Screening and Characterization of Fructosyl-Valine–Utilizing Marine Microorganisms

Koji Sode,* Fumimasa Ishimura, and Wakako Tsugawa

Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei,

Tokyo 184-8588, Japan

Abstract: We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from

the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val),

a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically

in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast

strain identified as Pichia sp. N1-1, produced a Fru-Val–oxidizing enzyme. The enzyme was purified in its active

form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H2O2 with the

oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase.

This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment

and of fructosyl-amine oxidase from budding yeast.

Key words: fructosyl-valine, Amadori compounds, glycated hemoglobin (Hb A1c), diabetes, biosensors.

INTRODUCTION

One major pathogenesis of diabetic complication has been

determined to be triggered by the formation of nonenzy-

matic glycated proteins. A nonenzymatic condensation re-

action of the free amino groups with glucose, called glyca-

tion to distinguish it from enzymatic glycosylation, result-

ing from the formation of a Schiff base, the aldimine,

followed by the Amadori rearrangement, consequently

forms a ketoamine, the so-called Amadori products (Figure

1) (Hodge and Rist, 1953). As the result of further Maillard

reactions in vivo, these Amadori products form the precur-

sors of the major advanced glycation end product (AGE).

Therefore, the Amadori compounds in vivo have been con-

sidered significant as clinical markers (Beisswenger et al.,

1993).

Among the various Amadori compounds, much atten-

tion is currently being paid to glycated hemoglobin (Hb

A1c) as the most important indicator of the degree of dia-

betic control. Since the amino-terminal residue of b-globin

is valine, fructosyl-valine (Fru-Val) is formed as the result

of Amadori rearrangement, and the resultant hemoglobin

molecules with glycated b-globin are called Hb A1c. Owing

to the in vivo lifetime of hemoglobin, the ratio of glycation

directly reflects the past blood glucose levels. Therefore, the

amount of Hb A1c is a good indicator of glycemic control

for a period of 2 to 3 months, and is more reliable than

rapidly fluctuating blood glucose levels (Gabbay et al.,

1977).

In order to determine Hb A1c levels in the blood, con-

siderable attention has been devoted to developing selectiveReceived October 21, 1999; accepted September 12, 2000

*Corresponding author.

Mar. Biotechnol. 3, 126–132, 2001DOI: 10.1007/s101260000065

© 2001 Springer-Verlag New York Inc.

and sensitive methods suitable for clinical diagnostics. Ion

exchange and affinity chromatography and immunoassay

methods have been developed and utilized for clinical di-

agnosis (Stenman et al., 1984; Standing and Taylor, 1992;

Wilson et al., 1993). However, the development of a bio-

chemical measurement system, which utilizes enzyme-

recognizing Hb A1c, is still in demand considering its prac-

tical application for automatic analyzers conventionally uti-

lized for clinical diagnosis.

A group of enzymes called fructosyl-amine oxidases

(FAOs) or Amadoriases, are known to recognize and oxi-

datively degrade fructosyl-amine compounds. The isolation

of FAOs has been reported mainly from fungi (Watanabe et

al. 1990; Horiuchi and Kurokawa, 1991; Sakai et al., 1995;

Yoshida et al., 1995; Takahashi et al., 1997), with some

reported from bacteria (Horiuchi et al., 1989), but not from

the budding yeast. Amadori-product–binding protein and

non-H2O2-liberating–type FAO-like enzymes have also

been reported (Gerhardinger et al., 1995). However, screen-

ings of microorganisms utilizing Amadori compound and

FAOs have been limited to terrestrial organisms; no one has

ever attempted to screen marine organisms. Considering

that the glycation of proteins has proceeded in various liv-

ing organs, the Amadori compounds should already be dis-

tributed in the marine environment; consequently, a variety

of organisms utilizing Amadori compounds are expected to

exist in the marine environment.

