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Improvement of the Catalytic Performance of a Talaromyces leycettanus #-Amylase by Changing the Linker Length Duoduo Zhang, Tao Tu, Yuan Wang, Yeqing Li, Xuegang Luo, Fei Zheng, Xiaoyu Wang, Yingguo Bai, Huoqing Huang, Xiaoyun Su, Bin Yao, Tong-Cun Zhang, and Huiying luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00838 • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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Page 1 of 36

Journal of Agricultural and Food Chemistry 1

1

Improvement

of

the

Catalytic

Performance

of

a

Talaromyces

2

leycettanus α-Amylase by Changing the Linker Length

3

Duoduo Zhanga,1, Tao Tub,1, Yuan Wangb, Yeqing Lib, Xuegang Luoa, Fei Zhengb,

4

Xiaoyu Wangb, Yingguo Baib, Huoqing Huangb, Xiaoyun Sub, Bin Yaob, Tongcun

5

Zhanga*, Huiying Luob*

6 7

a

8

Education and Tianjin Key Laboratory of Industrial Microbiology, College of

9

Biotechnology, Tianjin University of Science and Technology, Tianjin 300457,

Key Laboratory of Industrial Fermentation Microbiology of the Ministry of

10

China

11

b

12

Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081,

13

China

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed

14 15

16

Running title: Catalytic improvement of a GH13 α-amylase

17 18 19

1

Both authors contributed equally to this work.

20 21

*

Correspondence. [email protected] (T. Zhang); [email protected] (H. Luo)

22

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 36 2

23

ABSTRACT

24

A novel α-amylase, Amy13A, was identified in Talaromyces leycettanus

25

JCM12802 that consists of these domains: catalytic TIM-barrel fold, domain B,

26

domain C, Thr/Ser-rich linker region, and C-terminal CBM20 domain. The wild type

27

and three mutant enzymes were then expressed in Pichia pastoris GS115 to identify

28

the

29

(Amy13A-CBM) in catalysis. All enzymes had similar enzymatic properties,

30

exhibiting optimal activities at pH 4.5−5.0 and 55−60 °C, but varied in catalytic

31

performance. When using soluble starch as the substrate, Amy13A21 and

32

Amy13A33 showed higher specific activities (926.3 and 537.8 U/mg vs. 252.1 U/mg)

33

and catalytic efficiencies (kcat/Km, 25.7 and 22.0 mL/s⋅mg vs. 15.4 mL/s⋅mg) than the

34

wild type, while Amy13A-CBM had declined performance in catalysis. This study

35

reveals the key roles of the CBM and linker length in the catalysis of GH13

36

α-amylase.

37

KEYWORDS: α-amylase, catalytic efficiency, CBM, linker region, Talaromyces

38

leycettanus

roles

of

linker

length

(Amy13A21

and

Amy13A33)

39


and

CBM20

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40

INTRODUCTION

41

α-Amylase (EC 3.2.1.1) is an enzyme that cleaves the α-1,4-glycosidic bonds of

42

carbohydrates and oligosaccharides. It is a preeminent enzyme with wide

43

applications across the food, textile, pulp, and bioenergy industries.1 α-Amylases

44

have been derived from fungi, bacteria, archaea, plants, and animals. Among them,

45

those from fungi and bacteria dominate the industrial sectors.2-6

46

Based on the sequence similarity of catalytic domains, α-amylase belongs to

47

family 13 of glycoside hydrolases (GH; http://www.cazy.org).7 GH13 members are

48

diverse in functions and perform several functions, including hydrolysis,

49

transglycosylation, condensation and cyclization.8,9 Moreover, enzymes of GH13

50

frequently harbor a carbohydrate-binding module (CBM), most of which are

51

confined to families 20, 21, 25, 26, 34, 41, 45, 48, 53, 58, 68, 69, and 74

52

(http://www.cazy.org/Carbohydrate-Binding-Modules.html).10,11

53

CBM, or starch-binding domain (SBD),12-14 can brings the substrate into the active

54

pocket of

55

enzyme.15 Another role of CBM is to ‘‘unwind” α-glucan helices on the granule

56

surface,16 thus improving the hydrolytic efficiency.17 There is a linker region that

