HIGH YIELD PURIFICATION OF DEXTRANSUCRASE FROMLEUCONOSTOC MESENTEROIDES NRRL B-512F BY

PHASE PARTITIONING

MANISHA NIGAM1, ARUN GOYAL2,4 and SARVAGYA S. KATIYAR3

1Department of ChemistryIndian Institute of Technology Kanpur

Kanpur- 208 016 (UP)India

2Department of BiotechnologyIndian Institute of Technology Guwahati

Guwahati- 781 039AssamIndia

3Chhatrapati Shahu Ji Maharaj UniversityKanpur- 208 020 (UP)

India

Received for Publication March 10, 2005Accepted for Publication April 28, 2005

ABSTRACT

The extracellular dextransucrase (sucrose: 1,6-a-D-glucan 6-a-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRLB-512F was purified by phase partitioning using poly(ethylene) glycol (PEG)and dextran generated by dextransucrase. Three steps of repeated phasepartitioning by PEG 6000 and PEG 400 showed that the purification ofdextransucrase by PEG 6000 was much greater than that obtained by PEG400. Both the specific activity of 42.1 U/mg and the overall yield of 84% ofdextransucrase activity obtained after the third step of phase partitioning byPEG 6000 were significantly higher than that of 23.8 U/mg and 46% overallyield, respectively, by PEG 400 or of that previously reported. The successivethree steps, two-phase partitioning with a final concentration of 5% PEG6000, reproducibly yielded a homogeneous preparation of dextransucrase.

4 Corresponding author. TEL: 361-258-2208; FAX: 361-269-0762; EMAIL: arungoyl@iitg.ernet.in

Journal of Food Biochemistry 30 (2006) 12–20. All Rights Reserved.© 2006, The Author(s)Journal compilation © 2006, Blackwell Publishing

12

INTRODUCTION

Extracellular dextransucrase is well known for its commercial applica-tions and has attracted worldwide attention because of the need for a low-cost,rapid and efficient purification method. The dextransucrases have beenincluded in glycoside hydrolases and based on their sequence homologies,have been grouped into 99 families (Henrissat 1991; http://afmb.cnrs-mrs.fr/CAZY/). The enzyme dextransucrase belongs to the glycoside hydrolasefamily 70. Owing to its wide clinical and commercial applications, efforts areongoing to improve and optimize dextransucrase production (Goyal andKatiyar 1997; Behravan et al. 2003; Rodrigues et al. 2003). A wide variety oftechniques have been used for the purification of Leuconostoc mesenteroidesNRRL B-512F dextransucrase (Robyt and Walseth 1979; Kaboli and Reilly1980; Kobayashi and Matsuda 1980; Monsan and Lopez 1981; Paul et al.1984; Miller et al. 1986; Monsan et al. 1987; Fu and Robyt 1990; Rhee andLee 1991; Goyal and Katiyar 1994). Several researchers have reported thatdextransucrase exists in either single or multiple forms having molecularweights in the range of 64,000–245,000 (Kobayashi and Matsuda 1980, 1986;Miller et al. 1986; Willemot et al. 1988; Fu and Robyt 1990; Goyal andKatiyar 1994). The enzyme remains in an aggregated form in the presence ofdextran resulting in a high molecular weight. High molecular weight proteinshave been purified by precipitation using the nonionic hydrophilic polymerpoly(ethylene) glycol (PEG) (Honig and Kula 1976; Miekka and Ingham1978; Goyal and Katiyar 1994). Dextransucrases from Streptococcus mutans(Russell 1979) and from Leuconostoc mesenteroides NRRL B-512F (Barkeret al. 1987; Ajongwen et al. 1993; Goyal and Katiyar 1994) have been purifiedby precipitation using PEG.

The extracellular dextransucrase has also been purified by the phase-partitioning method (Paul et al. 1984; Monsan et al. 1987). The present articleis an outcome of the exploration of purification by two-phase partitioningusing different PEGs with certain modifications of optimized pH, temperatureand other experimental conditions. Phase partitioning occurs between dextranand polyethylene glycol. The dextran is generated by a solution of the substratesucrose and dextransucrase. The addition of a PEG solution to a dextran-richaqueous solution, when both are in suitable concentration in the medium, leadsto the formation of two phases; the top phase is rich in PEG while the bottomone is rich in dextran. Dextransucrase preferentially goes in to the dextran-richphase. The covalent association of dextransucrase and dextran in particular,results in the presence of dextransucrase in the dextran-rich bottom phase. Inthe present study dextransucrase was purified by the phase-partitioningmethod using PEGs of different molecular weights. We report that thepurification of dextransucrase by phase partitioning using PEG 6000

13PURIFICATION OF DEXTRANSUCRASE BY PHASE PARTITIONING

results in higher activity and higher overall yield of 84%, than those reportedpreviously.

