Identification of an Î±-(1,4)-Glucan-Synthesizing Amylosucrase from

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Identification of an #-(1, 4)-glucan-synthesizing amylosucrase from Cellulomonas carbonis T26 Yongchun Wang, Wei Xu, Yuxiang Bai, Tao Zhang, Bo Jiang, and Wanmeng Mu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05667 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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Page 1 of 38

Journal of Agricultural and Food Chemistry

Identification of an α-(1, 4)-glucan-synthesizing amylosucrase from Cellulomonas carbonis T26 Yongchun Wang,† Wei Xu,† Yuxiang Bai,† Tao Zhang,† Bo Jiang,† Wanmeng Mu†,§,*

†

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China

§

Ministry of Education, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China

*

Corresponding author. Tel: +86 510 85919161. Fax: +86 510 85919161.

E-mail address: [email protected] (W. Mu).

1

ACS Paragon Plus Environment


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Abstract

2

Amylosucrase, catalyzing the synthesis of α-(1, 4)-glucan from sucrose, has been

3

widely studied and used in carbohydrate biotransformation because of its versatile

4

activities. In this study, a novel amylosucrase was characterized from Cellulomonas

5

carbonis T26. The recombinant enzyme was overexpressed in Escherchia coli and

6

purified by nickel affinity chromatography. It was determined to be a monomeric

7

protein with molecular mass of 72 kDa. The optimum pH and temperature for

8

transglucosylation were measured to be pH 7.0 and 40 °C. The transglucosylation

9

activity was significantly higher than the hydrolytic activity. The main product

10

generated from sucrose was structurally determined to be α-(1, 4)-glucan. A small

11

amount of glucose was produced by hydrolysis and sucrose isomers including

12

turanose and trehalulose were generated as minor products. The ratio of hydrolytic,

13

polymerization, and isomerization reactions was calculated to be 5.8 : 84.0 : 10.2. The

14

enzyme favored to produce long-chain insoluble α-glucan at lower temperature.

15

Amyloscucrase ·

Cellulomonas carbonis ·

16

Keywords:

17

Polymerization · Transglucosylation

2


α-(1,

4)-Glucan ·

Page 3 of 38


18

INTRODUCTION

19

Homopolysacchride biosynthesis from sucrose attracts increasing attention in

20

recent years. Some microbial glycosyltransferases (EC 2.4.1) are able to polymerize

21

the D-glucose and D-fructose moieties of sucrose to synthesize fructans and glucans,

22

respectively. Inulosucrase (EC 2.4.1.9) and levansucrase (2.4.1.10), which occur in a

23

wide range of bacteria, catalyze the polymerization of sucrose to inulin-type and

24

levan-type fructan, with β-(2, 1) and β-(2, 6) fructosyl-fructose linkages, respectively.1

25

Lactic acid bacteria may use sucrose to synthesize a diversity of long-chain α-glucans

26

with different linkages by various glycoside hydrolase family 70 glucansucrases (or

27

glucosyltransferases).2 For instance, dextransucrase (EC 2.4.1.5) generates α-glucans

28

mainly composed α-(1, 6) linkages,3 mutansucrase (EC 2.4.1.125) catalyzes the

29

biosynthesis of α-(1, 3)-glucan, termed mutan;4 alternansucrase (EC 2.4.1.140)5 and

30

reuteransucrase (EC 2.4.1.-)6 catalyze the D-glucosyl residue polymerization from

31

sucrose

32

α-(1, 6)-α-(1, 4)-glucan termed reuteran, respectively. In addition, a glycoside

33

hydrolase family 13 enzyme, amyloscurase (AS, sucrose: 1, 4-α-D-glucan

34

4-α-D-glucosyltransferase, EC 2.4.1.4) converts sucrose to α-glucan with only α-(1, 4)

35

linkages.7

to

produce

α-(1,

6)-α-(1,

3)-glucan

termed

alternan

and

36

AS catalyzes the α-D-glucosyl residue transfer from sucrose to the 4-position of

37

non-reducing terminal residue of an α-glucan generating an insoluble α-(1, 4)-glucan

38

accompanied with the release of D-fructose from sucrose. Like some glucansucrases,

39

AS also has sucrose hydrolytic activity to release D-glucose and D-fructose molecules

40

and may catalyze the transglycosylation reaction from sucrose to many acceptor

41

molecules. In addition, in contrast to glucansucrases, AS uniquely produces a small

