Expression and Characterization of Levansucrase from Clostridium

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Expression and characterization of levansucrase from Clostridium acetobutylicum Song Gao, Xianghui Qi, Darren J. Hart, Herui Gao, and Yingfeng An J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05165 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Journal of Agricultural and Food Chemistry

1

Expression and characterization of levansucrase from

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Clostridium acetobutylicum

3

Song Gao a,b§, Xianghui Qi c§, Darren J. Hart d, Herui Gao a, Yingfeng An a §

§

*

4 5 6 7

a

College of Biosciences and Biotechnology, Shenyang Agricultural University,

Shenyang 110161, China b

College of Food Science, Shenyang Agricultural University, Shenyang

8

110161, China

9

c

School of Food and Biological Engineering, Jiangsu University, Zhenjiang

10

212000, China

11

d

12

Grenoble 38044, France

Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes,

13 14 15 16 17 18

* Corresponding author: Yingfeng An

19

Email: [email protected]

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Tel: +86-24-88487163.

Fax: +86-24-88487163

21 22

1

ACS Paragon Plus Environment


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Abstract

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The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase

25

was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287

26

bp and encodes 428 amino acid residues, which could convert 150 mmol/L

27

sucrose to levan with the liberation of glucose. The optimum pH and

28

temperature of this enzyme for levan formation were pH 6 and 60 ℃ ,

29

respectively. Levansucrase activity of Ca-SacB was completely abolished by 5

30

mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were

31

calculated to be 64 mmol/L and 190 µmol/min/mg, respectively. Interestingly,

32

Ca-SacB

33

fructooligosaccharide was identified in the product, indicating that Ca-SacB

34

may be valuable for industrial production of levan. In addition, Ca-SacB is the

35

first characterized levansucrase isolated from an anaerobic bacterium, which

36

should be valuable for exploring new enzyme resources and deepening the

37

understanding of the catalytic mechanisms of levansucrases.

was

found

to

have

high

product

specificity

38 39

Key words:

40

Ca-SacB; Clostridium acetobutylicum; levan; levansucrase

41 42 43 44

2


and

no

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Introduction

46

Levansucrase (EC 2.4.1.10), one of the fructosyltransferases, belongs to

47

glycoside hydrolase family 68 (GH68) 1 and catalyzes the production of levan,

48

composed of β-(2-6)-linked fructose residues.2 Levan has varieties of

49

applications in the fields of foods, cosmetics, and pharmaceuticals.3,4

50

Levansucrases are produced by various microorganisms belonging to the

51

genera Bacillus, Acetobacter, Lactobacillus, Geobacillus, Leuconostoc,

52

Zymomonas, Pseudomonas, etc.5 Levansucrase activity is involved in varieties

53

of processes including survival of bacteria in soil (e.g., B. subtilis), symbiosis

54

(e.g., Paenibacillus polymyxa) and phytopathogenesis (e.g., Pseudomonas

55

and Erwinia species) of plant interactive bacteria.6 Levansucrases catalyze at

56

least three different reactions: polymerization of fructose derived from sucrose,

57

hydrolysis of sucrose and hydrolysis of levan. 7

58

The 3D structures

of levansucrases 10

from Bacillus subtilis,8 B.

59

megaterium,9 Lactobacillus johnsonii

60

resolved. These levansucrases share a β-propeller fold consisting of five

61

antiparallel β-strands and a central negatively charged cavity, which are also

62

the significant characteristics of members of GH68.9

and Erwinia amylovora

11

were

63

Until now, all the reported levansucrases are from aerobic bacteria and

64

microaerobes, but no levansucrases from anaerobic bacteria have been

65

characterized. Therefore, isolating and characterizing of levansucrases from

66

anaerobic bacteria should be valuable for exploring new enzyme resources 3



67

and deepening the understanding of their catalytic mechanisms. C.

68

acetobutylicum is a very important anaerobic bacterium which can be used for

69

producing acetone, ethanol, and butanol from starch. Although levansucrase

70

Ca-SacB from C. acetobutylicum can be predicted by BLAST, no detailed

71

information about this enzyme has been reported. In the present study, we

72

report the first-time molecular cloning and expression of C. acetobutylicum

73

levansucrase (Ca-SacB) in E. coli. This result will give information for better

74

understanding of the catalytic strategies of Ca-SacB and laying the

75

foundations for industrial applications of this enzyme for the production of

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levan.

