In a similar study, cleavage of C-N bond of choline by Candida tropicalis was

104

accompanied by formation of trimethylamine and ethylene glycol (Mori et al., 1988). 3-

Hydroxypropionate is of special interest in view of that the biodegradable polymers of

it can potentially replace lot kinds of traditional petrochemistry-based polymers and be

used in some new fields such as surgical biocomposite materials and drug release

material (Jiang et al., 2009).

4.4 CONCLUSIONS

This study was carried out to investigate the enzymatic activities in the degradation

pathway of homocholine by the isolated strains. Screening of homocholine oxidation

activity in the isolated strains by replica staining and spectrophotometer assays showed

that NAD+-dependent dehydrogenase enzymes are predominant in the isolates.

Furthermore, dried cell reaction of Pseudomonas sp. strain A9 cells with homocholine in

the presence and absence of NAD+ demonstrated that enzymes responsible for the

metabolism of homocholine are alcohol and aldehyde dehydrogenases that require

NAD+ as electron acceptor.

Moreover, in the cell free extract of Pseudomonas sp. strain A9 an inducible

NAD+-dependent homocholine dehydrogenase waS detected. The crude enzyme has

broad substrate specificity and was unstable in vitro which makes the purification of the

enzyme is a challenge.

Furthermore, an inducible NAD+-dependent 3-hydroxypropionate dehydrogenase

activity was also detected in the cell free extract of Pseudomonas sp. strain A9. This

result indicated the presence of 3-hydroxypropionate as an intermediate metabolite in

the degradation pathway ofhomocholine by Pseudomonas sp. strain A9.

105

Overall, it could be concluded that in Pseudomonas sp. strain A9, homocholine is

oxidized to TMAPaldehyde by a NAD+ -dependent homocholine dehydrogenase, and

consequently, TMAPaldehyde oxidized to B-alanine betaine by a NAD+ -dependent

aldehyde dehydrogenase. Thereafter, cleavage of B-alanine betaine C-N bond yielded

trimethylamine and 3-hydroxypropionate (C-3 moiety). 3-Hydroxypropionate was

further oxidized to malonate semi-aldehyde by a NAD+ -dependent 3-

hydroxypropionate dehydrogenase (Fig. 4.12).

(CH3)3N~CH2CH2CH20H Homocholine

NAD"'~ Homocholine dehydrogenase NADH

(CH 3hN"'"CH2CH2CHO 3-N-Trimethylaminopropionaldehyde

NAD+~

NADH A TMAPaldehyde dehydrogenase

(CH3hN-CH2CH2COOH

Jl-alanine betaine

/ ~ (CH3)3N

Trimethylamine

~ ~

~ Biomass

II' /

HOCHCH2COOH 3-Hydroxypropionate

NAD+J 3-Hydroxypropionate dehydrogenase

NADH

OHCCH2COOH Malonate semialdehyde

Fig. 4. 12 Proposed degradation pathway of homocholine by Pseudomonas sp. strain A9

106

CHAPTERS

GENERAL CONCLUSIONS

Quaternary ammonium compounds (QACs) are either naturally distributed in the

biosphere with more than 100 reported examples including well-known representatives

such as choline, glycine betaine and ~-alanine betaine or widely used in commercial and

consumer applications as disinfectant, fabric softeners, hair conditioners arid

emulsifying agents. The massive production and utilization of QACs has led to their

extensive discharge into the environment, raising concern globally. Biological treatment

has been found to be an effective way to degrade QACs and especially aerobic

treatment processes can provide rapid biodegradation via bacteria. Although extensive

research has been conducted on the microbial degradation of choline and its structurally

related compounds, no research has been done on homocholine, a compound that

resemble choline in many aspects of cholinergic metabolisms. Furthermore, the reaction

catalyzes the oxidation of choline to glycine betaine is of considerable medical and

biotechnological applications, because the accumulation of glycine betaine in the

cytoplasm of many plants and human pathogens enables them to counteract

hyperosmotic environments. Similarly, the study of homocholine degradation enzymes

may has potential for the improvement of the stress resistance of plant by introducing an

efficient biosynthetic pathway for ~-alanine betaine in genetically engineered

economically relevant crop plant. Therefore, this research was conducted to investigate

the degradation of homocholine by soil microorganisms and to elucidate its degradation

pathway.

