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Improved thermostability of maltooligosyltrehalose synthase from Arthrobacter ramosus by directed evolution and site-directed mutagenesis Chun Chen, Lingqia Su, Fei Xu, Yongmei Xia, and Jing Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01123 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 24, 2019

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Journal of Agricultural and Food Chemistry

1

Improved thermostability of maltooligosyltrehalose synthase from

2

Arthrobacter ramosus by directed evolution and site-directed

3

mutagenesis

4 5

Chun Chena,b,c, Lingqia Sua,b,c*, Fei Xu b, Yongmei Xiaa, Jing Wua,b,c,*

6 7 8

aState

9

Lihu Avenue, Wuxi, 214122, China

Key Laboratory of Food Science and Technology, Jiangnan University, 1800

10

bSchool

11

of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

12

cInternational

13

Avenue, Wuxi, 214122, China

of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry

Joint Laboratory on Food Safety, Jiangnan University, 1800 Lihu

14 15

*Corresponding author: [email protected] and [email protected]

16 17 18 19 20 21 22

ACS Paragon Plus Environment


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Abstract

24

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in trehalose production.

25

MTSase from Arthrobacter ramosus has poor thermostability, limiting its industrial use.

26

In this study, mutant G415P was obtained by directed evolution, and S361R/S444E was

27

subsequently generated based on a structure analysis of the region around G415. The

28

t1/2 of G415P and S361R/S444E at 60 °C increased by 3.0- and 3.2-fold, respectively,

29

compared with the wild-type enzyme. A triple mutant (G415P/S361R/S444E) was

30

obtained through a combination of the above mutants, and its t1/2 significantly increased

31

by 19.7-fold. Kinetic and thermodynamic stability results showed that the T50 and Tm

32

values of the triple mutant increased by 7.1 and 7.3 °C, respectively, compared with

33

those of the wild-type enzyme. When the triple mutant was used in trehalose production,

34

the yield reached 71.6 %, higher than the 70.3 % achieved with the wild-type. Thus, the

35

mutant has a potential application for industrial trehalose production.

36 37 38 39 40 41

Keywords: maltooligosyltrehalose synthase, thermostability, Arthrobacter ramosus,

42

directed evolution, site-directed mutagenesis


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43

Introduction

44

Trehalose consists of two glucopyranoses linked by an α-1,1-glycosidic bond. As a

45

naturally occurring nonreducing disaccharide, trehalose can be found in

46

microorganisms, plants, and insects 1. Trehalose can stabilize proteins in living

47

organisms and participates in the formation of some microbial cell walls, thus playing

48

a specific role in the protection of organisms 2-4. In recent years, studies have found that

49

trehalose has a wide range of applications in cosmetics 5-6, medicine 7-12, and the food

50

industry

51

Administration in 2000 5.

52

There are three enzymatic routes for trehalose production: (1) trehalose-6-phosphate is

53

produced from glucose-6-phosphate and UDP-glucose by trehalose-6-phosphate

54

synthase (EC 2.4.1.15) and is then dephosphorylated to trehalose by trehalose-6-

55

phosphate phosphatase (EC3.1.3.12)

56

trehalose synthase (EC 5.4.99.16) 17; and (3) maltooligosyltrehalose is produced from

57

starch or maltodextrin by maltooligosyltrehalose synthase (MTSase; EC 5.4.99.15) and

58

is then converted to trehalose by maltooligosyltrehalose trehalohydrolase (MTHase; EC

59

3.2.1.141)

60

materials that can be obtained from a wide range of sources.

61

At present, MTSase can be divided into two types according to its source: thermophilic

62

and mesophilic MTSase. According to our current understanding of the literature, when

63

using Escherichia coli as an expression host, thermophilic MTSase is difficult to

13-15.

18.

Trehalose was generally recognized as safe by the US Food and Drug

16;

(2) trehalose is produced from maltose by

The third method is more commonly used due to the inexpensive raw



64

produce due to low soluble protein levels 19-21. However, the mesophilic MTSases from

65

Arthrobacter ramosus and Corynebacterium glutamicum have a good expression levels,

66

with the target soluble protein accounting for 36.2 % and 45.3 % of the total cellular

67

proteins 22-23, respectively. Higher production temperatures can accelerate the reaction

68

rate, prevent starch retrogradation, and inhibit microbial contamination during

69

industrial production

70

poor thermostability limits their use in industrial applications. Nevertheless, no studies

71

on improving the thermostability of mesophilic MTSases have been reported so far.

72

There are two methods to improve thermostability: directed evolution and rational

73

design. Directed evolution, proposed by Frances H. Arnold in the 1990s, mimics the

74

process of natural evolution, and valuable mutants can be screened from a large number

75

of mutant libraries

76

decarboxylase from 8000 independent variants using this method, and this mutant

77

exhibited a 4.2 °C increase in T50 27. Rational design is based on an understanding of

78

the relationship between protein structure and function. Ying Yang et al modified the

79

thermostability of α-L-arabinofuranosidase by rational design with the PoPMuSiC web

80

server and obtained a mutant in which the t1/2 was increased by 11-fold 28.

81

Previous work from our laboratory revealed that MTSase from A. ramosus exhibits a

82

high level of expression and good application performance but poor thermostability 23.

83

In this study, the molecular modification of MTSase was carried out by directed

84

evolution and site-directed mutagenesis to improve its thermostability, and enzymatic

24.

