production and single step purification of cyclodextrin

Accepted Manuscript

Title: Production and single step purification of CyclodextrinGlucanosyltransferase from alkalophilic Bacillus firmus byion exchange chromatography

Authors: Laxman S. Savergave, Santosh S. Dhule, Vitthal V.Jogdand, Sanjay N. Nene, Ramchandra V. Gadre

PII: S1369-703X(07)00407-XDOI: doi:10.1016/j.bej.2007.09.020Reference: BEJ 4638

To appear in: Biochemical Engineering Journal

Received date: 26-3-2007Revised date: 29-9-2007Accepted date: 30-9-2007

Please cite this article as: L.S. Savergave, S.S. Dhule, V.V. Jogdand, S.N. Nene, R.V.Gadre, Production and single step purification of Cyclodextrin Glucanosyltransferasefrom alkalophilic Bacillus firmus by ion exchange chromatography, BiochemicalEngineering Journal (2007), doi:10.1016/j.bej.2007.09.020

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

dx.doi.org/doi:10.1016/j.bej.2007.09.020

dx.doi.org/10.1016/j.bej.2007.09.020

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Fig. 1. Decrease in phenolphthalein absorbance with time. Profile of blank

readings observed by: () reproducing the method of Goel and Nene and ()

modified method.

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105

0 10 20 30 40 50 60 70

Time (min)

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Fig. 2. Growth profile and CGTase production by Bacillus firmus.

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Fig. 3. Binding and elution profile of CGTase using DEAE-Sepharose on AKTA

purifier.

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Fig. 4. SDS-PAGE. Lane 1 Crude CGTase, Lane 2 Molecular Weight Marker,

Lane 3 Purified CGTase from DEAE-Sepharose, Lane 4 Purified CGTase from

Phenyl-Sepharose.

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Production and single step purification of Cyclodextrin

Glucanosyltransferase from alkalophilic Bacillus firmus by

ion exchange chromatography

Laxman S. Savergave, Santosh S. Dhule, Vitthal V. Jogdand*, Sanjay N. Nene,

Ramchandra V. Gadre

Chemical Engineering and Process Development Division,

National Chemical Laboratory, Pune – 411 008, India.

*Corresponding author: [email protected], Tel. +91-20-25902347, Fax +91-20- 25903041)

Abstract

Production and purification of starch digesting CGTase from alklophilic Bacillus firmus

was investigated. Fermentation was carried out in 14 L bioreactor at 28 oC using a

medium containing dextrin, yeast extract, peptone, (NH4)H2PO4 and MgSO4 7H2O. The

extra-cellular enzyme was concentrated by tangential flow ultrafiltration. The

concentrated enzyme was chromatographed using DEAE-Sepharose and Phenyl

Sepharose. DEAE-Sepharose could be used to purify CGTase in a single step with 23.1

fold purification and 80.6 % recovery. The enzyme obtained had homogeneity and the

molecular weight was 76 kDa confirmed by SDS-PAGE.

Keywords: CGTase; Bacillus firmus; Cyclodextrin; Alkalophilic; Purification

* Manuscript

mailto:[email protected]

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1. Introduction

Cyclodextrin glycosyl transferase (CGTase), EC 2.4.1.19, is an extracelluar enzyme that

converts starch into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins

(CDs). It is an important hydrolytic enzyme that carries out reversible intermolecular as

well as intramolecular transglycosylation and performs cyclization, coupling and

disproportionation of malto-oligosaccharides [1]. Cyclodextrins have their systematic

names of cyclic α-D- (1,4)-linked D-glucose oligisaccharides consisting of 6, 7, 8

glycosyl units, well known as a-, b- and g- CDs [2]. CD molecules have the ability to

form inclusion complexes with a variety of compounds therefore they are used in a wide

range of application in food, pharmaceutical, cosmetic and agricultural industries [3, 4].