57

connects the catalytic domain and CBM. This linker is ﬂexible and its length is

58

related to enzyme structure and function.18,19 For example, the predominance of

59

negatively charged residues and the presence of short, disulfide-bridged loops in the

60

linker region lead to extended conformations of the cellulase Cel5G from

The

α-amylase

catalytic domain, thereby improving the hydrolytic activity of the



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61

Pseudoalteromonas haloplanktis;20 while the glucoamylase GA1 from Aspergillus

62

niger showed better thermostability than its mutant with a longer linker.21

63

In this study, a GH13 α-amylase, Amy13A, was obtained from Talaromyces

64

leycettanus JCM12802. The enzyme consists of five domains: catalytic TIM-barrel

65

fold, domain B, domain C, linker region, and C-terminal CBM20 domain. To

66

investigate the roles of the CBM and linker regions, three mutants were constructed.

67

Their enzymatic properties and catalytic performance were then compared with the

68

wild type. The findings may contribute to the rational design of GH13 α-amylases.

69 70

MATERIAL S AND METHODS

71

Strains, Culture Conditions, Plasmids, and Chemicals. T. leycettanus

72

JCM12802 (the Japan Collection of Microorganisms RIKEN BioResource Center,

73

Tsukuba, Japan) was cultivated in a culture medium22, consisting of 15 g L−1 wheat

74

bran, 15 g L−1 corncob, 15 g L−1 soy bean meal, 5 g L−1 NaCl, 5 g L−1 (NH4)2SO4, 1

75

g L−1 KH2PO4, 0.5 g L−1 MgSO4·7H2O, 0.2 g L−1 CaCl2, and 0.01 g L−1 FeSO4·7H2O,

76

at 45 °C on a rotary shaker at 180 rpm for 2 days.

77

Escherichia coli Trans1-T1 and the pEASY-T3 plasmid from TransGen (Beijing,

78

China) were purchased for gene cloning and DNA sequencing. Heterologous

79

expression was conducted using plasmid pPIC9 and Pichia pastoris GS115

80

(Invitrogen, Carlsbad, CA, USA). Maltooligosaccharides, soluble starch, amylose,

81

dextran, amylopectin, γ-cyclodextrin, α-cyclodextrin, β-cyclodextrin glycogen, and

82

pullulan from Sigma-Aldrich (St. Louis, MO, USA), The T4 DNA ligase and


Page 5 of 36


83

restriction endonucleases were supplied by New England Biolabs (Hitchin, UK) and

84

TaKaRa (Otsu, Japan), respectively. All chemicals were of analytical grade and

85

commercially available.

86 87

Gene Cloning of the α-Amylase. Total RNA was extracted from 2-day old

88

mycelia and purified according to the manufacturer’s instructions of Omega Fungal

89

DNA Mini kit (Norcross, GA, USA). Reverse transcription was completed with the

90

First Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan). A primer set specific for

91

fungal GH13 α-amylase (GH13F and GH13R; Table 1) was used to amplify the core

92

region. The 5′- and 3′-flanking regions were cloned by thermal, asymmetric,

93

interlaced (TAIL)-PCR using the TaKaRa genome walking kit and assembled with

94

the known sequence. The cDNA fragment of amy13A without the signal

95

peptide-coding sequence was amplified using specific primers Amy13A-F and

96

Amy13A-R with restriction sites (Table 1). The PCR product was cloned into vector

97

pEASY-T3 and sequenced.

98 99

Sequence

Analysis

and

Mutant

Design.

BLAST

programs

100

(http://www.ncbi.nlm.nih.gov/BLAST/) were used to compare the nucleotide and

101

amino acid sequences. Vector NTI Advance 10.0 software (Invitrogen) was used to

102

assemble sequences and forecast the molecular mass and pI value of the mature

103

protein.