42

amount of sucrose isomers including turanose and trehalulose when the D-glucosyl

3



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43

moiety of sucrose is transferred onto the released D-glucose and D-fructose,

44

respectively.8 Because of the versatile activities, AS has been widely used for

45

production of various carbohydrate-based bioactive compounds, such as modified

46

starch,9-13 sucrose isomers,14 α-glucans,15 and some bioactive α-glucosides, including

47

dihydrochalcone glucosides,16 α-D-glucosyl glycerol,17 (+)-catechin α-glycosides,18

48

salicin glycosides,19 arbutin-α-glucoside,20 and rutin derivatives.21 AS can be used to

49

convert sucrose to cyclodextrin,22 cycloamyloses coupled with 4-α-glucanotransferase

50

reaction23

51

synthase-trehalohydrolase.24 In addition, AS can be used for the synthesis of amylose

52

microparticles,25 amylose nanocomposite microbeads26 and amylose magnetic

53

microparticles27 through a self-assembly process of biosynthesized amylose.

and

trehalose

coupled

with

maltooligosyltrehalose

54

Seventy years ago, Hehre and coworkers first found that sucrose can be converted

55

to a glycogen-like polysaccharide by certain bacteria of Neisseria genus, without

56

dependence of D-glucose-1-phospahte as an intermediate substance, and they named

57

this responsible enzyme AS.28 Then, AS from Neisseria perflava (NPr-AS)29 and

58

Neisseria polysaccharea ATCC 43768 (NPo-AS)30 were identified in native and

59

recombinant form, respectively. In the new century, six more recombinant AS

60

enzymes have been identified from Deinococcus radiodurans ATCC 13939

61

(DRd-AS),31 Deinococcus geothermalis DSM 11300 (DG-AS),32,

62

macleodii KCTC 2957 (AM-AS),34 Arthrobacter chlorophenolicus A6 (AC-AS),35

63

Methylobacillus flagellatus KT (MF-AS),17 and Deinococcus radiopugnans ATCC

64

19172 (DRp-AS),7 respectively.

33

Alteromonas

65

Unlike other members of glycoside hydrolase family of enzymes, AS is not found

66

in a broad range of microorganisms.7 So far, this distinct enzyme has only been

67

characterized from less than 10 microorganisms abovementioned. In this work, a

4


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68

novel AS was identified from a gram-positive aerobic strain, Cellulomonas carbonis

69

T26, with a high α-(1, 4)-glucan-producing activity. The recombinant C. carbonis AS

70

(CC-AS), heterologously expressed in Escherichia coli, was purified and

71

characterized, and its enzymatic properties were investigated and compared with the

72

reported ones from other bacteria. To our best knowledge, it is the first report on the

73

AS identification from a Cellulomonas species strain.

74 75

MATERIALS AND METHODS

76

Cloning, expression, and enzyme purification. Full-length nucleotide sequence

77

of the AS-encoding gene (locus_tag: N868_11335) from C. carbonis T2638 was

78

commercially synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China).

79

The gene fused with a 6×histidine-tag sequence at 3′-terminal was inserted into

80

pET-22b(+) vector with NdeI and XhoI restriction sites. The generated reconstructed

81

plasmid, termed pET-CC-AS, was transformed into host E. coli BL21(DE3).

82

A selected colony of recombinant E. coli BL21(DE3) harboring pET-CC-AS was

83

inoculated in 200 mL Luria-Betani (LB) medium (5 g/L yeast extract, 10 g/L tryptone,

84

and 5 g/L NaCl) supplemented with 100 µg/mL ampicillin for growth at 37 °C and

85

200

86

isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1

87

mM to induce the expression at 28 °C for another 6 h.

rpm.

When

the

optical

density

at

600

nm

reached

0.6,

88

The pelleted cells were collected by centrifugation at 4,900 × g for 20 min,

89

resuspended in Lysis buffer (50 mM Tris-HCl buffer, 100 mM NaCl, pH 7.5), kept in

90

ice bath and then disrupted by sonication for 15 min (pulse on for 1 s and pulse off for

91

3 s) using a Vibra-Cell 72405 Sonicator (Bioblock, Illkirch, France). After being

92

centrifugated at 19,000 × g for 30 min, the supernatant dissolved AS was filtered

5



93

through a 0.45-µm filter, and then was loaded onto a Ni2+-chelating Sepharose Fast

94

Flow column (Uppsala, Sweden) for nickel affinity chromatography. The column was

95

equilibrated with a binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl,

96

pH 7.0). Followed by using washing buffer (50 mM sodium phosphate buffer, 500

97

mM NaCl, 50 mM imidazole, pH 7.0) and elution buffer (50 mM sodium phosphate

98

buffer, 500 mM NaCl, 500 mM imidazole, pH 7.0) to remove miscellaneous or

99

unbound proteins and obtain recombinant AS. All the purification steps were carried

100

out at 4 °C. The purified enzyme was then dialyzed against 50 mM sodium phosphate

101

buffer (pH 7.0).