107

Pure cultures are indispensable resources to develop an understanding of the

degradation pathway of homocholine and the enzymes involved in. In order to explore

the degradation ability of homocholine by soil microorganisms, enrichment cultures

were prepared from soil samples taken from various locations in Tottori University and

around Tottori City. The findings of this study demonstrate that the ability to degrade

homocholine is widespread among aerobic microorganisms since representatives of the

genus Arthrobacter, Rhodococcus and Pseudomonas were found to grow with

homocholine as a sole source of carbon and nitrogen. All these microorganisms are

widely distributed in the biosphere, and particularly in soil, they probably play an

important role in the degradation of homocholine in the nature. Although there are very

few reports on the presence of homocholine in mammalian brains and there is no direct

evidence on the presence of such compound in plants. The widespread utilization of

homocholine by soil microorganisms provides indirect evidence that the compound is

widely exists in the nature and may be in the plant kingdom. In this study, we reported

the isolation of four new homocholine degrading microbial species as a first study.

Strains belongs to the genus Arthrobacter, Rhodococcus and Pseudomonas were

isolated, identified and characterized for their ability to grow with homocholine and its

related analogues. With few exceptions, most of the bacteria isolated and identified so

far that degrade quaternary ammonium compounds were from the genus Pseudomonas

and Arthrobacter. However, Rhodococcus species capable of degrading quaternary

ammonium compounds have not yet been reported despite their versatility and ability

to degrade numerous organic compounds, including some difficult and toxic compounds.

In the present study we are able to isolate Rhodococcus strains that metabolized

homocholine as a sole source of carbon and nitrogen.

108

The study in chapter 3 was attempted to characterize and identify the metabolites

ofhomocholine degradation by the isolated strains as well as to elucidate the degradation

pathway of the compound by these isolates. During the degradation ofhomocholine by

growing and resting cells of the isolated strains, the amount of homocholine decreased

concomitantly with the mcrease of metabolites, identified as

trimethylarninopropionaldehyde, B-alanine betaine and trimethylamine. These findings

demonstrate the sequential oxidation of homocholine by these isolates. Thus, the

degradation pathway of homocholine was revealed to be through consequent oxidation

of alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH), and thereafter cleavage

of C-N bond of B-alanine betaine to give trimethylamine and alkyl chain (C3-moiety).

It is particularly notable that the detection of B-alanine betaine as an intermediate

metabolite during homocholine degradation by the isolated strains is important from a


of B-alanine betaine might be useful in biotechnology for the engineering of osmotic

stress tolerant crop plants.

This study in chapter 4 was carried out to investigate the enzymatic activities in

the degradation pathway of homocholine by the isolated strains. Screening of

homocholine oxidation activity in the isolated strains by replica staining and

spectrophotometer assays showed that NAD+-dependent dehydrogenase enzymes are

predominant in all isolates. Furthermore, dried cell reaction of Pseudomonas sp. strain

A9 cells with homocholine in the presence and absence ofNAD+ demonstrated that the

enzymes responsible for the metabolism of homocholine are alcohol and aldehyde

dehydrogenases that require NAD+ as electron acceptor. Moreover, in the cell free

extract of Pseudomonas sp. strain A9 an inducible NAD+ -dependent homocholine

109

dehydrogenase was detected. The crude preparation of this enzyme has broad substrate

specificity. Although various buffering conditions and stabilizing reagent were applied

to stabilize the enzyme activity, the enzyme was found to be unstable in vitro and lose

its activity soon after and during the purification processes. This phenomena makes the

purification of the enzyme is a challenge. Furthermore, an inducible NAD+-dependent

3-hydroxypropionate dehydrogenase activity was also detected in the cell free extract of

Pseudomonas sp. strain A9. This result indicated the presence of 3-hydroxypropionate

as an intermediate metabolite in the degradation pathway of homocholine by this strain.

Thus, in Pseudomonas sp. strain A9, homocholine is oxidized to

trimethylaminopropionaldehyde by a NAD+ -dependent homocholine dehydrogenase,

and consequently, trimethylarninopropionaldehyde oxidized to ~-alanine betaine by a

NAD+ -dependent aldehyde dehydrogenase. Thereafter, cleavage of j3-alanine betaine C

N bond yielded trimethylamine and 3-hydroxypropionate (C-3 moiety). Thereafter, 3-

hydroxypropionate was further oxidized to malonate semi-aldehyde by a NAD+

dependent 3-hydroxypropionate dehydrogenase.