25-26.

Therefore, although mesophilic MTSases have advantages,

Lemuel M. J. Soh et al screened a mutant of keto acid


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85

properties of the different mutants were compared with the wild-type enzyme.

86 87

Materials and Methods

88

Materials

89

E. coli JM109 was utilized for gene cloning, and E. coli BL21 (DE3) was utilized for

90

enzyme expression. The plasmid pET24a(+)-treY was constructed from previous work

91

in our laboratory. PrimeSTAR polymerase, rTaq DNA polymerase, Nde I, Hind III and

92

Dpn I were from Takara (Dalian, China). Other reagents were from Sinopharm

93

Chemical Reagent (Shanghai, China).

94 95

Construction of Random Mutagenesis Library

96

Error-prone PCR was performed to introduce random mutations into the MTSase gene

97

(2.2 kb) using plasmid pET24a(+)-treY as the template. The reaction volume was 50

98

µL and contained the following: 0.5 mM MgCl2, MnCl2 (three different concentrations:

99

0.05, 0.1, or 0.2 mM), 20 ng of template, 0.5 µL of primers, as shown in Table S1, 5

100

µL of 10×rTaq buffer, 4 µL of dNTP mix, and 0.5 µL of rTaq DNA polymerase. The

101

reaction program was as follows: 1 cycle of 300 s at 94 °C, 30 cycles of 10 s at 98 °C,

102

15 s at 55 °C, and 150 s at 72 °C, and 1 cycle of 10 min at 72 °C. After digestion by

103

Nde I and Hind III for 2 h, the PCR products were ligated into pET24a(+), which was

104

also digested by the same restriction enzymes. The ligation products were introduced

105

into E. coli JM109 for cloning. The resulting plasmids were then introduced into E. coli



106

BL21 (DE3). A total of 50 transformants from each library corresponding to different

107

concentrations of MnCl2 were randomly selected for DNA sequencing to determine

108

nucleotide change rates.

109 110

Screening of MTSase Variants

111

Independent clones from the mutant library were picked into 96-well plates filled with

112

500 μL of Terrific broth (TB) medium containing kanamycin (100 µg mL -1). The plates

113

were incubated at 32 °C for 24 h. The cells were lysed using the commercial Bacterial

114

Protein Extraction Kit from Cwbiotech (Beijing, China) at 37 °C for 3 h. Crude enzyme

115

was obtained by centrifugation at 4000 g at 4 °C for 20 min. A 96-well plate containing

116

200 µL of 0.2 % (wt/vol) maltodextrin (DE 16) and 50 µL of crude enzyme was

117

incubated at 45 °C for 10 min. The residual maltodextrin was measured by using the

118

3,5-dinitrosalicylic acid (DNS) reagent 29. The crude enzyme was incubated at 58 °C

119

for 10 min, and residual enzyme activity was measured as described above.

120 121

Homology Modeling and Structure Analysis

122

ClustalX was utilized for protein sequence alignment 30. The SWISS-MODEL server

123

was utilized to obtain theoretical structures of the wild-type and mutant MTSase by

124

homology modeling 31, with the crystal structure of Sulfolobus acidocaldarius MTSase

125

(PDB ID 1IV8) 32 as template. The quality of the modeled structures was validated with

126

“Structure Assessment” in the SWISS-MODEL server. The compatibility of the atomic


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127

model (3D) with the amino acid sequence (1D) was evaluated by VERIFY-3D 33. The

128

structures were presented and analyzed with the PyMOL Molecular Graphics System,

129

and the interactions between the amino acid residues were determined.

130 131

Construction of Mutants

132

The mutant S361R/S444E was obtained using whole-plasmid PCR. The template was

133

pET24a(+)-treY, and the primers are shown in Table S1. The reaction program was as

134

follows: 1 cycle of 300 s at 94 °C, 19 cycles of 10 s at 98 °C, 15 s at 55 °C, and 470 s

135

at 72 °C, and 1 cycle of 10 min at 72 °C. After digestion by Dpn I at 37 °C for 2 h, the

136

PCR products were introduced into E. coli JM109 for cloning. The correct variant genes

137

were confirmed by DNA sequencing, and then plasmids containing the correct variant

138

genes were introduced into E. coli BL21 (DE3). The mutant S361R/S444E/G415P was

139

constructed as described above, using pET24a(+)-treY S361R/S444E as the template;

140

the primers are shown in Table S1.

141 142

Expression and Purification of MTSase Enzymes

143

E. coli BL21 (DE3) harboring recombinant plasmid was cultured in TB medium

144

containing kanamycin (100 µg mL -1) at 32 °C for 24 h. The cells were centrifuged

145

(12000 g, 20 min) and then resuspended in sodium phosphate buffer (pH 7.0). After

146

ultrasonic homogenization and centrifugation at 12000 g for 20 min, the crude enzyme

147

was obtained.



148

The crude enzyme was subjected to salt fractionation and dialyzed against buffer A (20

149

mM sodium phosphate buffer, pH 7.0). A MonoQ 10/100 column and an AKTA-FPLC

150

system (GE Healthcare, Germany) were utilized for enzyme purification. The sample

151

was loaded onto a MonoQ 10/100 column that was pre-equilibrated with buffer A. The

152

adsorbed proteins were eluted by a linear gradient of buffer A and buffer B (20 mM

153

sodium phosphate buffer, 1 M NaCl, pH 7.0). The purified proteins were analyzed by

154

SDS-PAGE, and protein concentrations were quantitated with Bradford’s method 34.