MATERIALS AND METHODS

Materials and Methods

Leuconostoc mesenteroides NRRL B-512F was procured from MicrobialType Culture Collection (MTCC), Institute of Microbial Technology(Chandigarh, India). PEG 400 and PEG 6000 were obtained from BDHChemicals (Delhi, India). 3,5�-Dinitrosalicylic acid (DNS) and Folin’s reagentwere purchased from Sigma Chemical Company (St. Louis, MO). Sucrose waspurchased from Hi-media Pvt. Ltd, India.

Culturing of Microorganism and Production of Enzyme

Leuconostoc mesenteroides NRRL B-512F was maintained and subcul-tured in modified-DeMan, Rogosa, Sharpe (MRS) medium as described pre-viously by Goyal and Katiyar (1996). The enzyme was produced using anenzyme-production media as described earlier (Goyal et al. 1995). The saidmedia contained (in g/L), sucrose 20, yeast extract 20, K2HPO4 20,MgSO4·7H2O 0.2, MnSO4·4H2O 0.01, FeSO4·7H2O 0.01, CaCl2·2H2O 0.01,NaCl 0.01 and the pH was adjusted to 7.0. The fermentation was carried out ina 250-mL Erlenmeyer flask incubated in static condition in an incubator at 23Cfor 14–16 h. The cells were removed by centrifugation at 12,000 rpm for15 min at 4C and the clear supernatant obtained as cell free extract was usedfor purification of dextransucrase.

Assay of Dextransucrase Activity

The enzyme assay was performed at 30C in 0.2 m sodium acetate buffer(pH 5.2). Activity was determined by measuring the production rate of reduc-ing sugar. The reaction mixture (3.0 mL) contained 0.5 mL of 1.75 m sucrosedissolved in 0.2 m sodium acetate buffer (pH 5.2) and 0.5 mL of enzymesolution. The reaction mixture was incubated for 15 min at 30C. Aliquots0.1–0.2 mL from the reaction mixture were taken in 3.0 mL DNS (3,5-Dinitrosalicylic acid) reagent and the amount of reducing sugar was deter-mined by the method as described by Sumner and Sisler (1944).

Protein Concentration Determination

The protein concentration was estimated by the method of Lowry et al.(1951) using bovine serum albumin as the standard.

14 M. NIGAM, A. GOYAL and S.S. KATIYAR

Purification of Dextransucrase by Phase Partitioning

In the cell-free extract, the dextran concentration is very low and it is notpossible to separate PEG-dextran phase. Some amount of dextran is generatedin the cell-free extract. To the cell-free extract containing the crude enzyme(50 mL), 5% sucrose was added and incubated at 4C for 4 h, after which, theviscous extract was subjected to phase separation. A cold solution of 50%(w/v) PEG 6000 in water was slowly added while stirring until the turbidityappeared and cooled at 4C in a water bath. The final percentage of PEG 6000in crude extract was 5%. For phase separation by PEG 400, 50 mL of crudeenzyme was subjected to dextran generation by adding 5% sucrose asdescribed earlier. The PEG 400 was added to the dextran-rich extract till itturned turbid and the final concentration of PEG 400 was 25% (v/v).

After the phase partitioning, the mixture was centrifuged at 10,000 rpmfor 20 min at 4C. The PEG-rich top layer was discarded while the dextran-richbottom layer was analyzed for enzyme activity and subjected to further puri-fication by repeating the process three times. Each time the volume of thebottom phase was adjusted to the initial volume of 50 mL using 0.2 m sodiumacetate buffer (pH 5.2). Both the enzyme activity of dextransucrase and theprotein concentration were determined as described previously.

SDS-Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed according to the method of Laemmli (1970) using 7.5% poly-acrylamide gel.

RESULTS AND DISCUSSION

Purification of Dextransucrase by Phase Partitioning

Table 1 summarizes the results of dextransucrase purification by phasepartitioning using PEG 400. After the three successive steps of phase parti-tioning, a specific activity of 23.8 U/mg protein was obtained, resulting in30-fold purification with an overall percent yield of 46. The overall percentrecovery of dextransucrase protein was 1.5%. Table 2 shows the results of thepurification by PEG 6000. The phase partitioning by PEG 6000 resulted in aspecific activity of 42.1 U/mg with a 70-fold purification with an overall yieldof 84% (in terms of total enzyme units recovered). The overall protein recov-ery was 1.2%. The specific activity of 42.1 U/mg and the overall yield of 84%of dextransucrase activity obtained after the third step of phase partitioning byPEG 6000 was significantly higher than those reported previously (Kobayashiand Matsuda 1980; Miller et al. 1986; Fu and Robyt 1990; Goyal and Katiyar1994).