104

GENSCAN Web Server (http://genes.mit.edu/GENSCAN.html) were used to predict

The

online

SignalP

(http://www.cbs.dtu.dk/services/SignalP/)


and


Page 6 of 36 6

105

the signal peptide sequence, transcription initiation sites, introns, and exons. ClustalW

106

software (http://www.clustal.org/) generated the alignment of multiple protein

107

sequences, followed by rendering completed using the ESPript3.0 program

108

(http://espript.ibcp.fr/ESPript/cgi-bin/ESPriptcgi). Three mutant enzymes were then

109

designed to harbor the linker sequences of α-amylases asAA (BAA22993.1,

110

Amy13A21) and EPS (AEH03024.1, Amy13A33) or truncate the CBM20

111

(Amy13A-CBM). The mutagenesis was generated by using the overlap extension

112

PCR with specific primers (Table 1).

113 114

Expression and Purification of Recombinant Amy13A and Its Mutants. The

115

PCR products of amy13A, amy13A21, amy13A33 and amy13A-CBM were digested

116

with EcoRI and NotI and ligated into EcoRI-NotI-digested pPIC9 to produce

117

recombinant plasmids. The plasmids were linearized with BglII, followed by

118

transformation into P. pastoris GS115 competent cells through electroporation.

119

According to the Pichia expression protocols (Invitrogen), positive transformants

120

were screened in the minimal dextrose medium (2% glucose and 2% agarose), and

121

those with highest α-amylase activities were cultured at 30 °C, 200 rpm for 48 h in

122

1-L Erlenmeyer flasks containing 400 mL of buffered glycerol-complex medium

123

(BMGY). Cells were harvested and resuspended in 200 mL buffered,

124

methanol-complex medium (BMMY) containing 0.5% (v/v) methanol for 48 h

125

induction. The cell-free cultures were collected by centrifugation (12,000 g and 4 °C

126

for 10 min), and concentrated using an ultrafiltration membrane with a molecular


Page 7 of 36


127

weight cut-off of 10 kDa (Vivascience, Hannover, Germany). The crude enzymes

128

were loaded onto the HiTrap Q HP anion exchange column (Amersham Biosciences,

129

Uppsala, Sweden) which was equilibrated with buffer A (20 mM phosphate, pH 6.0).

130

Proteins were eluted with a linear NaCl gradient (0−1.0 M) in the same buffer at a

131

flow rate of 4 mL min−1. Fractions were collected, assayed for α-amylase activity,

132

and subjected to SDS-PAGE analysis. The protein concentration was then

133

determined using the Bradford method.

134

N-glycosylation was removed by incubation of the purified recombinant enzyme

135

and endo-β-N-acetylglucosaminidase H (Endo H, New England Biolabs) at 37 °C for

136

2 h. The identity of Amy13A was verified by using a matrix-assisted laser

137

desorption/ionization-time off light-mass spectrometry (MALDI-TOF-MS) at the

138

Institute of Apiculture Research, Chinese Academy of Agricultural Sciences (Beijing,

139

China).

140 141

Biochemical Characterization of Amy13A and Its Mutants. The DNS

142

method23 was used to determine the α-amylase activity by measuring the amount of

143

reducing sugar (glucose) per min under the assay conditions. Each measurement was

144

repeated three times.

145

The pH optima were determined by measuring the α-amylase activities at 60 °C

146

for 30 min in the following buffers: 100 mM citric acid-Na2HPO4 for pH 3.0−7.0, 100

147

mM Tris-HCl for pH 8.0−9.0, and 100 mM glycine-NaOH for pH 9.0−12.0. The pH

148

stability of each enzyme was determined by pre-incubating the enzyme at 37 °C for 1



Page 8 of 36 8

149

h in appropriate buffers of pH 2.0−11.0 and measuring the residual activities under the

150

standard conditions (pH 4.5 or 5.0 and 60 °C for 30 min). The temperature optima

151

were determined at the pH optimum of each enzyme and at 40−80 °C for 30 min.

152

Thermal stability was monitored by assessing the residual α-amylase activities under

153

standard conditions after incubation at 50 °C or 60 °C for various periods without

154

substrate.

155

The tolerance of Amy13A towards different metal ions and chemical reagents was

156

determined in the presence of 5 mM Mn2+, Na+, K+, Ag+, Cu2+, Mg2+, Pb2+, Ca2+, Ni2+,

157

Zn2+, Cr3+, Fe3+, EDTA, SDS, and β-mercaptoethanol. The enzyme activity without

158

any addition were treated as the control.