155 156

Determination of Kinetic Stability

157

To determine the half-life (t1/2) of the wild-type enzyme and mutants, samples were

158

incubated at 60 °C for up to 180 min. Samples were withdrawn given time points, and

159

the residual enzyme activities were measured as described above. The first-order rate

160

constants (kd) were obtained by linear regression of the ln (residual activity) versus

161

incubation time. The t1/2 and the changes in transition state free energy (ΔΔG) for

162

inactivation between the mutants and wild-type were calculated as follows: t1/2 = ln2/kd,

163

ΔΔG = -RTln (kd mutant /kd wild-type), where R represents the gas constant (8.314 J·mol-

164

1·K-1)

165

inactivation (T50), samples were incubated at different temperatures, ranging from 52

166

to 65 °C, for 10 min, and residual enzyme activities were measured as described above.

and T represents temperature

35.

To determine the temperature of half-

167 168

Determination of Thermodynamic Stability


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169

To determine the temperature at which half of the protein is in the unfolded state (Tm),

170

differential scanning calorimetry (DSC) was performed using a Nano DSC III (TA

171

Instruments, New Castle, DE). The samples were diluted to 0.8 mg mL -1 with sodium

172

phosphate buffer (pH 7.0). The phosphate buffer was used as a baseline reference. The

173

scanning was run from 50 to 80 °C at a rate of 1 °C min -1 after an equilibration at 50 °C

174

for 15 min at a pressure of 0.3 MPa. The results were analyzed using TA Instruments

175

software.

176 177

Determination of Optimal Temperature and pH

178

To determine the optimal temperature and pH, the enzyme activity of each sample was

179

assayed over a temperature range of 30–65 °C and over a pH range of 5.5–8.0.

180 181

Enzyme Kinetics

182

Kinetic parameter characterization was performed with different maltopentaose

183

concentrations (3, 6, 9, 12, 18, 24, 30, 36, 42 and 50 mM). Mixtures at pH 7.0 containing

184

10 µL of enzyme and 190 µL of maltopentaose were incubated at 45 °C for 10 min.

185

The reaction was terminated by the addition of 1 M NaOH, and residual maltopentaose

186

was determined with DNS reagent 29. The values of Vmax and Km were calculated by

187

nonlinear regression fitting with GraphPad Prism software (GraphPad Software Inc.,

188

San Diego, CA).

189



190

Production of Trehalose from Maltodextrin

191

The mutant S361R/S444E/G415P had the highest thermostability among all mutants;

192

therefore, we evaluated the application performance of S361R/S444E/G415P in

193

contrast with the wild-type enzyme. Mixtures (pH 5.5) containing 15 % (wt/vol)

194

maltodextrin (DE 16), MTSase (16 U per gram of maltodextrin), MTHase 18 (16 U per

195

gram of maltodextrin), pullulanase (5 U per gram of maltodextrin, Novozymes,

196

Denmark) and cyclodextrin glycosyltransferase 36 (2 U per gram of maltodextrin) at pH

197

5.5 were incubated at 45 °C for 36 h. The reactions were terminated by incubating in

198

boiling water for 20 min. Commercial glucoamylase (Novozymes, Denmark) was used

199

to hydrolyze the residual maltodextrin at 60 °C for 24 h. The reaction mixture was then

200

reheated in boiling water for 20 min to inactivate the glucoamylase. The quantity of

201

trehalose was determined by HPLC, according to Tsuei-Yun Fang et al 37.

202 203

Results

204

High-Throughput Screening

205

In this study, three mutant libraries were constructed by altering the concentration of

206

manganese chloride (0.05, 0.1, and 0.2 mM), and 50 transformants from each library

207

were randomly selected for DNA sequencing. The nucleotide change rates were 0–2,

208

3–6, and 7–12 bp/kb, respectively, for the three manganese chloride concentrations. To

209

ensure an appropriate rate, a manganese chloride concentration of 0.1 mM was selected.

210

Overall, 60–70 % of the clones were determined to exhibit enzymatic activity,


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211

indicating that the concentration of manganese chloride was appropriate. In total, 1500

212

clones were screened and one positive clone was obtained, which was confirmed to be

213

a G415P mutant by DNA sequencing. Following incubation at 58 °C for 10 min, the

214

residual enzyme activities of the G415P mutant and the wild-type enzyme were

215

determined to be 80.7 % and 43.4 %, respectively.

216 217

Homology Modeling

218

The crystal structure of S. acidocaldarius MTSase (PDB ID 1IV8), which shares 36 %

219

identity with A. ramosus MTSase, was selected as template for homology modeling to

220

obtain theoretical structures of A. ramosus MTSase using the SWISS-MODEL server.

