Synthesis of Isomalto-Oligosaccharides by Pichia pastoris Displaying

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Synthesis of isomalto-oligosaccharides by Pichia pastoris displaying the Aspergillus niger #-glucosidase Nannan Zhao, Yanshan Xu, Kuang Wang, and Suiping Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04140 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Journal of Agricultural and Food Chemistry

Synthesis of isomalto-oligosaccharides by Pichia pastoris displaying the Aspergillus niger α-glucosidase Nannan Zhao1, Yanshan Xu1, Kuang Wang, Suiping Zheng*

1. Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China 2. Guangdong research center of Industrial enzyme and Green manufacturing technology, School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, P. R. China

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

We explored the ability of an Aspergillus niger α-glucosidase displayed on P. pastoris

3

to act as a whole-cell biocatalyst (Pp-ANGL-GCW61) system to synthesize

4

isomalto-oligosaccharides (IMOs). IMOs are a mixture that includes isomaltose (IG2),

5

panose (P) and isomaltotriose (IG3). In this study, the IMOs were synthesized by a

6

hydrolysis-transglycosylation reaction in an aqueous system of maltose. In a 2-mL

7

reaction system, the IMOs were synthesized with a conversion rate of approximately

8

49% in 2 h when 30% maltose was utilized under optimal conditions by

9

Pp-ANGL-GCW61. Additionally, 0.5-L reaction system was conducted in a 2-L

10

stirred reactor with a conversion rate of approximately 44% in 2 h. Moreover, the

11

conversion rate was relatively stable after the whole-cell catalyst was reused three

12

times. In conclusion, Pp-ANGL-GCW61 has a high reaction efficiency and

13

operational stability, which makes it a powerful biocatalyst available for industrial

14

scale synthesis.

15

Aspergillus

niger

α-glucosidase,

16

Keywords:

17

Transglycosylation, IMOs, Scale synthesis

Yeast

2


whole-cell

catalyst,

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18

INTRODUCTION

19

The isomalto-oligosaccharides (IMOs) are a group of glucans that consist of glucosyl

20

saccharide units linked by α-1, 6-glycosidic linkages. Its main functional components

21

include isomaltose (IG2), panose (P) and isomaltotriose (IG3). The most direct

22

physiological function of IMOs is to promote the growth of probiotics. On this basis,

23

its indirect effects include regulating intestinal flora, inhibiting the growth of

24

pathogenic bacteria, improving constipation1-3, and increasing the synthesis of

25

essential vitamins and mineral absorption4. Wang Yu et al. found that IMOs can

26

regulate lipid metabolism2. In addition, IMOs have approximately 45-50% of the

27

sweetness of sucrose as well as having lower calories. Thus, it is an ideal sugar

28

substitute for a person with diabetes. IMOs have already been used in

29

pharmaceuticals2, 5 and food products6-7.

30

The production and application of IMOs first originated in Japan in 1985.

31

Commercial Isomalt-500 is an enzymatic product that contains approximately 50%

32

IMOs8. During the industrial scale production of IMOs, starch is first liquefied with a

33

thermostable bacterial α-amylase (e.g., Termamyl SC, Novozymes) to produce limit

34

dextrins, which are further saccharified by fungal α-amylase (e.g., Fungamyl 800L,

35

Novozymes) and transglucosylated using α-glucosidase (e.g., Transglucosidase L,

36

Amano Enzymes Inc, Japan) in a separate process to produce IMOs9.

37

α-Glucosidase (EC 3.2.1.20), a well-known hydrolyzing enzyme, can be used to carry

38

out the transglucosylation of maltose10. It is the key to producing IMOs. The

39

hydrolyzing reaction of α-glucosidase releases α-D-glucose from the non-reducing 3



40

end of oligosaccharides. The transfer occurs most frequently at the 6-OH group of the

41

non-reducing glucose unit, producing isomaltose from D-glucose or panose from

42

maltose11.

43

Many α-glucosidases from different sources have been found, and their characteristics

44

were determined12-15. The α-glucosidase from Aspergillus niger (ANGL) has a high

45

level of transglycosylation activity, even at quite low substrate concentrations16. The

46

mutagenesis, immobilization and heterologous expression of the α-glucosidase from

47

Aspergillus niger became research hotspots. The complete amino acid sequence of the

48

α–glucosidase from Aspergillus niger has been reported in 199217. Until now, ANGL

49

has been expressed in and secreted from E. coli, A. nidulans and P. pastoris18-20.

50

Fermentation, to produce a large amount of stable, high-quality α–glucosidase, is

51

extremely important. At the present, the most mature Aspergillus niger technology is

52

in Amano Enzymes Inc, and the α–glucosidase IMOs products account for almost all

53

imports from Japan into China. However, the Amano enzymes are free enzymes, and

54

because of their import and difficulty in recycling, the cost of IMOs production is

55

high in China.

56

Whole-cell catalysts are a kind of unique immobilized technology that do not need the

57

complicated free enzyme purification process. It can be recycled and reused by

58

centrifugation, which reduces the free protein contamination in the process of product

59

collection and purification. Moreover, it can also be used in fluidized beds to develop

60

the continuous production of lots of IMOs. The approach of displaying an enzyme

61

protein on the P. pastoris cell surface by using the GPI-modified cell-wall protein 4


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62

(GCW) has been successful in our laboratory21. It could be a cost-effective

63

bioconversion process that could be applied in manufacturing with a simpler

64

preparation.