159 160

Substrate Specificity and Kinetic Parameters. The enzymatic activities of

161

Amy13A and its mutants against soluble starch, amylose, amylopectin, glycogen, raw

162

starch, pullulan, dextrin, α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin were

163

measured as described above to determine their substrate specificity.

164

The enzymatic activities of Amy13A and its mutants were also measured in the

165

100 mM citric acid-Na2HPO4 containing 0.5−10 mg mL−1 of soluble starch at 60 °C

166

and pH 4.5 for 15 min for kinetic analysis. All experiments included three replicates.

167

Km and Vmax values were determined according to the Michaelis-Menten equation by

168

using the GraphPad Prism 7.01 software, and the kcat and kcat/Km values were then

169

calculated.

170

Analysis of the Hydrolysis Products. The enzymes were incubated at 50 °C with


Page 9 of 36


171

2% soluble starch for 24 h. Over the 24 h duration, samples were collected at 1, 2, 3, 4,

172

5, 12, 16, and 24 h, and boiled for enzyme inactivation. The reaction mixtures were

173

centrifuged at 12,000 g for 10 min, and the culture supernatants were analyzed with

174

high-performance, anion-exchange chromatography (HPAEC, model 2500, Dionex,

175

Sunnyvale, CA, USA) equipped with a 250 mm × 3 mm CarboPac PA200 guard

176

column. The hydrolysis products were eluted by a mobile phase (0.5 mL min−1) of 1

177

M NaOH. When using 10 mg mL−1 maltooligosaccharides as the substrates, the

178

reactions were incubated at 50 °C for 30 min, and the hydrolysis products were

179

determined as described above.

180 181

Nucleotide Sequence Accession Number. The nucleotide sequence of the GH13

182

α-amylases gene (amy13A) from T. leycettanus JCM12802 was deposited in

183

GenBank database under the accession number KY496326.

184 185

RESULTS AND DISCUSSION

186

Gene Cloning. The α-amylase gene, amy13A, was cloned from T. leycettanus

187

JCM12802 by TAIL-PCR method. amy13A contains 2316 bp, which is interrupted by

188

8 introns. The cDNA of amy13A contains 1833 bp and encodes a polypeptide of 610

189

amino acid residues. Amy13A belongs to the subfamily GH13_1. Typically, GH13

190

members have a modular structure of three domains: the catalytic TIM-barrel fold

191

(domain A), a sheet of the four antiparallel β-strands and two antiparallel β-strands

192

separated by loops of considerable length (domain B), and the C-terminal Greek key



Page 10 of 36 10

193

motif (domain C).24,25 Deduced Amy13A has a similar modular structure and contains

194

a putative N-terminal signal peptide of 20 residues, a Thr/Ser-rich linker region

195

(residues 496–502), and a C-terminal CBM20 domain (residues 503–602)(Fig. 1). It is

196

predicted that Glu250 acts as the proton donor and Asp226 as the catalytic nucleophile,

197

while Asp317 stabilizes the transition-state of Amy13A (see Fig. S1 in the

198

supplementary material). 26,27 The molecular mass of the mature protein was estimated

199

to be 64.7 kDa. Deduced Amy13A shares the highest sequence similarity of 77% to

200

the α-amylase from Rasamsonia emersonii CBS 393.64 (XP_001560614.1), 71% to

201

the asAA (BAA22993.1) from Aspergillus kawachii,28 and 55% to the EPS

202

(AEH03024.1) from Aureobasidium pullulans.29 Multiple sequence alignment of

203

Amy13A and other sequences demonstrated that Amy13A has a short and

204

un-conserved linker sequence (Fig. 2).

205

CBM20, the three mutants (Amy13A21 and Amy13A33 (Fig. 1B) with replacement

206

of the linker regions of close homologs asAA and EPS respectively, and

207

Amy13A-CBM with the removal of CBM20) were constructed.30 Amy13A21,

208

Amy13A33, and Amy13A-CBM consist of 1875 bp, 1914 bp, and 1506 bp that code

209

for polypeptides of 68.1 kDa, 69.3 kDa, and 45.4 kDa, respectively.