221

Subsequently, the results of Ramachandran plots and Qualitative Model Energy

222

Analysis (QMEAN) were obtained from the “Structure Assessment” module of

223

SWISS-MODEL. The Ramachandran plot (Supporting Information Figure S1) showed

224

that 92.7 % of residues were in favored regions, and 1.59 % of residues (P431, L433,

225

E344, G510, N404, W526, Q382, P2, P271, P272, and P402) were in outlier regions,

226

which were far away from position 415 in the modeled structure. The result of QMEAN,

227

which uses statistical potentials of the mean force to generate a global quality estimate

228

38,

229

3D was also used to evaluate the correctness of a 3D protein model, and the results

230

showed that 90.99 % of the residues had averaged 3D-1D scores ≥ 0.2, which is higher

231

than the minimal requirement of 80 %. These findings indicated that the predicted

was -2.44, which is higher than the minimal requirement of -4. Moreover, VERIFY-



232

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model was suitable.

233 234

Site-Directed Mutagenesis

235

Changing amino acids near the active site is often considered an effective strategy to

236

improve thermostability 35. A modeled structure shows that G415 is located near the

237

active site, and there are three long loops around it. Loops usually deviate from their

238

original positions due to their own sway, which makes it easy for water molecules to

239

enter the internal hydrophobic center, resulting in an unstable protein structure

240

Therefore, this region may be related to the thermostability of the enzyme. In this study,

241

two potential sites, S361 and S444, in A. ramosus MTSase were proposed by comparing

242

the amino acid sequences of the loops around G415 between thermophilic MTSase and

243

mesophilic A. ramosus MTSase, as well as by analyzing the crystal structure of

244

thermophilic MTSase and the modeled structure of A. ramosus MTSase. The amino

245

acid in thermophilic MTSase corresponding to S361 is aspartic acid or lysine, while the

246

amino acid in thermophilic MTSase corresponding to S444 is arginine or histidine

247

(Figure 1). The amino acids in thermophilic MTSase are charged amino acids, thus the

248

stability of the loops in this region is enhanced by the formation of salt bridges and

249

hydrogen bonds in these charged amino acids (and by extra electrostatic forces in S.

250

acidocaldarius MTSase). Therefore, we considered replacing S361 and S444 in the A.

251

ramosus MTSase with these charged amino acids to strengthen the stability of this

252

region. At the same time, both S361 and S444 are nonconserved amino acids, and the


39.

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253

distances from them to the active site are 11.4 and 12.9 Å, respectively; therefore, the

254

substitutions may not have a great impact on enzyme activity. The side chain steric

255

hindrance of W315 in AEα3 near S444 precludes the replacement of S444 with arginine

256

or histidine, so S444 was mutated to glutamic acid and S361 to arginine. The modeled

257

structure showed that a salt bridge could be formed between these two amino acids,

258

thus strengthening the interaction between the loops where the two amino acids are

259

located.

260

Based on the above analysis, the S361R/S444E mutant was generated. The mutant was

261

obtained as mentioned in the Materials and Methods. The residual activity of the

262

S361R/S444E mutant enzyme increased to 83.5 % following a 10 min incubation at 58

263

°C, indicating that the thermostability of the S361R/S444E enzyme was indeed

264

enhanced.

265 266

Combination of Mutations

267

Combinations of different mutants are likely to further increase the thermostability of

268

the enzyme 24. The triple mutant S361R/S444E/G415P was obtained by introduction of

269

the G415P substitution into the S361R/S444E double mutant, and the thermostability

270

of the triple mutant was further improved. The enzyme activity was almost unchanged

271

following incubation at 58 °C for 10 min, indicating that G415P had a synergistic effect

272

with S361R/S444E.

273



274

Purification of MTSase Enzymes

275

Fermentation broths of the wild-type enzyme and different mutants were centrifuged,

276

and the precipitated cells were ultrasonically disrupted and centrifuged. The supernatant

277

was precipitated with ammonium sulfate (60 % saturation), followed by dialysis, and

278

the enzymes were purified through a MonoQ anion exchange column. As shown in

279

Table 1, there were no significant differences in the purification of the mutant and wild-

280

type enzymes. The recovery rates were approximately 40 %. The specific activity of

281

the wild-type, G415P, S361R/S444E, and S361R/S444E/G415P enzymes were 152.2,

282

135.3, 161.5, and 158.6 U mg -1, respectively (Table 1). Electrophoresis results showed

283

that the mutations did not affect the molecular masses of enzymes, which were the same

284

as that of the wild-type enzyme (83 kDa) (Supporting Information Figure S2).

285 286

Kinetic Stability of the MTSase Enzymes

287

Kinetic stability refers to the thermal tolerance of enzymes when they experience

288

irreversible denaturation 40. The t1/2 is the time it takes for an enzyme to lose half of its

289

activity and is the most commonly used parameter to evaluate kinetic stability 40. The

290

industrial production of trehalose is usually carried out at 60 °C, so we chose this

291

temperature to investigate the t1/2 of the wild-type and mutant enzymes. As shown in

292

Table 2, the t1/2 of the wild-type enzyme was 4.6 min, while the t1/2 values of the G415P,

293

S361R/S444E, and S361R/S444E/G415P mutant enzymes were 13.7, 14.9, and 90.8

294

min, respectively, corresponding to 3.0-, 3.2-, and 19.7-fold higher values than that of


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295

the wild-type, respectively. At the same time, the transition state free energy (ΔΔG)

296

was calculated using the equation mentioned above in the section “Determination of

297

Kinetic Stability”. The ΔΔG values corresponding to the G415P, S361R/S444E, and

298

S361R/S444E/G415P mutants were 3.1, 3.4, and 8.2 kJ mol -1, respectively (Table 2).