102 103

Protein concentration and molecular mass. The protein concentration was

104

calculated according to the method of Bradford.36 The molecular mass of subunit and

105

native enzyme were examined by sodium dodecyl sulfate polyacrylamide gel

106

electrophoresis (SDS-PAGE) and gel filtration, respectively. For SDS-PAGE, a 5%

107

stacking gel and a 12% separating gel were used, and the separate protein bands were

108

fixed with trichloroacetic acid (12%, w/v), stained with Coomassie Brilliant Blue

109

R250, and finally destained until the background was colorless. For gel filtration, the

110

total molecular mass of the protein was estimated using a gel filtration

111

chromatography [Column: TSK G2000SWxl (Tosoh Bioscience LLC, Minato-ku,

112

Tokyo, Japan); Mobile phase: 100 mM phosphate buffer (pH 6.7) containing 100 mM

113

Na2SO4 and 0.05% (w/v) NaN3; Flow rate: 1 mL/min; Detection: UV at 280 nm;

114

Standard samples: thyroglobulin (porcine thyroid gland, MW: 669 kDa), β-amylase

115

(200 kDa), alcohol dehydrogenase (150 kDa), albumin: (66 kDa), and carbonic

116

anhydrase (29 kDa)].

117

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Page 7 of 38


118

Enzyme assay. Enzymatic activity was assayed at 40 °C with 100 mM sucrose as a

119

sole substrate in 50 mM phosphate buffer (pH 7.0) for 20 min. Total activity was

120

measured based on the release of fructose from sucrose, since fructose generation

121

reflects total consumption of sucrose. Glucose is produced from sucrose due to

122

sucrose hydrolysis with water as an acceptor and thus hydrolytic activity was

123

determined by calculating the release of glucose. Transglycosylation activity was

124

measured as total activity minus hydrolytic activity and calculated by subtracting the

125

amount of glucose from that of fructose.34 One unit of total activity and hydrolytic

126

activity were defined as the amount of enzyme catalyzing the release of 1 µmol

127

fructose and glucose per min, respectively. And transglycosylation activity was

128

defined as total activity minus hydrolytic activity. In this work, the enzyme activity

129

was described as total activity, unless otherwise specified.

130 131

Effect of pH, temperature, and metal ions on enzymatic activity. Effect of pH

132

on AS activity was investigated within a range of pH 4.5 – 8.5 at 40 °C. Three

133

different buffer systems were used including acetate buffer (50 mM, pH 4.5 – 6.0),

134

sodium phosphate buffer (50 mM, pH 6.0 – 7.5), and Tris-HCl (50 mM, pH 7.5 – 8.5).

135

Effect of temperature on the enzymatic activity was studied in 50 mM sodium

136

phosphate buffer (50 mM, pH 7.0) ranging from 30 to 50 °C. To investigate the effect

137

of metal ions on enzymatic activity, metal ions (in the form of CuSO4, FeSO4, ZnSO4,

138

MgSO4, MnSO4, and NiSO4) were used at a final concentration of 1 mM.

139 140

Effect of temperature on the enzyme stability. Thermostability was observed by

141

pre-incubating the purified enzyme at different temperatures and examined by

142

measuring the residual activity after various durations of pre-incubation.

7



143

The melting temperature (Tm) was determined by differential scanning calorimetry

144

(DSC) using a TA Instruments Nano DSC with platinum capillary cell (New Castle,

145

PA, USA). The enzyme was re-dialyzed against sodium phosphate buffer overnight,

146

and dialyzed buffer was collected to serve as reference. The enzyme solution was

147

degassed under vacuum (635 mmHg) for 10 min and loaded into the DSC cell. The

148

cell was heated from 25 to 100 °C at 3 atmospheric pressure with a temperature ramp

149

of 1 °C/min. Sodium phosphate buffer was used as corresponding reference. DSC data

150

were analyzed using TA Instruments NanoAnalyze software and the observed

151

thermograms were baseline-corrected.