Overall, this study provides basic information on the microbial degradation

pathway of homocholine and illustrates its degradation metabolites and the enzymes

involved in. This information is important in order to explore these metabolites and

enzymes in biotechnology to overcome hyperosmotic environmental stresses. Further

research will be focused on isolation, characterization and possible application of

homocholine-degrading enzymes.

110

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Zhang J, Okubo A and Yamazaki S. 1997. Determination of betaine aldehyde in plants

by low-pH capillary electrophoresis. Bunseki Kagaku (in Japanese) 46: 509-511.

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.J\.CXJVO)V .£ 'EV(j JVl TNT

:first and foremost, I thank :ALL.A:Jf the a{mighty for giving me yower, suyyort,yatience andyeoyfe's hefp to yursue this study.

I wou{d {ike to take this oyyortunity to exyress my deeyest gratitude and sincerest thankfu{ness to my Ph.'D major suyervisor Professor Nobuhiro :Jvlori, Laboratory of :Jvlicrobia{ Biotechno{ogy, :facu{ty of .Agricu{ture, Tottori 'University, for his de{icate suyervising, his enthusiasm in science and work, his afways we{coming attitucfe for discussion, and fiis exce{{ent guidance throughout my graduate studies. Without his va{uab{e guidance, thoughtju{ advise, continuous encouragement and end{ess yatience this thesis wou{d not have come into being.

I extencfed my deey ayyreciation and thanks to my Ph.'D co-suyervisor Professor }joshihiro Sawa, 'Deyartment of Life Science and Biotechno{ogy, Shimane 'University, for his a{ways we{coming attitude for discussion on the yrogress of this study.

:Jvly thanks and ayyreciation is extencfed to my Ph.'D co-suyervisor 'Dr. Tsuyoshi Ichiyanagi, 'Deyartment of Bioresource Sciences, Tottori 'University, for his kind hefp and thoughtju{ advises in the synthesis of the substrates and metabo{ites detection.

:Jvly deey ayyreciation and thanks to my Ph.'D co-suyervisor 'Dr. ]iro .Arima, 'Deyartment of .Agricu{tura{, Bio{ogica{, and 'Environmenta{ Sciences, :facu{ty of .Agricu{ture, Tottori 'University, for his hefp and disscission on the genetic identification of the strains in this study.

I extended my deey ayyreciation and thanks to 'Dr. 'Emi Sakuno, Nationa{ Institute for .Agro-'Environmenta{ Sciences, Biodiversity 'Division, Tsukuba, Ibaraki, for her hefp and assistance in the metabo{ites detection and identification.

:Jvly thanks and ayyreciation is extended to Professor :Jfisashi Tsujimoto, Laboratory of P{ant (jenetics and Breeding Sciences, :facu{ty of :Agricu{ture, Tottori 'University, for his hosyita{ity and access to his {aboratory microscoye. VVe afso thank Professor Takeshi Sanekata, Laboratory of l!eterinary Infectious 'Diseases, :facu{ty of :Agricu{ture, Tottori 'University, for his assistance in gram staining and e{ectron microscoyy.

I wish to thank my former advisor Professor 'EfJadi{ 'E. Babiker of the 'Deyartment of :food Science and Techno{ogy, 'University of :Khartoum, Sudan, who recommended and encouraged me to study in ]ayan, for his

127

remarkah{e mentorsfiiy and adYice.

My deeyest tfiankfu{ness and ayyreciation is offer to JJr Izzat S . .Jt Tafiir, .Jl.gricu{tura{ 'Research Coryoration, Sudan, for adYice and fie(p and JJr Mazen .Jl.hua{tayef, for fiis ya{ua6{e effort in tfie formatting of tfiis thesis .

.Jl.{so, I wou{d Cike to exyress my thanks to JJr. (jfiazi J{. 'Badawi, VniYersity of Xfiartoum, Sudan and JJr. 'Esamedeen 'E. 'E{gorasfii, VniYersity of Pretoria, Soutfi .Jl.frica for their generous fie(p and encouragement.