77 78

Materials and methods

79

2.1. Strains, plasmids and media

80

C. acetobutylicum was anaerobically cultured in medium containing 3%

81

(w/v) glucose, 0.5% (w/v) yeast extract, 0.07% (w/v) (NH4)2HPO4, 0.2% (w/v)

82

CaCO3, pH7.0. E. coli JM109 strain (Promega, USA) was used for molecular

83

cloning and propagation of the plasmids, and E. coli BL21(DE3) strain was

84

used for expression of the recombinant levansucrase. LB agar plates

85

supplemented with 5% (w/v) sucrose, 50 mg/L kanamycin, and 0.1 mmol/L

86

isopropyl-β-d-thiogalactopyranoside (IPTG) were used for the identification of

87

the levansucrase phenotype.

88

2.2. Cloning, expression and characterization of Ca-SacB 4


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Genomic DNA was isolated from C. acetobutylicum and used as template

90

for PCRs. Ca-SacB gene was amplified by PCR using primers: sacB-For

91

(5’-CTAGG ACGTC GTTGA AAACA AGAAA AACTT ATAAA ATGAT ATCTT

92

CGC-3’, Aat II underlined) and sacB-Rev1 (5’-TACCA CTAGT ATGTG

93

CAGGC GTAAC TACTC CTTCC CCAAG-3’, Spe I underlined). The PCR

94

products were cloned into the corresponding restriction enzyme sites of

95

pETM11 to give pET-Ca-SacB. pET-Ca-SacB was transformed into E. coli

96

BL21(DE3).

97

supplemented with 50 mg/L kanamycin. DNA sequencing was carried out by

98

GENEWIZ Company in China. BLAST program was used for sequence

99

homology searches of GenBank (NCBI, Bethesda, MD, USA).

The transformants

were

replicated

on

LB

agar

plates

100

For protein expression and purification, the strain of E. coli BL21(DE3)

101

transformed with pET-Ca-SacB was cultured in TB media and protein

102

expression was induced by 0.1 mmol/L IPTG. The cells were pelleted by

103

centrifugation, resuspended in 50 mmol/L sodium phosphate buffer (pH 5.9),

104

and then disrupted by sonication. The expressed protein was purified using

105

Ni-NTA agarose (Qiagen, Germany) chromatography. The lysate was

106

incubated with Ni-NTA slurry at 4℃ for 10 min followed by loading to a column.

107

The sample was washed three times with washing buffer (20 mmol/L imidazole,

108

300 mmol/L NaCl, 50 mmol/L NaH2PO4, pH 8.0) and Ca-SacB protein was

109

then eluted with 0.5 ml of elution buffer (250 mmol/L imidazole, 300 mmol/L

110

NaCl, 50 mmol/L NaH2PO4, pH 5.9). To determine kinetic parameters, sucrose 5



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111

hydrolysis was analyzed in a reaction containing an appropriate amount of

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sucrose (10-1000mmol/L) and purified enzyme. The reactions were incubated

113

at 50℃, and the glucose content was determined by Glucose Assay Kit (HuiLi

114

Biotech Co., China).

115

2.3. Isolation and component analysis of fructan

116

Purified Ca-SacB was added to 50 mmol/L sodium phosphate buffer (pH

117

5.9) containing 10% (w/v) sucrose. The reaction mixture was incubated at 20℃

118

for 72 h. The soluble section of the products catalyzed by Ca-SacB was filtered

119

through 0.22-µm Millipore filters and analyzed by high-performance liquid

120

chromatography (HPLC). HPLC analysis was carried out on a Waters 1525

121

HPLC system (Milford, MA, USA) using a Waters Symmetry C18 column (250

122

mm × 4.5 mm). The standards contain fructose (F), glucose (G), sucrose (GF),

123

1-kestose (GF2), nystose (GF3), and fructofuranosyl-nystose (GF4) (Meiji

124

Seika Kaisha Ltd). Degassed 70% acetonitrile at 1.0 mL/min was used as

125

mobile phase. The eluate was monitored with a 2414 Refractive Index

126

Detector.

127

Then

13

C-NMR spectrometry was used to analyze linkage type of the

128

fructan. An equal volume of ethanol was added to the reaction mixture,

129

followed by incubation at 4℃ for 12 h to allow the precipitation of fructan.