152 153

Iodine binding properties. Reactions were performed in duplicate in 50 mM

154

sodium phosphate buffer (pH 7.0) containing 100 mM sucrose. After reaction at 40 °C

155

for 24 h, one was checked by aqueous iodine solution treatment; the other was

156

hydrolyzed by 20 µL amyloglucosidase-A7095 (Sigma-Aldrich, St Louis, MO, USA)

157

at 37 °C for 2 h and followed by iodine treatment. The iodine reactivity was

158

performed using 40 µL of aqueous iodine solution [2% (w/v) KI and 0.2% (w/v) I2].

159 160

Isolation of soluble products produced from sucrose by CC-AS. Reaction

161

products from sucrose by CC-AS were centrifuged at 13,000 × g for 15 min to remove

162

the precipitate. The supernatant was treated with Sevag reagent (n-butanol:

163

chloroform = 1:4, v/v) for 6 times to remove any proteins.37 After that, three volumes

164

of 95% ethanol were added at room temperature, and stored at 4 °C overnight. The

165

mixture was then centrifuged at 4 °C and 13,000 × g for 30 min to separate precipitate.

166

Then the precipitate was collected, dissolved in deionized water, and freeze-dried

167

using a 4.5 L FreeZone freeze-dry system (Labconco Corp, MO, USA).

8


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Page 9 of 38


168 169

Fourier-transform infrared spectroscopy (FT-IR) analysis. The basic functional

170

group of the reaction products was determined by FT-IR analysis. The product

171

powder was mixed with potassium bromide at a ratio of 1:100 and pressed into a

172

tablet. An intermediate infrared region from 400 to 4000 cm-1 was used for scanning

173

at 4 cm-1 resolution using a Thermo Nicolet NEXUS 470 FT-IR (Thermo Fisher

174

Scientific, USA).

175 176

Nuclear magnetic resonance spectroscopy (NMR) analysis. Lyophilized sample

177

(35 mg) was mixed with 650 µL deuterium oxide and maintained in water bath at

178

90 °C for 5 h to completely dissolve the sample. The

179

1

180

at 60 °C using an AVANCE III 400MHz Digital NMR Spectrometer (Bruker Biospin

181

International AG, Switzerland). Chemical shifts (δ), expressed in ppm, were

182

determined

183

4, 4-dimethyl-4-silapentane-1-sulfonate (DSS) (δH = δC = 0.00 ppm) dissolved in the

184

samples.

1

H,

13

C, and

H-13C heteronuclear single quantum coherence (HSQC) NMR spectra were recorded

with

respect

to

the

signals

for

sodium

185 186

High performance anion exchange chromatography (HPAEC) analysis.

187

HPAEC with pulsed amperometric detection (PAD) was used for carbohydrate

188

analysis. Samples were filtered by a 0.45-µm membrane filter and then injected into

189

the HPAEC-PAD system (Dionex DX 600) equipped with an ED 50 electrochemical

190

detector with a gold working electrode, GP 50 gradient pump, LC 30 chromatography

191

oven, and AS 40 automated sampler (Dionex Corporation, Sunnyvale, CA, USA).

192

Dionex CarboPac™ PA1 and PA200 columns (Dionex Corporation) were used for

9



193

analysis of short-chain and relatively long-chain oligosaccharides, respectively. Three

194

eluents were used, including 1 M sodium acetate, ultrapure water, and 250 mM NaOH

195

as eluent A, B, and C, respectively. Relatively long-chain oligosaccharides were

196

eluted with a linear gradient (flow rate, 0.5 mL/min; column temperature, 30 °C;

197

elution procedure: 38% elution C and 58% elution B at 0.0 min, 24% elution C and 36%

198

elution B at 40.0 min, and 38% elution C and 58% elution B at 40.1 min). Short-chain

199

oligosaccharides were eluted by elution C at 1.0 mL/min and 30 °C.

10


Page 10 of 38

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200

RESULTS AND DISCUSSION

201

Sequence analysis. C. carbonis T26 was a Gram-positive, aerobic, motile, and

202

rod-shaped bacterium isolated from subsurface soil of a Chinese coal mine.38 Its

203

whole genomic sequence has just recently been determined and deposited at

204

DDBJ/EMBL/GenBank under accession number AXCY00000000.39 The genome

205

annotation showed the presence of a putative gene (locus_tag: N868_11335) encoding

206

the hypothetical protein (protein ID: KGM11272.1).