299

The larger the ΔΔG value, the higher the energy required for thermal denaturation. The

300

ΔΔG value of S361R/S444E/G415P was the highest, indicating that this mutant enzyme

301

was most resistant to high temperatures and had the highest thermostability.

302

The T50, another important parameter used to evaluate kinetic stability, represents the

303

temperature at which an enzyme loses half of its activity 40. The samples were incubated

304

at different temperatures for 10 min, and the T50 values were obtained by measuring

305

residual enzyme activities. As shown in Table 2, the T50 of the wild-type enzyme was

306

57.3 °C, while the T50 values of the G415P, S361R/S444E, and S361R/S444E/G415P

307

enzymes were 60.2, 60.5, and 64.4 °C, respectively, corresponding to 2.9, 3.2, and

308

7.1 °C higher T50 values than that of the wild-type enzyme.

309 310

Thermodynamic Stability of MTSase Enzymes

311

Unlike kinetic stability, thermodynamic stability is typically used to describe the trend

312

of protein unfolding

313

stability is the temperature at which half of the protein is unfolded (Tm). The Tm values

314

of the wild-type and mutant enzymes were obtained using differential scanning

315

calorimetry (DSC). As shown in Table 3, the Tm values of the wild-type, G415P,

40.

The most commonly used parameter for thermodynamic



316

S361R/S444E, and S361R/S444E/G415P enzymes were 60.8, 63.7, 63.3, and 68.1 °C,

317

respectively. The Tm values of the mutant enzymes were all higher than that of the wild-

318

type enzyme. In particular, the Tm of the S361R/S444E/G415P enzyme increased by

319

7.3 °C compared to that of the wild-type enzyme.

320

In the process of unfolding, the enthalpy (ΔH) of each sample was calculated by

321

nonlinear curve fitting. The value of ΔH represents the energy required for enzyme

322

unfolding. The higher the ΔH, the higher the energy required for enzyme unfolding,

323

and the more stable the enzyme

324

G415P, S361R/S444E, and S361R/S444E/G415P enzymes were 1364.2, 1553.5,

325

1645.2, and 2238.8 kJ mol -1, respectively. The ΔH of the S361R/S444E/G415P enzyme

326

was the highest, followed by S361R/S444E and G415P, while the ΔH of the wild-type

327

enzyme was the lowest, indicating that it was the most difficult for the

328

S361R/S444E/G415P enzyme to unfold at high temperatures, followed by the

329

S361R/S444E and G415P enzymes. It was relatively easy for the wild-type enzyme to

330

unfold, thus indicating that the wild-type enzyme had the lowest thermostability.

35, 41.

As shown in Table 3, the ΔH of the wild-type,

331 332

Effects of Temperature and pH on the Activity of MTSase Enzymes

333

The optimum temperatures (Topt) of the wild-type and mutant enzymes were

334

investigated. As shown in Figure 2A, the Topt values of the G415P and S361R/S444E

335

enzymes were both 40 °C—the same as that of the wild-type enzyme—while the Topt

336

of the S361R/S444E/G415P enzyme increased to 45 °C. Over the temperature range of


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337

45–60 °C, the relative activities of all mutant enzymes were higher than that of the wild-

338

type. The relative activity of the wild-type enzyme decreased sharply at temperatures

339

higher than 45 °C, followed by the G415P and S361R/S444E enzymes, while the

340

relative activity of the S361R/S444E/G415P enzyme decreased relatively slowly. For

341

example, relative activities of the wild-type, G415P, S361R/S444E, and

342

S361R/S444E/G415P enzymes at 55 °C were 54.7, 76.8, 74.2, and 83.3 %, respectively,

343

while the S361R/S444E/G415P enzyme retained most of its activity.

344

The optimum pH of the wild-type and mutant enzymes was also investigated. As shown

345

in Figure 2B, the optimum pH values of the wild-type, G415P, S361R/S444E, and

346

S361R/S444E/G415P enzymes were 7.0, 6.5, 6.5, and 7.0, respectively. The

347

S361R/S444E enzyme was active over a wider range (pH 6.0–7.5) than the wild-type

348

enzyme. The relative activities of the G415P and S361R/S444E/G415P enzymes were

349

higher than that of the wild-type enzyme at pH 5.5–6.5.

350 351

Kinetics of MTSase Enzymes

352

Kinetic parameters of the wild-type and mutant enzymes were determined using

353

maltopentaose at pH 7.0 and 45 °C. The results in Table 4 show that the Km of

354

S361R/S444E increased by 7.5 % compared with the wild-type enzyme, while its

355

kcat/Km was similar to that of the wild-type, indicating that the catalytic efficiency of

356

S361R/S444E was not affected by the mutations. The Km values of the G415P and

357

S361R/S444E/G415P enzymes decreased by 19.7 % and 15.1 %, respectively,



358

compared with the wild-type enzyme, while the kcat/Km values were 10.4 % and 30.4 %

359

higher than that of the wild-type enzyme, indicating that the catalytic efficiencies of

360

G415P and S361R/S444E/G415P were improved.

361 362

Production of Trehalose Using Maltodextrin as Substrate

363

The ability of the wild-type and S361R/S444E/G415P enzymes to produce trehalose

364

using maltodextrin was investigated. Trehalose was produced using the triple mutant or

365

wild-type enzyme in combination with mesophilic MTHase at 45 °C and pH 5.5. The

366

conversion rates for the triple mutant and wild-type enzymes were 71.6 % and 70.3 %,

367

respectively. The conversion rate of the triple mutant enzyme increased by 1.3 %,

368

consistent with its lower Km and higher kcat/Km values.