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Fructan was recovered by centrifugation (200,000×g) and resuspended in

131

water. The fructan pellet was washed twice by precipitations as described

132

above, and then the pellet was dehydrated by lyophilization. Then 6


13

C-NMR

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133

spectrometry was run at 125 MHz on AMX-500 (Bruker, Germany).

134

Assignment of peaks was based on the report of Shimamura et al.12

135

2.4. Effect of temperature, pH, metal ions and chemicals

136

Effects of temperatures between 20 and 80℃ on stability and activity of

137

the Ca-SacB were studied. Thermostability was determined by incubating the

138

enzyme (1133 U/mg) for 30 min at a designated temperature, where 1 U was

139

defined as the amount of enzyme required to release 1 µmol of glucose per

140

minute under standard conditions. After incubation, the residual enzyme

141

activity was assayed under the standard reaction condition at 50℃. The effect

142

of pH on enzyme activity was assayed by varying pH between 3.0 and 8.0.

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McIlvaine buffer (prepared by mixing 0.1 mol/L Na2HPO4 and 0.1 mol/L citric

144

acid) and borax buffer (prepared by mixing 0.05 mol/L borax and 0.2 mol/L

145

boric acid) were used for pH 3.0-7.0 and pH 8.0-9.0, respectively. Then

146

Ca-SacB was incubated at the indicated pH for 30 min at 50℃, and at each pH

147

the activity prior to incubation was used as positive control to determine pH

148

stability via assaying the residual activity after incubation. The effect of various

149

metal ions [CuCl2, AlCl3, Hg(NO3)2, MnCl2, MgSO4, KCl, LiCl, NaCl, ZnSO4,

150

CaCl2, BaCl2, NiSO4, CoCl2, SnCl2, RbCl, AgNO3, and FeSO4] and chelating

151

agents (EDTA, Urea and SDS) on levansucrase activity were studied by

152

incubating Ca-SacB solution with the respective chemicals for 30 min under

153

optimized conditions of 50℃ and pH5.9.

154

3. Results and discussion 7



155

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3.1. Cloning, expression and characterization of Ca-SacB

156

The Ca-SacB gene was composed of 1287 bp nucleotides encoding 428

157

amino acid residues. The deduced amino acid sequence of Ca-SacB gene

158

was compared with some reported levansucrases from other microorganisms.

159

It showed identity with amino acid sequences of levansucrases from

160

Brevibacillus formosus (53%),13 Streptomyces olindensis (44%),14 Rahnella

161

aquatilis (37%),15 Zymomonas mobilis (36%),16 Pseudomonas syringae

162

(36%),17 B. subtilis (29%).1 Ca-SacB originates from a gram-positive strain,

163

and its gene sequence was most homologous to those of other gram-positive

164

strains, such as B. formosus and S. olindensis.

165

Sequence alignment of Ca-SacB and levansucrase from B. subtilis

166

(Bs-SacB) based on structural superimposition was generated by ESPript 3.0

167

(Fig. 1). Secondary structure elements were labelled using the structure of

168

Bs-SacB as template. The conserved regions are suggested to be important

169

for the activities, e.g., sucrose hydrolysis and transfer of fructose to the proper

170

acceptors.18,19 There are totally seven conserved regions in the reported

171

levansucrases from gram-positive strains,20 six of which (i.e., II to VII) are

172

conserved in Ca-SacB. In addition, three crucial amino acid residues (i.e., D in

173

conserved region II, D in region IV, and E in region V) that function together at

174

the center of the active site (i.e., catalytic triad) in reported levansucrases

175

were also conserved in Ca-SacB.

176

21

To further understand the structure and functions of Ca-SacB, a protein 8


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177

model was built by SWISS-MODEL using the crystal structure of Bacillus

178

subtilis levansucrase (PDB ID: 1oyg) as template (Fig. 2). According to the

179

model, Ca-SacB has the typical structure of β-propeller fold consisting of five

180

blades with antiparallel β-strands (Fig. 2-a), and the β-propeller of each

181

structure forms a central negatively charged cavity, which is essential for

182

activity (Fig. 2-a, b). Although the protein sequences of CA-SacB and Bs-SacB

183

have a low identity (29%), the alignment of the model of CA-SacB and the

184

solved Bs-SacB structure shows that their structures might have high identity

185

(Fig. 2-c).