207

In this work, this hypothetical protein was identified as an AS with high α-(1,

208

4)-glucan-producing activity, termed CC-AS. Among all the eight reported AS

209

enzymes, only NPo-AS was from the wild microorganism with unknown amino acid

210

sequence information. Overall, various reported AS enzymes showed 35-75% of

211

identities with one another (Table 1). Herein, CC-AS was found relatively

212

homologous in amino acid sequence with AC-AS (GenBank accession No.

213

ACL41561.1) and NPo-AS (CAA09772.1) with 67.03% and 58.85%, respectively,

214

and shared less than 50% identity with other ones including NPo-AS (CAA09772.1),

215

AM-AS (BAG82876.1), DRd-AS (NP_294657), DG-AS (ABF44874.1), DRp-AS7,

216

and MF-AS (ABE50875.1) (Table 1).

217

The crystal structures of NPo-AS,40 DG-AS,41 and DRd-AS42 have recently been

218

determined and deposited in Protein Database Bank (PDB) with No. 1G5A, 3UCQ,

219

and 4AYS, respectively. All the structure information supported the enzyme as a

220

glycoside hydrolase family 13 member containing a catalytic (β/α)8-barrel domain.

221

The active sites were highly conversed in all reported ASes (Fig. 1). Two strictly

222

reserved residues Glu334 and Asp292 in the (β/α)8-barrel domain of CC-AS were

223

hypothesized as catalytic residues as the general acid/base and the nucleophile,

224

respectively, and played an important role in formation of the β-glucosyl enzyme

11



225

intermediate through an α-retaining mechanism. These two residues were found to be

226

completely conserved as corresponding residues Glu328 and Asp286 in NPo-AS,40

227

Glu326 and Asp284 in DG-AS,41 and Glu318 and Asp276 in DRd-AS,42 respectively.

228 229

Expression and purification of the recombinant CC-AS. The CC-AS encoding

230

gene, which was annotated using locus_tag: N868_11335 in C. carbonis T26 genome,

231

was identified as an open reading frame of 1,935 base pairs of nucleotides encoding a

232

protein of 644 amino acids with a calculated molecular mass of 71,934 Da and a

233

theoretical isoelectric point of 5.39 calculated by the ExPASy Computer pI/Mw tool.

234

The full-length gene was synthesized commercially and cloned into a pET-22b(+)

235

expression vector containing an in-frame 6×histidine-tag sequence at the 3’-terminus.

236

The recombinant plasmid was transformed into host cell E. coli BL21(DE3) and the

237

foreign gene was expressed by IPTG induction. As shown in Fig. 2A, IPTG

238

significantly induced the recombinant CC-AS expression, forming a strong protein

239

band at approximately 70 kDa on the SDS-PAGE gel, and the expression amount

240

reached the maximum at 6 h.

241

Because of fusion with a 6×histidine-tag, the recombinant CC-AS was easily

242

purified to homogeneity by one-step nickel affinity chromatography. Shown in Fig.

243

2A, the purified enzyme showed a molecular mass of approximately 70 kDa, which

244

was close to the predicted molecular mass based on the known amino sequence. In

245

previous works, some recombinant ASes, including NPo-AS,43 DRd-AS,31 DG-AS,32,

246

33

247

(GST) tag. But GST fusion probably affected the catalytic activity of AS and the

248

purified recombinant GST-fused AS generally required GST removal step for further

249

characterization. GST-free DG-AS showed 2.4 times higher specific activity than

and AM-AS,34 were expressed in fusion with a 26-kDa glutathione S-transferase

12


Page 12 of 38

Page 13 of 38


250

GST-fused one.32 The cleavage of GST-tag also improved the activity of GST-fused

251

DRd-AS31. In addition, GST fusion probably affected the recombinant AS expression

252

level. It was reported that GST-fused DG-AS was mainly produced as insoluble and

253

inactive protein forming inclusion bodies.32 Jeong et al. mentioned that AS expression

254

level expressed in a pGEX system with GST fusion recombination was lower than

255

that in pET and pHCE expression system.17 Recently, AC-AS,35 DRp-AS,7 and

256

MF-AS17 were expressed in fusion with a 0.7-kDa 6×histidine-tag using pET vectors,

257

with relatively high expression level and no need of treatment for the fusion tag. In

258

this work, a remarkable expression level of the recombinant CC-AS was achieved

259

using the pET-22b(+) vector under the T7 promoter, and the enzyme was easily

260

purified by affinity chromatography for further investigation.