Here we report on the isolation and characterization of

microorganisms utilizing Amadori compounds and their

enzymes from the marine environment. In this study we

have used Fru-Val as the Amadori compound, considering

further applications for Hb A1c diagnosis.

MATERIALS AND METHODS

Chemicals

All the Amadori compounds used in this study were pre-

pared according to the methods of Keil et al. (1985). Horse-

radish peroxidase was obtained from Wako Pure Chemical

(Osaka, Japan). Other chemicals used in this study were of

reagent grade.

Screening Procedure

Fru-Val–utilizing microorganisms were isolated from sea-

water samples as follows. Coastal seawater samples collected

from various independent sites on the Izu Peninsula, Shi-

zuoka Prefecture, Japan, were concentrated 200-fold by fil-

tration using nitrocellulose membranes (f is 47 mm; pore

size, 0.45 µm, Advantech, Tokyo, Japan). Membrane filters

with concentrated seawater samples were directly inocu-

lated in the following selection media, a minimal medium

(0.6% Na2HPO4, 0.3% KH2PO4, 3.0% NaCl, 0.012%

MgCl2, 0.011% CaCl2) containing Fru-Val either as the ni-

trogen source (0.52% with 1.0% of D-glucose) or as the

carbon source (0.4% with 0.1% NH4Cl). The primary se-

lection was carried out in the above-mentioned media aero-

bically at 30°C for 3 days. The primary selection was re-

peated 3 times for each sample, and the growing microor-

ganisms in each medium were then inoculated in an agar

plate with complex medium (1% polypeptone, 0.2% yeast

extract, 0.1% MgSO4, 2.5% NaCl, 1.5% agar), in order to

isolate single colonies. The colonies that appeared in the

agar plates were again inoculated in the minimal media

containing Fru-Val as the nitrogen source and incubated

aerobically at 30°C for 3 days. The visible strains during this

isolation procedure were chosen as the Fru-Val–utilizing

microorganisms.

The Fru-Val–utilizing microorganisms were then tested

for their fructosyl-amine–oxiding ability using whole cells,

according to our previous report on the detection of bac-

terial sugar–dehydrogenase activity (Tsugawa et al., 1998) as

follows. Cells cultivated until their stationary phase in each

minimal medium with Fru-Val were centrifuged (at 5000 g

for 20 minutes), washed twice with a 3% NaCl solution, and

resuspended in a 10 mM potassium-phosphate buffer of pH

7.0. To 90 µl of thus-prepared cell suspension, 210 µl of

Figure 1. The mechanism of glycated

protein formation.

Fructosyl-Valine–Utilizing Marine Microorganisms 127

a reaction buffer containing 10 mM Fru-Val, 0.86 mM

phenazine methosulfate (PMS), and 0.086 mM 2,6-

dichlorophenol indophenol (DCIP) was added, and it was

incubated at room temperature. The absorbance decrease

due to the reduction of DCIP was monitored at 600 nm as

an indication of FAO activity.