I indebted to my co{{eagues in tfie {a6oratory of Microbia{ hiotecfino{ogy for tfieir fie(p, friendiess, attitude and suyyort esyecia{{y, Mr Xita, Mr 'Raju{6ari, Ms Takeada, Mr :Nakao, Mr Sato, Ms Tsukamoto, Ms Miyazaki, Mr Cfii6a, Mr Morimoto, Ms Ito and Ms Xono.

I wou{d Cike to tfiank my entire friend, co{{eagues, and tfie academic, tecfinica{, and administrationa{ staff of Tottori VniYersity Library, staff of tfie (jraduate Scfioo{, and staff of tfie Internationa{ Student .Jl.ffair.

My gratitude and thanks to tfie Ministry of 'Education, Science, Syorts and Cu{ture of ]ayan for yroYidlng tfie financia{ suyyort tfiat effective{y contributed in tfie smooth comy{etion of tfiis study.

My deeyest {oye and ayyreciation goes to my fami{y for tfieir suyyort not on{y tfirougfi my Pfi.JJ study, hut afso tfirougfi tfie years tfiat yreyared me for tfiis stage. My yarents fiaye serYed as tfie eyitome of inYo{Yement, edification, and {oye tfirougfiout my Cife. Tfiank you now and a{ways. My fiearife[t {oye to my brother Mohamed .Jl.fimed and sisters 'Butfiina, J'atifiia, :Nawa{, 'Egfi6a{, 'Entisar, and 'Ekfi{ass for tfieir end{ess{y strugg{e tfirougfi tfie years to fie(p my own deye{oyment. You fiaye a{{ my {oye and gratitude.

Last{y, and most imyortant{y, my gratitude is extended to my 6e{oyed wife, Xfia{da, wfio fias insyired me witfi fier eYer-growing {OYe. :J{er neYer-endlng suyyort of mine fias been a source of strength and encouragement beyond wordS. I ayyreciate you more tfian you wi{{ eYer know. :My sweet thanks and {oye to my Citt{e son 'E{mujtaha wfio fiis cfiaos was en{igfitened my mind and arranged my ideas, and many thanks for fiis {oye{y signature on niost of my documents, wfieneYer fie get a chance.

Jsam .Jl.{i Mohamed .Afimed

Marcfi, 2010

128

ABSTRACT

Microbial degradation and metabolism of quaternary ammonium compounds such as

choline, TMA-butanol, and L-, and D-carnitine have been well investigated.

Furthermore, the degradation pathways of these compounds in microorganisms have

been studied. Although extensive research has been conducted on the microbial

degradation of choline and its structurally related compounds, no research has been

done on homocholine, a compound that resemble choline in many aspects of cholinergic

metabolisms. Therefore, this research was conducted to investigate the degradation of

homocholine by soil microorganisms and to elucidate its degradation pathway.

Chapter 1 defines the aim of the work, provides a literature review of the relevant

studies on the distribution and function of quaternary ammonium compounds, and also

presents the microbial degradation of quaternary ammonium compounds.

In chapter 2, the isolation, characterization and identification of homocholine

degrading microorganism were described. Thirty strains with high growth on

homocholine medium were selected from 142 strains grown on homocholine. From these

strains, four strains showed the highest growth and homocholine degrading rate were

selected. Strains belong to the genus Arthrobacter, Rhodococcus and Pseudomonas were

identified and characterized by morphological, biochemical and genetical analysis. With

few exceptions, most of the bacteria isolated and identified so far that degrade

quaternary ammonium compounds were from the genus Pseudomonas and

Arthrobacter. However, Rhodococcus species capable of degrading quaternary

ammonium compounds have not yet been reported, in the present study we are able to

129

isolate Rhodococcus strains that metabolized homocholine as a sole source of carbon and

nitrogen.

The study in chapter 3 was attempted to characterize and identify the metabolites

of homocholine degradation by Arthrobacter sp. strain E5, Rhodococcus sp. strain A2

and Pseudomonas sp. strain A9 as well as to elucidate the degradation pathway of the

compound by these isolates. The degradation of homocholine by the resting cell

suspensions and growing cell cultures of these strains and the detection of formed

metabolites were tested by capillary electrophoresis, GC-MS and F AB-MS methods.