186

Based on Bs-SacB structure1 and alignment of amino acid sequence of

187

Bs-SacB (Fig 1 and Fig 2-d), we could predict three fully conserved active site

188

amino acid residues (D71, D222 and E306). The D71 (corresponding to D86 in

189

Bs-SacB) and E306 (corresponding to E342 in Bs-SacB) might form the pair of

190

essential catalytic side chains, whereas D222 (corresponding to D247 in

191

Bs-SacB) might interact with hydroxyls of the fructosyl unit of substrate, and

192

form strong hydrogen bonds. E306 might be part of a complex network of

193

interactions that includes R221 (corresponding to R246 in Bs-SacB), H324

194

(corresponding to Arg360 in Bs-SacB) and Y371 (corresponding to Y411 in

195

Bs-SacB), and Q304 (corresponding to E340 in Bs-SacB). Although some of

196

the amino acid residues mentioned above are not fully conserved, their side

197

chains or properties are similar to that of Bs-SacB.

198

In order to characterize the enzymatic properties of Ca-SacB, His-tagged 9



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199

Ca-SacB was purified by Ni–NTA chromatography. The purified levansucrase

200

from E. coli lysate showed specific activity of 1133 U/mg. The Km of Ca-SacB

201

was 64 mmol/L sucrose and the Vmax was 190 µmol/min/mg. The Km value of

202

this enzyme was similar to the Km of levansucrase from R. aquatilis JCM-1683

203

(50 mmol/L).22 However, levansucrases from Z. mobilis

204

were reported to have Km of higher values: 160 and 122 mmol/L, respectively.

205

3.2. Analysis of sugar components

23

and P. syringae

24

206

The transfructosylation reactions and levan formation by Ca-SacB were

207

assayed in a standard reaction containing sucrose. As a result, Ca-SacB

208

expressed in E. coli could catalyze the production of turbid levan (Fig. 3-a).

209

The soluble section of the products catalyzed by Ca-SacB was analyzed by

210

HPLC. As a result, nearly no fructooligosaccharide was identified from this

211

section, indicating that levan was the only product of transfructosylation by

212

Ca-SacB (Fig. 3-b). According to the HPLC diagram, about 61% sucrose has

213

been converted into levan and glucose during the reaction.

214

The linkage type of the polymer was analyzed by

13

C-NMR spectrometry

215

(Fig 4). Assignment of peaks was based on the report of Shimamura et al.12

216

The results indicate that the polymer is levan of β-2, 6-fructan. In this study,

217

only the linkage type of the insoluble polymer with high molecular mass has be

218

analyzed by 13C-NMR spectrometry, because the soluble sucrose and

219

glucose in the reaction mixture have been eliminated by centrifugation.

220

3.3. Effect of temperature, pH, metal ions and chemicals 10


Page 11 of 28


221

As shown in Fig. 5, the optimum temperature of Ca-SacB was 60℃. The

222

activity was greatly reduced at temperatures below 30℃ or above 70℃ (Fig.

223

5-a). At temperatures higher than 70 ℃ , this enzyme was inactive. The

224

thermostability decreased sharply above a 70℃ threshold temperature. The

225

optimum pH of Ca-SacB was found to be 6.0 (Fig. 5-b). The activity was

226

greatly reduced at pH below 4.0 or above pH 7.0.

227

The effect of metal ions and other reagents on levansucrase activity of

228

Ca-SacB was determined by incubating Ca-SacB in the presence of reagents

229

at 50℃ for 30 min. The residual activity was assayed by the standard method.

230

As a result, the activity was strongly inhibited by CuCl2, Hg(NO3)2, AgNO3 and

231

SDS; while SnCl2 and MnCl2 increased levansucrase activity by 43% and 28%,

232

respectively (Table 1). SDS is a commonly used protein-denaturing agent in

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biology laboratories. The levansucrases from A. diazotrophicus,6 Bacillus sp.

234

TH4-2,20 and Leuconostoc mesenteroides

235

inactivated by Hg2+ and Ag+. Recently, Mn2+ has also been found to have

236

positive effect on the activity of levansucrase from B. subtilis, which was

237

postulated to be associated with the folding cofactor effect of this metal.25

238

Interestingly, in this study SnCl2 was found to have the strongest activation

239

effect on Ca-SacB, but it has never been reported to have similar effect on

240

other levansucrases.