To examine the effects of linker lengths and

210 211

Generation, Expression, and Purification of Amy13A and Its Mutants. The

212

recombinant Amy13A and its mutant enzymes were successfully generated and

213

expressed in P. pastoris GS115. Pure proteins were then collected through anion

214

exchange chromatography, and appeared to possess molecular masses of 45 to 69 kDa


Page 11 of 36


215

in SDS-PAGE. After treatment with Endo H, all enzymes showed a single band

216

corresponding to the theoretical masses (Fig. 3). MALDI-TOF-MS analysis identified

217

several peptides of Amy13A, which corresponded to the deduced sequence of

218

Amy13A. This result confirmed the purity of the band and the identity of Amy13A.

219 220

Enzymatic Properties of Amy13A and Its Mutants. Soluble starch was used as

221

the substrate for enzyme characterization. The purified Amy13A, Amy13A21, and

222

Amy13A33 displayed optimal activities at pH 4.5, while the pH optimum of

223

Amy13A-CBM measured at pH 5.0 (Fig. 4A). Their pH optima fall within the pH

224

range of fungal α-amylases (pH 4.0−7.0) and are similar to the α-amylases from

225

Aspergillus oryzae (pH 5.0)31 and Aspergillus awamori (pH 4.8)32. Similar to the

226

GH13 α-amylase from Penicillium expansum that has a temperature optimum of

227

60 °C,33 Amy13A and its mutants showed maximum activities at 60 °C (Fig. 4B).

228

This temperature optimum is higher than those of α-amylases from A. awamori

229

(50 °C)34 and Penicillium griseofulvum (40 °C)35. The mutant enzymes had similar,

230

pH-stability profiles as shown for Amy13A (Fig. 4C). After 1 h incubation at 37 °C, it

231

maintained residual activities of higher than 70% over the pH range of 3.0 to 8.0.

232

Analysis of the thermostability profiles (Fig. 4D) revealed the distinct differences

233

between the wild-type and mutant enzymes. Amy13A appeared to be highly stable at

234

50 °C, retaining 71% activity after 1 h incubation. When the incubation temperature

235

was increased to 60°C, only 66% residual activity was retained after 10 min

236

incubation. Amy13A33 and Amy13A-CBM showed worse thermostability, retaining



Page 12 of 36 12

237

57% and 76% activities at 50 °C for 1 h, respectively, and 50% and 32% activities at

238

60 °C for 10 min. In contrast, the thermostability of Amy13A21 clearly improved; it

239

retained 93% activity at 50 °C for 1 h and 72% at 60 °C for 10 min. The results

240

indicated that the linker length and CBM have effects on the thermostability of

241

Amy13A, as observed in previous studies.21 The linker length has been found to play

242

roles in thermal adaptation,36,37 such as Cel9A from the thermophile Termobifda

243

fusca38 and CotB-DSM from Clostridium thermocellum39. Chen et al. reported that

244

introducing a suitable linker between protein domains can reduce the stereospecific

245

blockade, facilitate correct folding, and increase enzyme stability. 39 We thus infer that

246

the improved thermostability of Amy13A21 might be ascribed to the enhanced,

247

structural stability due to the introduction of a longer, flexible linker. However, a

248

longer linker, as in the Amy13A33, may impair the conformational stability and result

249

in weakened thermostability.18,40 Moreover, Amy13A was found to be slightly more

250

thermostable than Amy13A-CBM. It is therefore suggested that the CBM20 of

251

Amy13A plays a vital role in the thermostability by maintaining the enzyme

252

conformation. These results were in agreement with those previously observed in the

253

α-amylases of Lactobacillus amylovorus41 and Cryptococcus sp. strain S-242.

254

The tolerance of Amy13A towards metal ions and chemical reagents were also

255

determined (Table 2). β-Mercaptoethanol and Ca2+ enhanced the enzyme activities

256

by 28.3% and 23.9%, respectively, while no effects on the activity of Amy13A were

257

found from other chemicals.

258


Page 13 of 36


259

Substrate Specificity and Kinetic Values of Amy13A and Its Mutants. It is

260

well known that the CBM domain is associated with binding to insoluble substrate.