CGTase is produced by species of Bacillus, Brevibacterium, Clostridium,

Corynebacterium, Klebsiella, Micrococcus, Pseudomonas, Thermoanaerobacter and

Thermoanaerobacterium [1]. Industrial production of CGTase became attractive only

when alkalophilic Bacillus species were introduced as production organism. All the

CGTase enzymes produce a-, b- or g-CDs from starch in different ratios. Enzymes that

can synthesize predominately one type of CD are preferred for industrial applications

because separation of individual CDs from their mixture is expensive [5].

The characteristics of CGTase to transglycosylate various aglycone molecules like

stevioside, rebaudioside etc. is gaining attention of researchers in the field of

biotransformation [6-9].

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β-Cyclodextrins form colorless inclusion complex with phenolphthalein therefore, this

property is used in the spectrophotometric method for estimation of β-CD concentration

and thereby β-CGTase activity. . The main problem of this method is that the color of the

reagent is unstable and its optical density decreases linearly with time. Thus the decrease

in colour intensity is both due to cyclodextrin concentration and time required for

determination of optical density, which lead to erroneous results [10].

Various unit operations used in downstream processing for getting pure protein from

fermentation broth constitutes the largest part of the production cost [11]. There are

many reports on the purification of the CGTase where it required multiple steps of

column chromatography after cell separation [12, 13]. Purification strategies used mainly

involves adsorption of CGTase on starch, followed by gel filtration. However, drawback

with the starch columns is that CGTase reacts with starch and produces cyclodextrins

during elution and thus requires an additional step to exclude the CDs. Due to the

additional step of removal of CDs very low recovery of the purified CGTases is reported.

In the present work, attempt is made to purify CGTase produced by an alkalophilic

Bacillus firmus, in a single-step using column chromatography.

DEAE-Sepharose and Phenyl Sepharose were used as chromatographic matrices and

evaluated for purification and recovery of purified CGTase. Based on these parameters,

DEAE-Sepharose was found to be more efficient for the separation than other reported

matrices. Homogeneity of the purified enzyme was confirmed by SDS-PAGE.

2. Material and Methods

2.1. Materials

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b-Cyclodextrin and bovine serum albumin (fraction V) were purchased from Sigma.

Soluble starch was purchased from E. Merck Mumbai, India and dextrin was from Laxmi

Starch, Coimbature, India. All media components were procured from Hi-Media,

Mumbai, India. Electrophoresis reagents were from SRL Pvt. Ltd., Mumbai, India.

Molecular weight marker, DEAE-Sepharose and phenyl Sepharose were procured from

Amersham Biosciences (GE Healthcare), Uppsala, Sweden. All other chemicals were of

analytical grade.

2.2 Strain:

Bacterial strain was Bacillus firmus 5119 from NCIM (National Collection of Industrial

Microorganism) Pune, India. [14].

2.3. Phenolphthalein reagent stability study

Phenolphthalein assay reagent was prepared according to Goel and Nene [15]. The

reagents prepared were phenolphthalein stock solution (4mM) in ethanol, 125 mM

Sodium carbonate, 0.05 M Tris-HCl buffer (pH 7.0) and 300 mg/ml b-CD in Tris-HCl

buffer. Advantage of making phenolphthalein stock solution in ethanol is that it could be

diluted with sodium carbonate buffer just before CD analysis where one ml this solution

was added to 4 ml ethanol and its volume was made to 100 ml with Na2CO3. To one ml

of appropriately diluted b-CD solution (to get 50 to 300 mg/ml b-CD) 4 ml of

phenolphthalein (corresponding to 60 mg phenolphthalein per assay) reagent was added

and its optical density was measured at 550nm. Percentage decrease in optical density

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was calculated with respect to a control that contained one ml Tris-HCl buffer instead of

b-CD solution.

In the modified method standard graph was prepared by diluting various volumes of b-

CD stock solution (500 mg/ml b-CD) to 5 ml with 50 mM Tris-HCl buffer (pH 7.0). To

this 5 ml of 125 mM sodium carbonate and 0.5 ml of phenolphthalein stock (25 mg

phenolphthalein / 100ml absolute ethanol) were mixed and used for b-CD estimation as

above and percent decrease in absorbance was calculated with respect to blank containing

Tris-HCl buffer instead of b-CD solution.