7

were also strongly inhibited or

241

To sum up, in the present study, we first describe the cloning,

242

heterologous expression and characterization of levansucrase gene Ca-SacB 11



243

from C. acetobutylicum, which should lay the foundation for further

244

modification of this enzyme for more efficient production of fructan. Further

245

studies aimed at better understanding the catalysis of transfructosylation by

246

Ca-SacB is now in progress.

247

Acknowledgments

248 249

The authors would like to thank Sergi Castellano and Promdonkoy Patcharee for helpful discussions and review of this manuscript.

250 251

§The authors Song Gao and Xianghui Qi contributed equally to this

252

work.

253 254

Funding Sources

255

This work was supported by National Natural Science Foundations of

256

China (grant numbers 31100045, 31270114, 31571806), Program for Liaoning

257

Excellent Talents in University (grant number LR2014018), and Liaoning

258

BaiQianWan Talents Program (grant number 2015-40).

259 260 261 262 263 264 265

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(21) Verhaest, M.; Van den Ende, W.; Roy, K.L.; De Ranter, C.J.; Laere,

333

A.V.; Rabijns, A. X-ray diffraction structure of a plant glycosyl hydrolase family

334

32 protein: fructan 1-exohydrolase IIa of Cichorium intybus. Plant J.

335

2005,41,400−411.

336

(22) Hernandez, L.; Arrieta, J.; Menendez, C.; Vazquez, R., Coego, A.;

337

Suarez, V.; Selman, G.; Petit-Glatron, M.F.; Chambert, R. Isolation and

338

enzymic properties of levansucrase secreted by Acetobacter diazotrophicus

339

SRT4,

340

1995,309,113−118.

a

bacterium

associated

with

sugar

cane.

Biochem.

J.

341

(23) Yanase, H.; Iwata, M.; Nakahigashi, R.; Kita, K.; Kato, N.; Tonomura,

342

K. Purification, crystallization and properties of the extracellular levansucrase

343

from Zymomonas mobilis, Biosci. Biotechnol. Biochem. 1992,56,1335−1336.

344

(24) Hettwer, U.; Gross, M.; Rudolph, K. Purification and characterization

345

of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola.

346

J. Bacteriol. 1995,177,2834−2839.

347

(25) Artur, S.; Kamila, G.; Małgorzata, G. Synthesis of ß-(2-6)-linked

348

fructan with a partially purified levansucrase from Bacillus subtilis. J. Mol. Catal.

349

B-Enzym. 2016,131,1−9.

350 351 352 353

16


Page 17 of 28


354

Fig. 1 Ca-SacB and Bs-SacB sequence alignment based on structural

355

superimposition. Secondary structure elements α-helices and β-strands are

356

indicated by squiggles and arrows, respectively. The α-helices (labelled α) and

357

β-strands (labelled β) are consecutively numbered. The regions considered as

358

important for activity are underlined and consecutively numbered from I to VII.

359

The catalytic triad at the expected center of the active site (i.e., D in conserved

360

region II, D in region IV, and E in region V) are marked with triangles.

361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 17



391

Fig. 2 Analysis of protein model of CA-SacB. (a) shows model of Ca-SacB.

392

α-helices are shown by solid lines in red, and β-strands in blue; (b) shows

393

surface of protein model of Ca-SacB. The central negatively charged cavity in

394

red shows the center of the active site; (c) shows alignment of Bs-SacB

395

structure (in green) and the model of CA-SacB (in blue); (d) shows alignment

396

of some important amino acid residues Bs-SacB structure (in blue) and in the

397

model of CA-SacB (in green). Numbering of amino acid residues is based on

398

Bs-SacB, with the numbering of CA-SacB in parentheses.