369 370

Discussion

371

MTSase from A. ramosus exhibited a high expression level and high specific activity,

372

but poor thermostability, with a t1/2 at 60 °C of only 4.6 min, limiting its use in industrial

373

applications.

374

At present, no research on thermostability modification of mesophilic MTSase from

375

any sources has been reported; thus, regions related to thermostability and amino acid

376

residues suitable for substitution remain unknown. Directed evolution is a commonly

377

used strategy to improve enzyme thermostability. Because mutant libraries are

378

abundant, there is a high probability of identifying positive mutants, so directed


Page 18 of 39

Page 19 of 39


379

evolution is a simple and efficient method. As a common means of directed evolution,

380

error-prone PCR is a mature method and is simple to carry out 42. The error rate of the

381

library is primarily determined by the concentration of manganese chloride in the error-

382

prone PCR system. If the concentration of manganese ions is too high, it can lead to

383

excessively high error rates, resulting in most clones having no enzyme activity. A

384

concentration of manganese ions that is too low leads to low error rates, and considering

385

the degeneracy of codons, it is possible that only nucleotides change, with the resulting

386

amino acids remaining unchanged. We controlled the rate of nucleotide change at 3–6

387

bp/kb by using a manganese chloride concentration of 0.1 mM, which resulted in 60–

388

70 % of the clones exhibiting enzymatic activity. In this way, the mutant G415P was

389

obtained by directed evolution, and the results of kinetic and thermodynamic stability

390

analyses indicated that the thermostability of the mutant was greatly improved.

391

The modeled structure of the G415P mutant showed that a proline is in the middle of

392

an α-helix. Analysis of the thermophilic S. acidocaldarius MTSase (PDB ID 1IV8) 32

393

and Sulfolobus tokodaii MTSase (PDB ID 3HJE) 43 crystal structures showed that the

394

amino acids corresponding to G415 are P385 and P383, respectively. Both prolines are

395

in the middle of the long helix AEα8 (Figure 3). We replaced P385 of the S.

396

acidocaldarius MTSase with glycine, and the residual activity of the mutant enzyme

397

decreased to 19.2 % following incubation at 85 °C for 20 min, compared to 47.3 %

398

residual activity of the wild-type enzyme in these conditions. The thermostability of the

399

mutant decreased, indicating that this position may be important for the thermostability



Page 20 of 39

400

of MTSase. Proline, which has a pyrrolidine ring side chain, is generally considered to

401

impose rigid constraints on the N-Cα rotation, thus enforcing the stability of protein

402

structure 44. For this reason, substitution with proline at suitable residues is usually an

403

effective strategy to improve the thermostability of enzymes. Trevino et al reported that

404

replacement of threonine in a -turn with proline improved the thermostability of

405

RNase Sa 44, and Tian et al found that replacement of glycine in a loop with proline

406

increased the thermostability of methyl parathion hydrolase 41. A possible reason for

407

the increased thermostability of the A. ramosus enzyme G415P could be that the

408

mutated P415 imposes conformational rigidity to backbone of the long AEα8 helix,

409

thus enforcing the stability of the protein structure (Figure 4B). Furthermore, since

410

glycine lacks a side chain and has more backbone conformational flexibility

411

replacement of proline in the thermophilic MTSase enzyme with glycine led to a

412

decrease in thermostability. Our results were consistent with these reports and provide

413

a special case of increased thermostability caused by the replacement of glycine with

414

proline in the middle of a helix. This finding may be useful in the modification of

415

mesophilic MTSase from other sources.

416

Although the thermostability of G415P was greatly improved, additional improvements

417

will be necessary for industrial use. To further improve the thermostability of

418

mesophilic A. ramosus MTSase, S361R/S444E was obtained based on rational design.

419

The results of kinetic and thermodynamic stability analyses showed that the

420

thermostability of S361R/S444E was greatly improved compared with that of the wild-


45,

Page 21 of 39


421

type enzyme.

422

In the wild-type enzyme, S361 and S444 are located on the protein surface. The water

423

molecules that bind to serine in the environment are easily released at high temperatures,

424

resulting in instability in the local structure where the protein and water molecules bind

425

46,

426

loop between AEα5 and AEα6, and S444 is present in a long loop between AEα8 and

427

Aα6. Loops usually wiggle and sway, resulting in protein structure instability

428

Additionally, S361 and S444 do not interact with the surrounding amino acids (Figure

429

4A). All of these factors may contribute to the poor thermostability of the wild-type

430

enzyme.

431

Regarding the thermophilic S. acidocaldarius MTSase, the amino acids corresponding

432

to S361 and S444 are D347 and R414, respectively. In general, two polar nonhydrogen

433

atoms (one with a hydrogen attached) would form a hydrogen bond if their distance is

434

less than 3.5 Å, and two full oppositely charged atoms within the same distance would

435

form a salt bridge. An electrostatic interaction is also formed by two full oppositely

436

charges atoms; however, with a longer distance than 3.5 Å between them 47. In this way,

437

R414 can form an electrostatic interaction and a salt bridge with D347 and D412,

438

respectively. These forces reinforce the stability of the loops in this region (Figure 3A).