Enzyme Preparation and Enzyme Assay

A Pichia sp., strain N1-1, was cultivated in a 500-ml culture

flask containing 100 ml of the minimal medium supple-

mented with Fru-Val as the sole nitrogen source aerobically

at 30°C for 3 days. Cells were then centrifuged (at 5000 g for

20 minutes), washed twice with a 3% NaCl solution, and

resuspended in a 10 mM potassium-phosphate buffer, pH

7.0. Cells were disrupted by a French pressure at 147 MPa

(Ootake-seisakusho, Tokyo, Japan). The homogenate was

centrifuged at 5000 g for 20 minutes to remove unbroken

cells. The supernatant was then ultracentrifuged at 69,800 g

for 90 minutes to remove cell debris. Then the sample was

dialyzed against a 10 mM potassium-phosphate buffer of

pH 7.0 at 4°C for 12 hours. The dialyzed sample was sub-

jected to anion exchange chromatography (DEAE-

Toyopearl 650M; f is 22 × 200 mm; Tosoh, Tokyo, Japan),

then equilibrated with a 10 mM potassium-phosphate

buffer of pH 7.0. The absorbed proteins were eluted under

a linear NaCl gradient (0–0.75 M). The active fraction

eluted at about 0.7 M was pooled and concentrated using

lyophilization. The thus-prepared sample was subsequently

subjected to gel filtration chromatography (TSK gel

G3000SW glass column, f is 8.0 mm × 300 mm; Tosoh),

using a 10 mM potassium-phosphate buffer containing 0.3

M NaCl. The active fraction was then analyzed by sodium

dodecylsulfate polyacrylamide gel electrophoresis (SDS-

PAGE) using a gradient gel (PhastGel 8–25, Amersham

Pharmacia Biotech, Uppsala, Sweden). The sample showing

a single band after silver staining was used for further ki-

netic experiments.

Enzymatic activity was determined as follows: 10 µl of

an enzyme sample dissolved in a 10 mM potassium-

phosphate buffer of pH 7.0 was mixed with 260 µl of a

reaction buffer containing 0.6 U of horseradish peroxidase,

1.5 mM 4-aminoantipyrine, and 2.0 mM phenol. The en-

zyme reaction was initiated by adding 30 µl of various con-

centrations of Fru-Val or other fructosyl-amine compounds

at room temperature, and the formation of a quinoneimine

dye (antipyrine dye) was measured at 505 nm using a spec-

trophotometer (Shimazu UV-1200, Kyoto, Japan).

Effect of Fructosyl-Valine on the Expressionof FAO

Cells were cultivated at 30°C in the following 4 different

media: complex medium (tryptone peptone, 0.5%; yeast

extract, 0.5%; NaCl, 0.5%; D-glucose, 0.4%); cominimal

medium (Na2HPO4, 0.6%; KH2PO4, 0.3%; NaCl, 0.05%;

MgSO4, 0.024%; CaCl2, 0.002%) containing 0.4% D-glucose

as a carbon source and 0.4% NH4Cl as a sole nitrogen

source; minimal medium containing 0.4% D-glucose as a

carbon source and 0.4% valine as a sole nitrogen source;

and minimal medium containing 0.4% D-glucose as a car-

bon source and 0.4% Fru-Val as a sole nitrogen source.

Cells harvested by centrifugation (7000 g, 10 minutes, 4°C)

at their stationary phase were washed twice with 0.85%

NaCl solution and resuspended in 10 mM potassium-

phosphate buffer, pH 7.0. Cells were disrupted by ultrasoni-

cation (VC 100, Sonics & Materials Inc., Conn.). These

homogenates were centrifuged at 18,500 g for 10 minutes to

remove unbroken cells. FAO activities of these cell extracts

were determined by detection of hydrogen peroxide as de-

scribed above.

RESULTS

Screening of Marine MicroorganismsUtilizing Fru-Val

From the coastal seawater samples, 12 microbial strains

were isolated that grew aerobically in a minimal medium

supplemented with Fru-Val as the sole nitrogen source

(Fru-Val-N-medium) (Table 1). None of the microbial

strains could grow in the minimal medium supplemented

with Fru-Val as a sole carbon source (Fru-Val-C-medium).

Table 1. Composition of Minimal Media

Chemical Fru-Val-N-medium (%) Fru-Val-C-medium (%)

Na2HPO4 0.6 0.6

KH2PO4 0.3 0.3

NH4Cl — 0.1

NaCl 0.05 0.05

MgSO4 0.01 0.01

CaCl2 0.01 0.01

Fru-Val 0.52 0.4

D-Glucose 1.0 —

128 Koji Sode et al.

Among them, 10 microbial strains isolated from the Fru-

Val-N-medium were eukaryotic cells, and the remaining 2

strains were prokaryotic cells. The cells isolated from the

Fru-Val-N-medium were not able to grow in the Fru-Val-

C-medium. FAO activity was seen in 9 of the eukaryotic

strains isolated from Fru-Val-N-medium, but none of the

prokaryotic strains. Among the strains showing FAO activ-

ity, the N1-1 strain grew rapidly, showing the highest

amount of FAO activity. Therefore, we further character-

ized the N1-1 strain as a source of FAO.