399 400 401 402 403 404 405 406 407 408 409 410 411 412

18


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Page 19 of 28


413

Fig. 3 Production of turbid levan introduced by Ca-SacB in the presence

414

of sucrose and analysis of the soluble section by HPLC. (a) shows the

415

production of turbid levan by transfructosylation activity of Ca-SacB. 1 and 2

416

refer to reactions be associated with E. coli BL21(DE3) harboring plasmid

417

pET-Ca-sacB and pETM11, respectively; (b) shows HPLC chromatogram of

418

the soluble section of the products catalyzed by Ca-SacB. G, GF, GF2, GF3

419

and

420

fructofuranosyl-nystose, respectively

GF4

refer

to

glucose,

sucrose,

1-kestose,

421 422 423 424 425 426 427 428 429 430 431 432 433 434

19


nystose

and


435

Fig. 4. Analysis of components of sugar synthesized using purified

436

Ca-SacB by

437

synthesized using Ca-SacB; (b) shows chemical shifts for C-NMR spectra of

438

levan and polymer synthesized using Ca-SacB.

13

C-NMR spectra. (a)

13

C-NMR spectra shows polymer

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456

20


Page 20 of 28

Page 21 of 28


457

Fig. 5. Effect of temperature (a) and pH (b) on activity and stability of

458

Ca-SacB. The activity (shown as solid circles) and stability (shown as solid

459

squares) were measured using 5% (w/v) sucrose as substrate. Error bars

460

represent means ± standard deviations (n=3).

461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 21



Table 1 Effect of metal ions and detergents (5mmol/L) on levansucrase activity Compound Relative activity(%) Control (without any metal ion) 100 AgNO3 2 Hg(NO3)2 2 55 AlCl3 BaCl2 107 FeSO4 50 LiCl 104 NaCl 100 NiSO4 109 SnCl2 143 RbCl 99 KCl 118 ZnSO4 41 CoCl2 104 CuCl2 5 CaCl2 113 128 MnCl2 MgSO4 115 EDTA 36 Urea 96 SDS 3

22


Page 22 of 28

Page 23 of 28


Fig. 1 Ca-SacB and Bs-SacB sequence alignment based on structural superimposition. Secondary-structural elements α-helices and β-strands are indicated by squiggles and arrows, respectively. The α-helices (labelled α) and β-strands (labelled β) are consecutively numbered. The regions considered as important for activity are underlined and consecutively numbered from I to VII. The catalytic triad at the expected center of the active site (i.e., D in conserved region II, D in region IV, and E in region V) are marked with triangles.

99x108mm (300 x 300 DPI)



Fig. 2 Analysis of protein model of CA-SacB. (a) shows model of Ca-SacB. α-helices are shown by solid lines in red, and β-strands in blue; (b) shows surface of protein model of Ca-SacB. The central negatively charged cavity in red shows the center of the active site; (c) shows alignment of Bs-SacB structure (in green) and the model of CA-SacB (in blue); (d) shows alignment of some important amino acid residues Bs-SacB structure (in blue) and in the model of CA-SacB (in green). Numbering of amino acid residues is based on Bs-SacB, with the numbering of CA-SacB in parentheses.

85x80mm (300 x 300 DPI)


Page 24 of 28

Page 25 of 28


Fig. 3 Production of turbid levan introduced by Ca-SacB in the presence of sucrose and analysis of the soluble section by HPLC. (a) shows the production of turbid levan by transfructosylation activity of Ca-SacB. 1 and 2 refer to reactions be associated with E. coli BL21(DE3) harboring plasmid pET-Ca-sacB and pETM11, respectively; (b) shows HPLC chromatogram of the soluble section of the products catalyzed by Ca-SacB. G, GF, GF2, GF3 and GF4 refer to glucose, sucrose, 1-kestose, nystose and fructofuranosyl-nystose, respectively

33x13mm (300 x 300 DPI)



Fig. 4. Analysis of components of sugar synthesized using purified Ca-SacB by 13C-NMR spectra. (a) 13CNMR spectra shows polymer synthesized using Ca-SacB; (b) shows chemical shifts for C-NMR spectra of levan and polymer synthesized using Ca-SacB. 57x38mm (300 x 300 DPI)


Page 26 of 28

Page 27 of 28


Fig. 5. Effect of temperature (a) and pH (b) on activity and stability of Ca-SacB. The activity (shown as solid circles) and stability (shown as solid squares) were measured using 5% (w/v) sucrose as substrate. Error bars represent means ± standard deviations (n=3).

82x80mm (300 x 300 DPI)



TOC Graphic 44x24mm (300 x 300 DPI)


Page 28 of 28

Expression and Characterization of Levansucrase from Clostridium

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