2.4. Modified – Cyclodextrin glycosyltransferase assay

1 ml of appropriately diluted enzyme sample was incubated at 60 oC for 15 min with 5 ml

of 1 % (w/v) gelatinized soluble starch in 50 mM, 7.0-pH Tris-HCl buffer. Reaction was

terminated by boiling the reaction mixture for 3 min and reaction volume was made to 10

ml with distilled water. Two ml of above reaction mixture was withdrawn and mixed

with 3 ml of Tris-HCl buffer, 5 ml of 125 mM Na2CO3, and 0.5 ml of phenolphthalein

(25 mg phenolphthalein / 100ml absolute alcolhol). Absorbance was measured at 550 nm.

The percent decrease of sample was calculated with respect to control containing 5 ml of

buffer, 5 ml of sodium carbonate and 0.5 ml of phenolphthalein.

100A

AA=absorbancein decrease%

control

testcontrol³

-

Where, Acontrol = Absorbance of control and Atest = Absorbance of sample.

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The amount of b-Cyclodextrin (b-CD) produced was estimated from the standard graph

of 0 -500 mg /ml b-CD concentration against % decrease in absorbance. One unit of

CGTase was defined as the amount of enzyme required to produce 1 mm of b-CD / min.

2.5. Production of CGTase

Gawande et al. [16] optimized media for Klebsiella pneumoniae pneunoniae AS-22. They

found soluble carbon in the form of dextrin was better than starch for cyclodextrin

glucanosyltransferase enzyme production. Use of dextrin as carbon source is also

advantageous as it can be used in higher concentration and its solution can be easily

pumped for operating the fermenter in fed batch mode. Therefore we used dextrin in the

medium. During inoculum build up, a tube containing 5 ml basal medium containing

dextrin 40 g/l, yeast extract 10 g/l, peptone 10 g/l, (NH4)H2PO4 4 g/l, MgSO4 .7 H2O 0.5

g/l, was autoclaved and separately autoclaved Na2CO3 10 g/l was added to it and

inoculated with a loop full of freshly prepared slant of B. firmus. The tube was incubated

at 28 oC on rotary shaker at 210 rpm for 48 h and 0.5 ml from culture broth was

transferred to 10 tubes each containing 4.5 ml of basal medium and incubated for12 h.

These ten tubes were transferred to ten 250 ml flasks containing 45 ml basal medium and

incubated for 12 h. All the flasks were pooled together and used as seed culture for 14 L

New Brunswick Scientific Bioflow-2000 fermenter.

Fermentation medium was also as in the inoculation medium and separately autoclaved

Na2CO3 10 g/l was added to it before inoculation. Fermentation was carried out at 28 oC

with 10 l working volume. Airflow rate of 0.5-vvm (volume of air per unit volume of

medium per minute) was used throughout the run. The pH of the medium was initially

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maintained at 9 ° 0.5 with 1% sodium carbonate. The dissolved oxygen (DO) was

maintained above 20 % of oxygen saturation using an automatic agitation control and

silicon oil was used as antifoam.

Fermentation was carried out in batch mode till the stationary phase was reached.

Samples were removed at regular interval to check the growth and CGTase activity. Cell

mass was measured as optical density at 600 nm. An aliquot was centrifuged at 10,000

rpm for 10 min and CGTase activity in the supernatant was determined. Protein was

estimated according to Lowry et al. [17].

2.6. Cell separation and CGTase concentration

Fermentation broth was cooled to 10 oC and centrifuged at 18,000 rpm using a Sharples

centrifuge having provision of continuous feeding. Supernatant was subjected to

microfiltration with hollow fiber membrane module with membrane area of 3.75 sq. ft

(AgTech U. S. A). Then the enzyme in the permeate was concentrated by ultrafiltration

using a 10 kDa MWCO [molecular weight cut-off] hollow fiber membrane module (Nitto

Denko Corporation, surface area of membrane 0.375 m2).