During the degradation of homocholine by growing and resting cells of these strains, the

amount of homocholine decreased concomitantly with the increase of metabolites,

identified as trimethylarninopropionaldehyde, ~-alanine betaine and trimethylamine.

These findings demonstrate the sequential oxidation of homocholine by these isolates.

Thus, the degradation pathway of homocholine was revealed to be through consequent

oxidation of alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH), and thereafter

cleavage of C-N bond of !3-alanine betaine to give trimethylamine and alkyl chain (C3-

moiety).

The study in chapter 4 was carried out to investigate the enzymatic activities in

the degradation pathway of homocholine by the isolated strains. Screening of the

homocholine oxidation activity in the isolated strains by replica staining and

spectrophotometer assays showed that NAD+-dependent dehydrogenase enzymes are

predominant in all isolates. Furthermore, dried cell reaction of Pseudomonas sp. strain

A9 cells with homocholine in the presence and absence ofNAD+ demonstrated that the

enzymes responsible for the metabolism of homocholine are alcohol and aldehyde

dehydrogenases that require NAD+ as electron acceptor. Moreover, in the cell free

130

extract of Pseudomonas sp. strain A9 an inducible NAD+ -dependent homocholine

dehydrogenase was detected. The crude preparation of this enzyme has broad substrate

specificity. Although various buffering conditions and stabilizing reagent were applied

to stabilize the enzyme activity, the enzyme was found to be unstable in vitro and lose

its activity soon after and during the purification processes. Furthermore, an inducible

NAD+ -dependent 3-hydroxypropionate dehydrogenase activity was also detected in the

cell free extract of Pseudomonas sp. strain A9. This result indicated the presence of 3-

hydroxypropionate as an intermediate metabolite in the degradation pathway of

homocholine by this strain. Thus, in Pseudomonas sp. strain A9, homocholine is

oxidized to trimethylaminopropionaldehyde by a NAD+ -dependent homocholine

dehydrogenase, and consequently, trimethylaminopropionaldehyde oxidized to B

alanine betaine by a NAD+ -dependent aldehyde dehydrogenase. Thereafter, cleavage of

B-alanine betaine C-N bond yielded trimethylamine and 3-hydroxypropionate (C-3

moiety). Thereafter, 3-hydroxypropionate was further oxidized to malonate semi

aldehyde by a NAD+ -dependent 3-hydroxypropionate dehydrogenase.

Overall, this study provides basic information on the microbial degradation

pathway of homocholine and illustrates its degradation metabolites and the enzymes

involved in. This information is important in order to explore these metabolites and

enzymes in biotechnology to overcome hyperosmotic environmental stresses. Further

research will be focused on isolation, characterization and possible application of

homocholine degrading enzymes.

131

摘要

コリン，トリメチルアミノブタノーノレ， L-カノレニチン， D・カノレニチン

などの第 4級アンモニウム化合物の分解・代謝の研究は詳細になされて

きた.コリンやその構造類縁体の微生物分解に関する研究は数多くある

が，ホモコジンはコリン作動性代謝の面から見てコリンと類似の化合物

であるにもかかわらず，その微生物分解に関する研究はなされていない.

そこで，本研究ではホモコリン分解菌を土壌微生物に探索し，その分解

経路を明らかにすることを自的とした.

第 1章では，本研究の目的を述べ，第 4級アンモニウム化合物の分布

やその機能についての研究及び第 4級アンモニウム化合物の微生物分解

に関する研究について概説した.

第 2章ではホモコリン分解微生物の単離と同定が述べられている.ホ

モコリンに生育できる 142菌株を土壌から分離し，生育が良好であった30

菌株を選抜した.さらに，ホモコロン分解速度が速い菌株として 4菌株

を選抜した.形態学的観察，生化学的性糞の検討及び分子生物学的分析

により，これら 4蕗株はArtltrobacter属，RJlOdococcuS属， Pseudomollas属

に属する紹菌であることが明らかとなった. 今までに報告されている第

4級アンモニウム化合物を分解できる菌のほとんどはArtltrobacter属ある

いはPseudomollas痛の細菌であり，本研究でも同様の結果であった.一方，

これまで第 4級アンモニウム化合物を分解できるRJlOdocoCCllS属細菌は報

告されておらず，本研究での分離が初めての例である.