Table 2 shows the taxonomic properties of the strain

N1-1. Considering that the strain showed an oval shape, no

ability to ferment sugars, formation of a mycocandida-type

pseudomycelium under the anaerobic condition, and a film

on the complex medium, this strain was identified as Pichia

species (Iizuka and Gotoh, 1980; Kurtzman and Fell, 1993).

Figure 2 shows the dependence of cell growth on the NaCl

concentration. With increases in the NaCl concentration,

the growth rate decreased. However, this strain was able to

grow in NaCl concentrations higher than 10%. Flannery

(1956) categorized yeast strains by their growth dependence

on salinity. According to this categorization, this is a salt-

tolerant yeast strain, able to grow at salinity higher than 2%.

Figure 3 shows the growth curve of the N1-1 strain in

either the Fru-Val-N-medium or the complex medium. The

N1-1 strain grew well in the complex medium, with a higher

specific growth rate than in the Fru-Val-N-medium. How-

ever, only slight FAO activity was detected in the cells pre-

pared in a complex medium—less than 13% of that ob-

served in the cells cultivated in the Fru-Val-N-medium. We

also investigated the impact of the presence of Fru-Val in

the culture medium on the expression of FAO. The cells

cultivated in either ammonium chloride or valine grew as

well as those in the Fru-Val–supplemented minimal me-

dium. However, the FAO activity observed in the cells cul-

tivated in the Fru-Val–supplemented minimal medium (2.0

× 10−4 U/mg protein), was 6.1-fold and 3.1-fold that of

cells cultivated in the ammonium chloride or valine-

supplemented medium. These results suggested that FAO

was induced by the presence of Fru-Val and not by the

other available nitrogen source. Therefore, for the further

preparation of the FAO sample, the N1-1 strain was culti-

vated in Fru-Val-N-medium.

Table 2. Characteristics of N1-1 Strain

Characteristic Description/value

Shape Short oval

Size (µm) (3.0–5.0) × (4.5–6.0)

DBB staining −

Ballistospore −

Mycelium Pseudomycelium

Fermentation

D-Glucose −

D-Galactose −

Sucrose −

Maltose −

Lactose −

Raffinose −

Assimilation

D-Glucose +

D-Galactose +

Sucrose −

Maltose −

Lactose +

Raffinose +

Nitrate −

Film formation

on liquid medium

Formed in the

complex medium

Nutrition requirement

Vitamin −

Fatty acid −

Pigment production −

Cultivation temperature ∼42°C

Optimal 32∼34°C

Genus Pichia sp.

Figure 2. Effect of NaCl concentration on the cell growth of N1-1

strain. Cell density of N1-1 strain was measured after 10 hours of

cultivation in complex medium containing 0.5% peptone, 0.3%

yeast extract, 0.3% malt extract, 1% D-glucose, and various con-

centration of NaCl at 30°C aerobically.

Fructosyl-Valine–Utilizing Marine Microorganisms 129

Characterization of the Fru-Val–Oxidizing Enzymefrom the Strain N1-1

Figure 4 and Figure 5 show the SDS-PAGE and molecular

weight estimation by gel filtration chromatography of the

final preparation of the Fru-Val–oxidizing enzyme isolated

from the strain N1-1. Only one protein band was detected,

with a molecular mass of approximately 54 kDa by SDS-

PAGE. As calibrated by the standard protein of gel filtration

chromatography, the molecular mass was about 57 kDa in

its native form. These results suggested that the enzyme

consisted of a single protein of about 54 kDa. This enzyme

was purified in its active form, and does not require cofac-

tors for PMS-DCIP–mediated Fru-Val oxidation. Further-

more, this enzyme liberates stoichiometric amounts of

H2O2 under oxidative conditions in the presence of Fru-

Val, but the absence of any mediators (results not shown).