2.7. Phenyl Sepharose Chromatography

AKTA purifier, a protein purification system from Amersham Biosciences, was used for

purification studies. Binding study was carried out at different ammonium sulphate

concentrations from 0.8 to 1.2 M. Tricorn 10/200 column with phenyl sepharose was

equilibrated with 25 mM, pH 7.0 Tris-HCl buffer containing 0.8 to 1.2 M (NH4)2SO4,

respectively. One ml of concentrated enzyme sample was supplemented with 1 M

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(NH4)2SO4 and loaded at linear flow rate of 38.2 cm/h. Elution was carried out by

stepwise decrease in the ionic strength of (NH4)2SO4 ranging from 0.25 M to 0 M, at

linear flow rate of 76.4 cm/h. Fractions were analyzed for CGTase activity and protein

concentration.

2.8. Purification of CGTase using DEAE - Sepharose

DEAE–Sepharose was packed in Tricorn 10/200 column and equilibrated with 25 mM,

pH 7.0 Tris-HCl buffer. One ml of concentrated enzyme sample with 70 Units and 7.87

mg of protein was loaded at linear velocity of 76.4 cm / h. The column was washed with

the equilibrating buffer to remove unbound proteins. Five ml fractions were collected.

For elution ionic strength of NaCl was increased and its linear velocity was 76.4 cm/h.

The collected fractions were analyzed for protein and CGTase activity.

2.9. SDS – PAGE

Homogeneity of protein in eluted fractions was checked by SDS-PAGE on a vertical slab

gel electrophoresis using 7.5 % acrylamide gel at constant current of 30 mA for 2 h. Gel

(8 cm×12 cm) was run according to the method of Laemmli [18]. Twenty-five ml of

appropriately diluted marker and samples were applied into the wells. Molecular weight

marker from Amersham Biosciences containing Myosin (220 kDa), a2-Macroglobulin

(170 kDa), b-Galactosidase (116 kDa), Transferrin (76 kDa) and Glutamic

dehydrogenase (53 kDa) were used. Protein bands were envisaged by silver staining

protocol.

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3. Results and Discussion

3.1. Modification in the phenolphthalein method

The phenolphthalein method is based on decrease in absorbance due to formation of

inclusion complex of phenolphthalein with b-Cyclodextrin. The percent reduction in

absorbance can be directly correlated with the concentration of CD. However,

phenolphthalein reagent is unstable and its colour intensity decreases with time [19]. This

is due to isomerization of the indicator [10] at higher pH, which was about 11 in the

method reported by Goel. In the modified method we were able to control the pH of the

reagent to 10 to 10.2, which increased the stability of the reagent Fig .1.

Decrease in pH was achieved either by increasing the volume of Tris-HCl buffer in the

assay mixture or by increasing Tris-HCl buffer concentration to 250 mM or decreasing

sodium carbonate concentration to 50 mM. However, it was observed that sodium

carbonate concentration also affected the absorbance of the reagent. M. Makela et al

observed 3.5 fold increase in the colour intensity by increasing buffer concentration from

0.004 M to 0.1 M [19]. Similarly increase in the Tris-HCl buffer concentration also

showed decrease in the colour intensity. Therefore, we used the modification where

volume of the buffer was increased so that blank value was more stable.

3.2. Production and concentration of CGTase

In most of the microorganisms, CGTases are extracellular enzymes and differ in their

amount and type of CDs produced. Conventionally, CGTase is produced by submerged

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fermentation and maximal production is observed in the stationary phase [20]. It is

reported that CGTase produced along with cellular growth [21]. During our experiments,

the culture growth took place rapidly and dissolved oxygen decreased correspondingly.

During initial period exponential growth of the culture was observed but there was hardly

any enzyme produced up to 9 h of fermentation. PH was automatically controlled in the

range of 8.5 to 9.5. After 12 h, an increase in enzyme activity was observed which

reached its maximum in late log phase as depicted in Fig. 2. Maximum CGTase activity

was 6.8 U/ml at 33 h and biomass O. D. reached 45. The fermentation broth was

centrifuged to remove the biomass and CGTase. The clear supernatant enzyme was

filtered using AgTech (U.S.A.) microfiltration hollow fiber module having pore size of

0.1 micron and area of 3.75 sq. ft. Then the enzyme was concentrated by ultrafiltration.