261

Deletion of the CBM domain usually resulted in a dramatic decrease in raw starch

262

hydrolysis, without a complete loss in activity.43,44 In comparison to Amy13A, which

263

had a specific activity of 44.7 U/mg towards raw starch, the activity of

264

Amy13A-CBM was decreased (0.8-fold), while those of Amy13A21 and Amy13A33

265

were significantly increased (5.9-and 4.0-fold, respectively). The results confirmed

266

the role of CBM in hydrolysis of insoluble substrate.

267

When using soluble starch as the substrate, Amy13A showed higher, specific

268

activity (252.1 U mg−1) than α-amylases from Paenibacillus sp. SSG-1 (113.89 U

269

mg−1)45 and Haloarcula japonica (24 U mg−1)46. The specific activities of mutant

270

enzymes towards soluble starch showed similar trends as towards raw starch.

271

Amy13A21 and Amy13A33 each saw activity increases of 2.7- and 1.2-fold, while

272

Amy13A-CBM had a decreased activity of 0.2-fold. Previous reports stated

273

shortening the linker region might cause a decrease of specific activity towards

274

macromolecular substrates.47 Our results confirmed the roles of linker region and

275

CBM, both of which may render effects on the protein structure, and demonstrated

276

they play roles in the hydrolysis of raw starch and soluble starch.

277

Amy13A, Amy13A21, Amy13A33, and Amy13A-CBM had great variations in

278

catalytic performance against various substrates. When defined the enzyme activity

279

towards soluble starch as 100%, Amy13A exhibited the highest specific activity on

280

γ-cyclodextrin (178%), moderate on dextrin (73%), raw starch (66%), and glycogen



Page 14 of 36 14

281

(42%), and no activity on amylose, amylopectin, α-cyclodextrin, β-cyclodextrin, and

282

pullulan. While Amy13A21 and Amy13A33 possessed the highest specific activity

283

on γ-cyclodextrin (140 and 151%), moderate on dextrin (77 and 48%), raw starch

284

(29 and 33%), and glycogen (37 and 27%), Amy13A-CBM showed decreased

285

activities (90%, 42%, 17% and 4%) against γ-cyclodextrin, dextrin, raw starch, and

286

glycogen, respectively. These results indicated that replacement of the linker regions

287

affects the hydrolytic capability of Amy13A towards different substrates. Previously,

288

researchers reported that the xylan-binding domain and linker sequence of ATx from

289

Thermomonospora fusca play important parts in the binding and hydrolysis of

290

insoluble substrates.48 Other studies have shown that the linker region has no effect

291

on the affinity to soluble or insoluble substrates.49 Our results indicated that the

292

linker region plays a role in the hydrolysis of soluble and insoluble substrates, while

293

the CBM domain is associated with substrate binding. Excessively long or short

294

linkers are detrimental to catalysis, thus a precise, inter-domain spacing is necessary

295

for efficient hydrolysis. Thus, the substituted linker regions may affect the swing of

296

CBM20 to increase or decrease the probability of substrate binding and thereafter the

297

activity towards different substrates.

298

As shown in Table 3, the Km values of Amy13A, Amy13A21, and Amy13A33

299

are similar, which is much lower than that of Amy13A-CBM. In comparison to

300

Amy13A, Amy13A21, and Amy13A33, Amy13A-CBM had increased Vmax values,

301

but decreased kcat values. As a result, the catalytic efficiencies (kcat/Km) of

302

Amy13A21 and Amy13A33 were improved (1.7- and 1.4-fold), while that of


Page 15 of 36


303

Amy13A-CBM was significantly decreased (0.1-fold). A previous study has shown

304

that the mutant enzyme of AmyP with CBM truncation has increased substrate

305

affinity to gelatinized rice starch (i.e. decreased Km), and, as a result, improved

306

catalytic efficiency (4.3-fold). It indicated that the CBM of AmyP may relate to

307

substrate binding, as well as soluble starch hydrolysis.49 In other studies, the CBM

308

even seems to hinder the degradation of soluble starch, because the truncated

309

enzyme displays increased activity on soluble starch.44, 50, 51 On the other hand, the

310

linker length showed effects on α-amylase catalysis. In comparison to Amy13A21,

311

Amy13A33, possessing a longer linker, showed less catalytic efficiency

312

improvement due to the relatively low turnover rate. This longer linker may enhance

313

the freedom degree of CBM, consequently affecting the product release. Therefore,

314

the presence of a CBM domain and a linker with favorable length is much beneficial

315

for GH13 α-amylases to hydrolyze soluble starch efficiently.