Therefore, this enzyme is considered to be an oxidase.

Table 3 shows the substrate specificity and kinetic pa-

rameters of the isolated Fru-Val–oxidizing enzyme. The Km

values for Fru-Val and for «-fructosyl-lysine were 2.0 mM

and 0.65 mM, respectively. At the substrate concentration

of 1 mM, this enzyme showed the highest level of activity

toward «-fructosyl-lysine. A trace reaction was observed to-

ward fructosyl-glycine. Fructosyl-leucine and fructosyl-

propylamine were not substrates of this enzyme. Since vari-

ous fructosyl-amine compounds were oxidized as the sub-

strate of this enzyme, the enzyme isolated from the strain

N1-1 can be categorized as a type of FAO.

DISCUSSION

In this article we describe the first study of the isolation of

microorganisms utilizing fructosyl-amine (Amadori com-

pounds) and of the fructosyl-amine oxidase from the ma-

rine environment. Our results showed that a variety of mi-

croorganisms able to utilize Amadori compounds can be

isolated from the marine environment. This is also the first

report on the isolation of FAO from budding yeast.

Other microorganisms utilizing Amadori compound

have been previously reported. The most extensive study

has been conducted on fungal FAOs. The fungal FAOs are

divided into two groups by molecular weight: dimeric en-

zymes (composed of two identical subunits) of about 40

kDa (Horiuchi and Kurokawa, 1991), and monomeric en-

zymes of about 50 kDa (Sakai et al., 1995; Yoshida et al.,

1995; Takahashi et al., 1997). Only one bacterial FAO has so

far been reported (Horiuchi et al., 1989), a dimeric enzyme

Figure 3. Growth curve of N1-1 strain in minimal medium and

complex medium. Cells were cultivated in complex medium (d)

containing 0.5% peptone, 0.3% yeast extract, 0.3% malt extract,

1% D-glucose, and in Fru-Val-N-medium (s) at 30°C aerobically.

Figure 4. SDS-PAGE analysis of fructosyl-amine oxidase from

N1-1 strain. Lane 1, molecular mass standards; lane 2, purified

frutosyl-amine oxidase from N1-1 strain.

130 Koji Sode et al.

similar to that of the above fungal enzyme. However, no

one has reported on FAO from budding yeast. All of these

FAOs contained flavin adenine dinucleotide (FAD) as their

cofactor. On the basis of molecular weight analyses, the

FAO from the newly isolated yeast strain N1-1 is similar to

the fungal enzymes in Aspergillus sp. (Takahashi et al.,

1997), Penicillium janthinellum (Yoshida et al., 1995), and

Fusarium oxysporum (Sakai et al., 1995).

On the basis of substrate specificity, FAOs can be cat-

egorized into 3 groups: the enzymes that preferably oxidize

a-keto-amine, those that preferably oxidize «-keto-amines,

like «-fructosyl-lysine, and those that oxidize both types of

fructosyl-amines. Since the N1-1 enzyme oxidized both

Fru-Val and «-fructosyl-lysine, this enzyme is categorized in

the third group, as those from Aspergillus sp. (Takahashi et

al., 1997). However, the substrate specificity of the N1-1

enzyme was different from those reported in Aspergillus sp.

Although these enzymes oxidize both a- and «-keto-amine

compounds, the N1-1 enzyme could not oxidize fructosyl-

propylamine and scarcely reacted with fructosyl-glycine,

which are the two best substrates of the Aspergillus sp. en-

zymes. Therefore, FAO isolated from the strain N1-1 is a

new enzyme showing unique substrate specificity.

We are currently investigating the application of N1-

1–derived FAO for the development of a biochemical analy-

sis system and an enzyme sensor system for glycated protein

measurements, such as those for fructosyl-albumin and

Hb A1c.

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