439

The corresponding amino acids in another thermophilic S. tokodaii MTSase are K340

440

and H412, respectively. H412 can form a salt bridge bond with D299, while K340 can

441

form a hydrogen bond with S253, strengthening the stability of loops in this region

especially when serine residues are present in the loops. S361 is present in a long


39.


442

(Figure 3B). These factors may contribute to the improved thermostability of the two

443

thermophilic MTSases compared with that of the mesophilic wild-type enzyme from A.

444

ramosus.

445

Analysis of the modeled S361R/S444E structure indicated that E444 can form a salt

446

bridge with R361, while R361 can form a salt bridge with E265 and D442 (Figure 4C).

447

These bridges form salt bridge networks that strengthen the interactions among the three

448

loops in this region, suggesting that the thermostability of S361R/S444E is improved

449

by the enhanced stability of loops in this local region, similar to the cases of S.

450

acidocaldarius and S. tokodaii MTSase. Meanwhile, S361K/S444E, S361Q/S444Q,

451

and S361Q/S444L were generated, and their residual enzyme activities were 65.9 %,

452

26.2 % and 5.4 %, respectively. Compared with the wild-type, S361Q/S444Q and

453

S361Q/S444L showed a decreased thermostability, while S361K/S444E showed an

454

improved thermostability. In mutant S361K/S444E, K361 could form salt bridge only

455

with E444 (Figure S5), so there is less stabilizing force in S361K/S444E compared with

456

S361R/S444E. This is in accordance with the results showing that the residual enzyme

457

activity of S361R/S444E was 83.5 %, which was higher than 65.9 % for S361K/S444E.

458

However, with the additional salt bridge, S361K/S444 still showed an improved

459

thermostability compared to the wild-type. In S361Q/S444Q, there is no interaction

460

between Q316 and Q444 (Figure S5), and glutamine is prone to cause instability of the

461

protein structure due to its easy deamidation at high temperatures 48, which may be the

462

reason that S361Q/S444Q showed a decreased thermostability. In S361Q/S444L, there


Page 22 of 39

Page 23 of 39


463

is also no interaction between Q316 and L444 (Figure S5). In addition to the easy

464

deamidation of Q316, the hydrophobicity of L444 and its location on the protein surface

465

and exposure to water combine to render a greater instability of the enzyme structure;

466

therefore, S361Q/S444L showed a dramatic decrease in thermostability.

467

The above results may further confirm that R361 and E444 form a salt bridge, and the

468

interaction between position 361 and 444 is important to improve the thermostability

469

of A. ramosus MTSase.

470

Several studies have reported that combinations of different mutants often further

471

improve the thermostability of enzymes

472

was obtained by combining S361R/S444E with G415P. The t1/2 at 60 °C of triple mutant

473

enzyme increased remarkably by 19.7-fold compared to the wild-type enzyme,

474

suggesting that G415P and S361R/S444E had an unexpected synergistic effect.

475

The modeled structure of the S361R/S444E/G415P mutant showed that E265, R361,

476

D442, and E444 form salt bridge networks, as in S361R/S444E. The E444 residue is in

477

a loop, termed “loopA”. R361 is in another loop, termed “loopB”. LoopA and loopB

478

protrude, respectively, from the C-terminal and N-terminal of helix AEα8 where P415

479

is located (Figure 4D). P415 imposes conformational rigidity on AEα8. The stability in

480

this local region of loopA, loopB and AEα8 could be reinforced as a whole based on

481

the combined effects of the salt bridge networks and the conformational rigidity, thus

482

resulting in a higher stability of the entire S361R/S444E/G415P protein structure

483

compared with that of G415P or S361R/S444E. This is consistent with the results of

24, 35, 49-50.

The S361R/S444E/G415P mutant



484

the kinetic and thermodynamic stability of the three mutants.

485

It is generally difficult to increase the stability of an enzyme while maintaining or

486

increasing its catalytic activity

487

S361R/S444E/G415P possessed enhanced thermostability without sacrificing catalytic

488

activity; there was even a small increase in application performance, making it more

489

advantageous than the wild-type enzyme for industrial applications.

490

In conclusion, raising the temperature of trehalose production is considered a primary

491

goal to inhibit microbial contamination, which often appears in trehalose production.

492

MTSase from Arthrobacter ramosus has a good application performance but poor

493

thermostability. Thus, the thermostability of MTSase must be improved. The

494

S361R/S444E/G415P mutant was obtained through a combination of directed evolution

495

and site-directed mutagenesis and exhibited a significant improvement in

496

thermostability. The t1/2 of the S361R/S444E/G415P enzyme was 90.8 min, while the

497

t1/2 of the wild-type enzyme was only 4.6 min. The reasons for the difference in

498

thermostability between the wild-type and mutants are discussed above. In general, the

499

415 position and the loop region around this residue are important for the

500

thermostability of MTSase and may be useful for future modifications of MTSase

501

thermostability.

39-40.

The enzyme kinetic parameters showed that

502 503

Supporting Information

504

Table S1, primers used for error-prone PCR and site-directed mutagenesis. Figure S1,


Page 24 of 39

Page 25 of 39


505

Ramachandran plot of A. ramosus MTSase model. Figure S2, SDS-PAGE analysis of

506

purified wild-type and mutant enzymes. Figure S3, residual enzyme activities of wild-

507

type and mutant enzymes after incubation at 60 °C for up to 180 min. Figure S4, DSC

508

analysis of wild-type and mutant enzymes. Figure S5, modeled structures of mutant

509

enzymes. A: mutant S361K/S444E; B: mutant S361Q/S444Q; C: mutant S361Q/S444L.