316 317

Analysis of the Cleavage Mode and Hydrolysis Products. The hydrolysis

318

products of Amy13A and its mutant enzymes against soluble starch and

319

maltooligosaccharides were examined by HAEPC. The major hydrolysis products of

320

soluble starch by Amy13A were maltose (G2) and maltotriose (G3) (Fig. 5A), which

321

accounted for 55.9% and 24.3%, respectively. This great maltose-producing capability

322

of Amy13A makes it attractive for applications in various fields.52 Amy13A21,

323

Amy13A33, and Amy13A-CBM showed similar product patterns to the wild type

324

(data not shown). The hydrolysis of various maltooligosaccharides by Amy13A was



Page 16 of 36 16

325

also determined (Fig. 5B). Amy13A acted only on maltotriose and larger polymers,

326

which makes it a viable catalyst for the production of maltose.

327

High maltose syrup is prevalent in food industries as an additive in the soft drink,

328

baking, brewing, canning, and confectionery.53 Pure maltose administered at high

329

concentrations can circumvent the insulin metabolism and doesn’t elevate blood

330

glucose levels, and thus has a potential to be used as an alternative to D-glucose for

331

intravenous feeding and diabetes injection. Besides the potential uses in food and

332

pharmaceutical industries, Amy13A can be used as a carrier of enzyme preparations to

333

increase resistance to adverse environments.

334 335

ACKNOWLEDGEMENTS

336

This research was supported by the National High Technology Research and

337

Development Program of China (863 program, no. 2013AA102803) and the National

338

Key Research and Development Program of China (no. 2016YFD0501409) and the

339

China Modern Agriculture Research System (no. CARS-42).

340 341

SUPPORTING INFORMATION

342

Sequence alignment of Amy13A and structure-resolved α-amylase from Aspergillus

343

niger (Figure S1).

344 345

REFERECNES

346


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identified

in

R.;

α-amylase

Suszkiewicz,

(AmyP)

K.;

represents

Blennow,


a

A.

new

A

family

novel

of

type

Page 25 of 36


Table 1. Primers used in this study Primers

Sequences (5′→3′)a

GH13-F

GMTKCCTWCCAYGGNTAYTGG

GH13-R

GTGTGGATNCGNAGNCCRTC

Amy13A-F/

GGGGAATTC TTGGCTCCAGCGGAATGGCGGAAAC

Amy13A21-F1/ Amy13A33-F1 Amy13A-R/

GGGGCGGCCGCTCATCTCCACGTCGCAACCACCGT

Amy13A21-R1/ Amy13A33-R1 Amy13A-CBM-F

GGGGAATTC TTGGCTCCAGCGGAATGGCGGAAAC

Amy13A-CBM-R

GGGGCGGCCGCGCCCGTCGTACCGGATCCACTGCAGAGC

Amy13A21-F2

AGCGGCACCCCGACCACCATTAAAACCAGCGCGGTGACG ACGGGCTGCACAGCGGCAACCT

Amy13A21-R2

GGTTTTAATGGTGGTCGGGGTGCCGCTGGTGCTACCGGAT CCACTGCAGAGCCCA

Amy13A33-F2

AGCGCGGCGGCGACCACCAGCAGCAGCTGCACCGCGAC CAGCACCACCGTCCCGGTGCTGTTTGAG

Amy13A33-R2

AGCTGCTGCTGGTGGTCGCCGCCGCGCTGCTGCTGCTGC TGCTGGTGGTCGCTTTGCTGGTGGAGGTTGCCGCTGTGC AG

a

The restriction sites are underlined.