261

The total molecular mass of the native recombinant CC-AS was measured to be

262

approximately 72 kDa based on the gel filtration results (Fig. 2B). Thus, it was

263

indicated that CC-AS should be a monomer. In previous studies, NPo-AS crystalized

264

as a monomer,40 however, both DG-AS41 and DRd-AS42 crystal structures showed the

265

homodimeric proteins. And the biochemical analysis indicated that the monomeric

266

NPo-AS and dimeric DG-AS were stable over a few weeks at 4 °C.41 AC-AS was a

267

monomeric protein like NPo-AS and CC-AS,35 and MF-AS17 and DRp-AS7 were

268

determined to be homodimeric proteins.

269

Based on the reported crystal structure information, DG-AS dimerizes via an

270

interface composed of seven regions shown in Fig. 1.41 The NPo-AS displays

271

relatively shorter loops in regions 4, 6, and 7 compared to DG-AS40 and thus probably

272

prevents the formation of dimer.41 By comparison, CC-AS showed similar regions 4,

273

6, and 7 with NPo-AS but different from DG-AS (Fig. 1), and this was the possible

274

reason that CC-AS was a monomeric protein like NPo-AS.

13



Page 14 of 38

275 276

Effect of pH and temperature on catalytic activity of the recombinant CC-AS.

277

The total, hydrolytic, and transglucosylation activities of the recombinant CC-AS

278

were measured at 40 °C with pH from 4.5 to 8.5. Shown in Fig. 3A, the highest total

279

and transglucosylation activities were shown at pH 7.0, however, the highest

280

hydrolytic activity was shown at pH 5.0. Overall, the enzyme showed remarkably

281

higher transglucosylation than hydrolytic activity above pH 5.5, with the ratio of

282

transglucosylation/hydrolysis

283

transglucosylation was just slightly higher than hydrolytic activity at pH 4.5 and 5.0,

284

with the T/H ratio of 1.7 and 1.6, respectively (Fig. 3B).

(T/H)

ranged

from

3

to

8,

however,

the

285

Effect of temperature on various activities of the recombinant CC-AS was also

286

determined (Fig. 3C). It showed the highest transglucosylation activity at 40 °C and

287

the highest hydrolytic activity at 45 °C. The transglucosylation activity was

288

significantly higher than the hydrolytic activity at all tested temperatures with the T/H

289

ratio ranged from 4 to 13 (Fig. 3D).

290

The total specific activity was calculated to be 4700 U/g at pH 7.0 and 40 °C, and

291

the specific activities for transglucosylation reaction and sucrose hydrolysis were

292

determined to be 4200 U/g at pH 7.0 and 40 °C and 1100 U/g at pH 5.0 and 40 °C,

293

respectively.

294

The previously reported AM-AS showed the same optimum pH (8.0) for hydrolytic

295

and transglucosylation activity, but prominently exhibited hydrolytic activity. It

296

showed T/H ratio close to 1 : 1 at optimum pH at 8.0, however, the T/H ratio was less

297

than 0.2 below pH 8.0 and above pH 9.0. In addition, AM-AS showed optimum

298

temperature at 45 °C for both transglucosylation and hydrolytic activity with T/H ratio

299

below 1.0. By comparison, CC-AS showed transglucosylation activity prominently

14


Page 15 of 38


300

and displayed remarkably higher T/H ratio than AM-AS, indicating that CC-AS had a

301

great potential for application.

302

In addition, DG-AS,33 AC-AS,35 MF-AS,17 and DRp-AS7 showed optimum pH and

303

temperature at 8.0 and 45 °C, 8.0 and 45 °C, 8.5 and 45 °C, and 8.0 and 40 °C,

304

respectively. But the T/H ratio of these enzymes was not studied in detail.

305 306

Effect of metal ions on catalytic activity of the recombinant CC-AS. The total

307

activity of recombinant CC-AS was measured in presence of various metal ions at the

308

final concentration of 1 mM (Fig. 3E). All the tested metal ions showed negative

309

effects on the activity. The Fe2+, Mg2+, and Mn2+ caused marginal decreases in the

310

enzyme activity (

Identification of an Î±-(1,4)-Glucan-Synthesizing Amylosucrase from

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