The ultrafiltration removed undesired low molecular weight proteins from the broth

which resulted in increased specific activity of CGTase from 1.0 U / mg to 8.8 U / mg of

protein.

3.3. Purification of CGTase

During the hydrophobic interaction chromatography, optimum concentration of

ammonium sulphate for hydrophobic interaction and purity of CGTase was found to be

1M. CGTase eluted at 0.095M (NH4)2SO4 from phenyl sepharose column with 8.9 fold

purification and percent recovery of 64.7, but single band on SDS-PAGE was not observe

(data not presented). Tachibana et al. purified the enzyme by applying it consecutively to

anion-exchange chromatography (Resource Q), hydrophobic interaction chromatography

(Phenyl Superose), and affinity chromatography (a-CD- (epoxy)–Sepharose 6B). After

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these three steps CGTase was purified 1,750-fold with a yield of only 10 % [22].

Volkova et al. used butyl-Toyopearl column followed by DEAE-Sephacel and purified

the CGTase with 13 fold concentration [23].

In our study DEAE-Sepharose was found to be more efficient for the purification of

CGTase of B. firmus. CGTase eluted with gradient elution from 0.1 to 0.2 M NaCl and

the corresponding chromatogram is shown in Fig. 3. Other tightly bound proteins were

eluted at 1M NaCl. Table 1 indicates the purification of CGTase by DEAE-Sepharose

with 80 % recovery and purification fold of 23. Yim et al. purified the CGTase using

DEAE-Sephadex A-50 followed by DEAE-Sepharose CL-6B and reported recovery of

26.6 % [24]. Cao et al. have reported increase in specific activity from an average of 603

U/mg of protein in crude broth to 5753 U/mg of protein after DEAE-cellulose and

Sepharose CL-6B gel filtration step [25].

3.4. Homogeneity and molecular weight of the enzyme

Purified enzyme from DEAE-Sepharose showed a single band (Fig. 4.) by SDS-PAGE

indicating one-step purification for CGTase whereas complete purification could not be

achieved using phenyl sepharose. Molecular weight of CGTase was estimated as 76 kDa.

Molecular weight of the previously purified CGTases from Bacillus firmus was reported

to be 80 kDa [26]. Kobayashi et al. reported that Bacillus macerans cyclodextrin

glucanotranferase could be dissociated into two subunits by SDS-PAGE [27]. However,

present study verified that the enzyme from Bacillus firmus was found to be monomeric

in nature.

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4. Conclusion

We have modified the CGTase assay, which excludes the variations due to

phenolphthalein color instability and provides a quick and reliable estimation of CGTase

activity. Starch adsorption chromatography is one of the popular methods for the initial

capture of the CGTase, but it demands gel filtration for the separation of CDs formed

during elution of enzyme from the column. We have successfully purified CGTase in a

single chromatographic step using DEAE-Sepharose. Between two chromatographic

techniques used, namely HIC and IEC, the later gave highly purified enzyme with better

recovery. Phenyl Sepharose gave partially purified enzyme with two more contaminating

proteins and low recovery. Enzyme was found to elute at 0.2 M NaCl and 0.095M

(NH4)2SO4 during ion exchange and hydrophobic interaction chromatography,

respectively. Purification protocol resulted in increased specific activity from 1 U/mg to

23.3 U/mg with purification fold of 23.1 and 80 % recovery. CGTase reported in this

study was monomeric with molecular weight of 76 kDa. Single band in SDS-PAGE

showed homogeneity of the purified CGTase.

Acknowledgement: The Department of Biotechnology (DBT), India, supported this

research.

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Table 1 Summary of CGTase purification from alkalophilic Bacillus firmus

StepTotal Volume (ml)

Total Protein(mg)

Total Units

Specific Activity (U / mg)

PurificationFold

Percent Yield

Crude enzyme

(microfiltered)7650 50949 51255 1.0 - 100

Ultrafiltration670 5274 46900 8.8 8.8 91.5

DEAE-Sepharose12.1 2.6 61.7 23.3 23.1 80.6

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