15PURIFICATION OF DEXTRANSUCRASE BY PHASE PARTITIONING

TAB

LE

1.PU

RIF

ICA

TIO

NO

FD

EX

TR

AN

SUC

RA

SEB

YT

HE

PHA

SE-P

AR

TIT

ION

ME

TH

OD

USI

NG

PEG

400

No.

ofSt

eps

PEG

400

%(v

/v)

Vol

.(m

L)

Dex

tran

sucr

ase

Prot

ein

(mg/

mL

)SA

*(U

/mg)

Fold

puri

ficat

ion

(U/m

L)

Tota

lU

nits

Ove

rall

%yi

eld

Cru

de0

501.

7587

.5–

2.20

0.89

–St

ep1

2550

1.65

85.5

940.

602.

753

Step

225

500.

9045

.052

0.11

8.20

10St

ep3

2550

0.80

40.0

460.

034

23.8

030

*Sp

ecifi

cac

tivity

.

TAB

LE

2.PU

RIF

ICA

TIO

NO

FD

EX

TR

AN

SUC

RA

SEB

YT

HE

PHA

SE-P

AR

TIT

ION

ME

TH

OD

USI

NG

PEG

6000

No.

ofSt

eps

PEG

6000

%(v

/v)

Vol

ume

(mL

)D

extr

ansu

cras

ePr

otei

n(m

g/m

L)

SA*

(U/m

g)Fo

ldpu

rific

atio

n

(U/m

L)

Tota

lU

nits

Ove

rall

%yi

eld

Cru

de0

501.

4070

.0–

2.28

0.6

–St

ep1

550

1.27

63.5

901.

101.

83

Step

25

501.

2462

.089

0.14

58.

614

Step

35

501.

1859

.084

0.02

842

.170

*Sp

ecifi

cac

tivity

.

16 M. NIGAM, A. GOYAL and S.S. KATIYAR

The purification of dextransucrase by phase partitioning using PEG 400and PEG 6000 showed that repeated phase partitioning by PEG 6000 led to adegree of purity much greater than that obtained by PEG 400. The purificationof dextransucrase by phase partition using PEG 6000 resulted in higher overallyield (84%) than those reported previously (Paul et al. 1984; Monsan et al.1987). It was further found that a three-step phase partitioning with a finalconcentration of 5% PEG 6000, reproducibly yielded a homogeneous prepa-ration of dextransucrase, showing a single band on analysis by SDS-PAGE(Fig. 1). Repeated phase partitioning by PEG 6000 resulted in purified dex-transucrase preparation. The purified enzyme could be obtained with higher

Mw 1 2kDa

205

11697.4

66

45

29

FIG. 1. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF DEXTRANSUCRASEPURIFIED BY PHASE PARTITIONING. MOLECULAR WEIGHT MARKER PROTEINS OF

SIZE IN kDA ARE (LANE MW) MYOSIN (205), b-GALACTOSIDASE (116),PHOSPHORYLASE B (97.4), BOVINE PLASMA ALBUMIN (66) OVALBUMIN (45) ANDCARBONIC ANHYDRASE (29). (LANE 1) SHOWS THE CRUDE CELL EXTRACT AND

LANE 2 SHOWS THE PURIFIED DEXTRANSUCRASE AFTER THREE-STEP PHASEPARTITIONING WITH PEG 6000

17PURIFICATION OF DEXTRANSUCRASE BY PHASE PARTITIONING

specific activity and higher overall yields. The continuous culture of L.mesenteroides NRRL B-512F coupled with this purification technique can beused for large-scale production of purified dextransucrase.

The PEG phase-partitioning method for purification of dextransucrasedescribed in the present study is simple, inexpensive and less time consumingas compared to the other purification methods involving ultrafiltration andchromatography (Kobayashi and Matsuda 1980; Miller et al. 1986; Monsanet al. 1987; Fu and Robyt 1990). Thus providing a useful means of producingthe purified enzyme with high yield of protein and activity. The two-phasepartitioning process using PEG and dextran is quick and easy and allows therapid concentration of crude dextransucrase having a positive effect of dextranon the enzyme stability (Goyal et al. 1995). The method described previouslycan be increased in scale to deal with large volumes and could be widely usedfor the preparation of dextransucrase. There is scope of achieving furtherhigher specific activities, higher yields and higher folds of purification withPEGs of other molecular weights. The present study shows that evolving acomprehensive purification methodology relying on phase partitioning byPEG requires a clear understanding of protein-dextran, protein-PEG anddextran-PEG interactions in the selective use of the process.

ACKNOWLEDGMENT

We are thankful to Dr. Leela Iyengar for helpful suggestions anddiscussions.

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20 M. NIGAM, A. GOYAL and S.S. KATIYAR