Page 26 of 36 26

Table 2. Effect of metal ions and chemical reagents (5 mM) on the Amy13A activity Chemicals

Relative activity (%) a

Chemicals

Control

100.0 ± 0.97

Ni2+

79.8 ± 2.22

K+

91.0 ± 2.01

Cr3+

74.5 ± 3.64

Mg2+

89.5 ± 1.64

Cu2+

56.6 ± 1.44

Na+

89.9 ± 1.69

Zn2+

71.2 ± 4.22

Ca2+

123.9 ± 0.67

Fe3+

Pb2+

61.0 ± 5.77

β-Mercaptoethanol

Ag+

72.2 ± 1.15

EDTA

86.3 ± 0.41

Mn2+

82.2 ± 2.86

SDS

84.8 ± 0.60

a

Relative activity (%)

64.5 ± 2.14 128.3 ± 0.88

Data are shown as mean ± SD (n=3); the enzymatic activity of Amy13A towards

soluble starch was defined as 100%


Page 27 of 36


Table 3. Substrate specificity of the wild type and mutant enzymes Enzymes

Amy13A

Amy13A-CBM

Amy13A21

Amy13A33

(U mg−1)

(U mg−1)

(U mg−1)

(U mg−1)

Soluble starch

252.1

204.9

926.3

537.8

Glycogen

106.6

8.3

346.7

147.8

Dextrin

183.4

85.8

717.2

256.2

γ-Cyclodextrin

449.0

183.9

1292.4

812.4

44.7

34.9

264.4

180.1

Raw starch



Page 28 of 36 28

Table 4. Kinetic parameters of the wild type and mutant enzymes Km

Vmax

kcat

kcat/Km

(mg mL−1)

(µmoL min−1 mg−1)

(s−1)

(mL s−1 mg−1)

Amy13A

0.2

217.2

3.6

15.4

Amy13A-CBM

2.9

251.3

4.2

1.4

Amy13A21

0.3

456.9

7.6

25.7

Amy13A33

0.3

336.6

5.6

22.0

Enzymes


Page 29 of 36


FIGURE LEGENDS

Figure 1. Rational design of Amy13A variants. (A) Sequences of the linker regions. (B) Representation of the chimeric proteins with linkers of variable lengths.

Figure 2. Multiple sequence alignment of Amy13A and six GH13 amylases that have linker regions. Identical and similar amino acids are highlighted in red.

Figure 3. SDS-PAGE analysis of the recombinant proteins. Lanes: M, the molecular weight markers; 1, the crude enzyme of Amy13A; 2, the purified Amy13A; 3, the deglycosylated Amy13A; 4, the crude enzyme of Amy13A33; 5, the purified Amy13A33; 6, the deglycosylated Amy13A33; 7, the crude enzyme of Amy13A21; 8, the purified Amy13A21; 9, the deglycosylated Amy13A21; 10, the crude enzyme of Amy13A-CBM; 11, the purified Amy13A-CBM; and 12, the deglycosylated Amy13A-CBM.

Figure 4. Biochemical characterization of the purified recombinant enzymes. (A) pH-activity profiles. (B) Temperature-activity profiles. (C) pH stability. (D) Thermostability.

Figure 5. HAEPC analysis of the hydrolysis products by Amy13A. (A) The major hydrolysis products of 2% soluble starch. 1, After incubation at 50 °C for 24 h; 2,



Page 30 of 36 30

After incubation at 50 °C for 1 h; and 3, maltooligosaccharide standards (5 µg mL−1). (B)

The

major

hydrolysis

products

of

maltooligosaccharides.

1,

maltooligosaccharide standards (5 µg mL−1); 2, The major hydrolysis products of DP7; 3, The major hydrolysis products of DP6; 4, The major hydrolysis products of DP5; 5, The major hydrolysis products of DP4, and 6 The major hydrolysis products of DP3.


Page 31 of 36


Figure 1 152x100mm (300 x 300 DPI)



Figure 2 48x9mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36


Figure 3 254x92mm (300 x 300 DPI)



Figure 4 190x142mm (300 x 300 DPI)


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Figure 5 254x190mm (300 x 300 DPI)



TOC Graphic 44x24mm (600 x 600 DPI)


Page 36 of 36

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