第 3章ではArtltrobactersp. strain E5， RltodocoCCllS SPl strain A2，

Pselldomollas Sp. strain A9によるホモ立リン分解の代謝産物の同定を試みた.

選抜した 3菌株のホモコリン培養液や休止菌体反応液中の代謝産物をキ

ャピラリー電気泳動， GC

養基質であるホニそそコリンの減少に{伴半い，ト門リメチルアミJ7。ロ吋tピ。才かンアル好テやヒトド守，日ー

アラニンベタイン，トリメチルアミンと同定された代謝産物が増加した.

このような結果から，ホモコリンの分解経路として，ホモコリン分子中

のアルコーノレ基がアノレデヒド基，カノレボキシノレ基へと酸化され，さらに，

133

3圃アラニンベタイン中の炭素圃窒素聞の結合が切断され，トリメチルアミ

ンとアルキノレ鎖 (C3部分)を生じるような経路が存在することを明らか

にした.

第 4章ではホモコリン分解経路に関与する酵素について検討した.ホ

モコリン分解障として分離した30菌株中のホモコリン酸化活性をレプリ

カ法と分光光度法でスクリ…ニングし，ほとんどの菌株にはNAD依存性

の脱水素酵素が存在することが明らかとなった.さらに，Pseudomollas sp.

strain A9の風乾菌体を用いて欝索活性を検討したところ，ホモコリンの酸

化iこはNAD依存性のアルコーノレ脱水素酵素とアノレデヒド脱水素酵素が関

与していることが明らかとなった.また， A9株の無細胞抽出液中にNAD

依存性のホモコリン脱水素酵素活性を見出し，本酵素は誘導酵素である

ことを証明した.粗酵素液を用いて基質特異性を検討したところ広い特

異性を示した.酵素精製に先立ちNAD依存性ホモコリン脱水素酵素活性

の安定化を検討したが，格段に安定化させる条件を見出すことはできな

かった.また， NAD依存性3・ヒドロキシプロピオン酸脱水素騨索活性を

Pseudomollas Sp. strain A9の締抱抽出液中に見出し，誘導的に生成すること

を明らかにした.このことは， 3・ヒドロキシプロピオン酸が本菌のホモ

コリン分解の代謝中間体であることを示している.以上の結果から以下

のような分解経路を推定した.Pseudomollas sp. strain A9では，ホモコリン

は卜?メチルアミ)7。世ヒ。オンアルデヒドを経由して日開アラニンベタインに酸化され

る.これらの反応を触媒する酵素はNAD依帯性ホモコリン脱水素酵素と

NAD依存性アノレヂヒド脱水素酵素である.次に，日欄アラニンベタインは

そのC-N結合が開裂を受けトリメチルアミンとみヒドロキシプロぜオン

酸が生成する.さらに，みヒドロキシプロピオン酸はNAD依存性みとド

ロキシプロピオン酸脱水素酵素により酸化され，マロン酸セミアルデヒ

ドとなる.

134

LIST OF ORIGINAL PUBLICATIONS

(1) Mohamed Ahmed， I.A.， Arima， 1.， Ichiyanagi， TラSakuno，E. and Mori， N.

Isolation and characterization of 3・.N-trimethy lamino-1・propanoldegrading

Arthrobacter sp. strain E5， Research Journal 0/ Microbiology， 4(2)・49・58ラ

(2009)

This paper partly covers Chapter 2 and 3 in the thesis.

(2) Mohamed Ahmed， I.A.ラArimaヲ JラIchiyanagiラT.， SakunoラE.and MoriラN.

Isolation and characterization of 3-N園 trimethylamino-トpropanoldegrading

Rhodococcus sp. strain A2. FEMS恥1icrobiologyletter 296(2): 219剛 225，(2009).


(3)問。hamedAhmed， I.A.ラArimaラ1.， Ichiyanagi， T.， SakunoラE.and MoriラN.

Isolation and characterization of homocholine degrading Pseudomonas sp. strain

A9 and B9b. World Journal of Microbiology and Biotechnology (In pressラ DOI:

10.1007/s11274刷 010綱 0320嗣 z).


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studies on the microbial degradation of homocholine …

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