510 511

Acknowledgements

512

This work received financial support from the National Natural Science Foundation of

513

China (31730067, 31771916), the National Science Fund for Distinguished Young

514

Scholars (31425020), the Natural Science Foundation of Jiangsu Province

515

(BK20180082), the National First-class Discipline Program of Light Industry

516

Technology and Engineering (LITE2018-03), and the 111 Project (No. 111-2-06).

517 518

Conflict of interest

519

The authors declare that they have no conflict of interest.

520 521

REFERENCES

522 523 524 525 526 527 528 529 530

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46. Nagendra, H. G.; Sukumar, N.; Vijayan, M., Role of water in plasticity, stability, and action of proteins: The crystal structures of lysozyme at very low levels of hydration. Proteins Structure Function & Bioinformatics 2015, 32 (2), 229-240. 47. Petsko, G. A.; Ringe, D., Bonds that Stabilize Folded Proteins. In Protein structure and function, Eleanor, L., Ed. New Science Press: London, UK, 2004; pp 10-11. 48. Daniel, R. M.; Dines, M.; Petach, H. H., The denaturation and degradation of stable enzymes at high temperatures. Biochemical Journal 1996, 317, 1-11. 49. Duan, X.; Chen, J.; Wu, J., Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis. Applied and Environmental Microbiology 2013, 79 (13), 4072-4077. 50. Yu, S.; Wang, X.; Zhang, T.; Jiang, B.; Mu, W., Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1. Journal of Agricultural and Food Chemistry 2016, 64 (31), 6188-6195.

668 669

FIGURES

670

Figure 1. Multiple sequences (loops around G415) alignment of MTSase from various

671

microorganisms. S361 and S444 in A. ramosus MTSase sequence were labeled with red

672

and purple box, respectively.

673 674

Figure 2. Effects of temperature and pH on the activity of wild-type and mutant

675

enzymes. A: The Topt of the wild-type and mutant enzymes. The reaction was carried at

676

pH 7.0 at different temperature. B: The optimum pH of the wild-type and mutant

677

enzymes. The reaction was carried at different pH at 45 °C.

678 679

Figure 3. The crystal structures of thermophilic MTSase. A: MTSase from S.

680

acidocaldarius, B: MTSase from S. tokodaii.

681



682

Figure 4. The modelled structures of the wild-type and mutant enzymes. Helix AEα8

683

was colored in green, while loopA, loopB were colored in cyan, and orange yellow,

684

respectively. A: wild-type; B: mutant G415P; C: mutant S361R/S444E; D: mutant

685

S361R/S444E/G415P.


Page 30 of 39

Page 31 of 39


TABLES Table 1. Purification scheme of wild-type and mutant enzymes. purification

enzyme

wild-type G415P crude enzyme S361/S444E S361/S444E/G415P

total protein (mg) 301 285 299 274

total activity (U) 8493 7283 9288 8347

specific purification activity fold (U mg -1) 28.2 1.0 25.5 1.0 31.1 1.0 30.5 1.0

yield (%) 100 100 100 100

ammonium sulfate fraction

wild-type G415P S361/S444E S361/S444E/G415P

61 54 64 56

4841 3860 5202 4591

78.9 71.5 80.8 82.4

2.8 2.8 2.6 2.7

57 53 56 55

monoQ anion exchange chromatography

wild-type G415P S361/S444E S361/S444E/G415P

24 21 23 20

3652 2840 3715 3172

152.2 135.3 161.5 158.6

5.4 5.3 5.2 5.2

43 39 40 38



Page 32 of 39

Table 2. Kinetic stability of the wild-type and mutant enzymes. enzyme wild-type G415P S361R/S444E S361R/S444E/G415P

t1/2 (min) 4.6 13.7 14.9 90.8

kd (min -1) 0.15068 0.05059 0.04652 0.00763


ΔΔG (kJ mol -1) 3.1 3.4 8.2

T50 (°C) 57.3 60.2 60.5 64.4

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Table 3. Thermodynamic stability of the wild-type and mutant enzymes. enzyme wild-type G415P S361R/S444E S361R/S444E/G415P

Tm (°C) 60.8 63.7 63.3 68.1


ΔH (kJ mol -1) 1364.2 1553.5 1645.2 2238.8


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Table 4. Kinetic analysis of the activities of wild-type and mutant enzymes using the maltopentaose as substrate. enzyme wild-type G415P S316R/S444E S316R/S444E/G415P

Km (mM) 6.6 ± 0.2 5.3 ± 0.3 7.1 ± 0.4 5.6 ± 0.2

kcat (S -1) 242.7 ± 215.4 ± 251.1 ± 268.6 ±

11 10 13 11

kcat/Km (S -1 mM -1) 36.8 40.6 35.4 48.0


Vmax (U mg -1) 175.1 ± 4 155.5 ± 4 181.1 ± 6 193.8 ± 7

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Figure 1.



Figure 2. A

B


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Figure 3. A

B



Figure 4.


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TOC Graphic For Table of Contents Only


Improved Thermostability of Maltooligosyltrehalose ... - ACS Publications

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