65

In this study, we constructed successfully a recombinant P. pastoris strain with ANGL

66

immobilized on the cell surface by GCW61. We explored the ability of the

67

ANGL-displaying P. pastoris whole-cell biocatalyst (Pp-ANGL) to synthesize IMOs

68

using maltose. We mainly focused on the synthesis of isomaltose (IG2), panose (P)

69

and isomaltotriose (IG3). Reaction parameters such as pH and temperature were

70

optimized, and the effect of the concentrations of the whole-cell biocatalyst and

71

substrate were determined. Subsequently, scaled-up reactions were performed in a 2-L

72

stirred reactor under optimized conditions. We also tested the the reusability of

73

Pp-ANGL-GCW61. In this paper, we report the first, synthesis of IMOs using the

74

whole-cell biotransformation approach by cell surface displaying the α-glucosidase

75

from Aspergillus niger.

76

MATERIALS AND METHODS

77

Strains and growth conditions

78

E. coli was used as the host strain for plasmid storage and amplification. P. pastoris

79

GS115 was used for cell surface display. The recombinant Dalbergia cochinchinensis

80

Pierre β-glucosidase (DCBGL) surface-displaying plasmid pKDCBGL-GCWn

81

（n=12,19,21,49,61）was previously constructed by Guo et al.22 in our laboratory. On

82

this basis, the recombinant plasmid pKANGL-GCW61 and ANGL-displaying P.

83

pastoris strain Pp-ANGL-GCW61 were constructed for this study. DNA sequences 5



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84

encoding mature ANGL (NCBI accession No. XP_001402053.1) were synthetized after

85

codon usage optimization by Genrey (Shanghai, China) and maintained in

86

Escherichia coli Top10 cells.

87

E. coli was cultivated on LB plates or in LB medium (1% NaCl, 0.5% yeast extract,

88

and 1% tryptone, plates also contain 2% agar) containing 50 µg/mL kanamycin or 25

89

µg/mL zeocin at 37 °C and 250 revolutions per minute (rpm). Yeast strains were

90

grown on either MD plates (2% glucose, 1.34% yeast nitrogen base, and 2% agar) or

91

in BMGY/BMMY media (1.34% yeast nitrogen base, 1% yeast extract, 2% peptone,

92

100 mM potassium phosphate (pH 6.0), and 1% glycerol or 2% methanol). P. pastoris

93

GS115 strain was cultured at 30 °C and 250 rpm. P. pastoris GS115 transformed with

94

pPIC9K (GS115/9K) was used as a negative control.

95

Standard chemicals including isomaltose, panose and isomaltotriose were purchased

96

from Sigma-Aldrich (St. Louis, USA).

97

Construction

98

recombinant yeast strain GS115/ pPIC9K-ANGL-GCW61

99

The target gene encodes the mature ANGL was amplified from the synthetized

of

the

pPIC9K-ANGL-GCW61

expression

plasmid

and

100

template by PCR, with the upstream 5’-

101

CCGGAATTCATGTACCCATACGATGTTCCAGATTACGCTTCTACAACTGCACC

102

AA -3’ and downstream 5’-

103

CGACGCGTCCATTCCAAAACCCAGT -3’ primers, while an HA peptide tag was

104

added at the N-terminus for further study. The PCR product and the plasmid

105

pKDCBGL-GCW61 were both digested with EcoRI and MluI, gel-purified and then 6


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106

ligated to form the recombinant plasmid pPIC9K-ANGL-GCW6122.

107

The recombinant plasmid pPIC9k-ANGL-GCW61 was linearized with Kpn2I and

108

integrated into the host strain P. pastoris GS115 by electroporation in homologous

109

recombination method23. The transformants were selected by incubation at 30 °C for 3

110

days on MD plates. The confirmed transformants were precultured first in 10 mL

111

BMGY medium at 30 °C and 200 rpm. After 24 h, the cells were collected by

112

centrifugation at 6000 rpm for 5 min and then resuspended in 25 mL BMMY medium

113

containing 2% (v/v) methanol to make sure that the initial OD600 was controlled to

114

be 1. To maintain the induced expression of the fusion proteins, 500 µL of methanol

115

was added to the culture every 24 h throughout the induction phase.

116

Hydrolysis activity of the ANGL-displaying P. pastoris cell

117

α-Glucosidase activity was determined by release of p-nitrophenol from

118

4-nitrophenyl-α-D-glucopyranoside (pNPG) solution. Briefly, the hydrolysis activity

119

was measured in a 500-µL reaction at 55°C for 10 min with 5 mM pNPG in 0.04 M

120

Britton-Robinson buffer (pH 5.0)24. The 500-µL reaction included the recombinant P.

121

pastoris cells collected from 250 µL fermentation broth, which washed and

122

resuspended with 250 µL Britton-Robinson buffer (pH 5.0). The reaction was

123

terminated and colored by adding 500 µL of 2 M sodium carbonate. Then, the

124

p-nitrophenol released in the reaction was determined by measuring its absorbance at

125

405 nm22. One unit of hydrolysis activity was defined as the amount of biocatalyst

126

required to release 1 µmol of p-nitrophenol from pNPG per min at 55 °C. Using this

127

method,

the

hydrolysis

activity

of

the

yeast

7


whole-cell

biocatalyst


128

(Pp-ANGL-GCW61) was 2.88 U/ (g dry cell).

129

Preparation of the ANGL-displaying P. pastoris whole-cell biocatalyst

130

After 5 days of methanol induction, the recombinant P. pastoris cells were collected,

131

washed, and then resuspended in a 0.04 M Britton-Robinson buffer (pH 4.0) with 0.2

132

M trehalose added as a protectant for freeze-drying25. Freeze-dried ANGL-displaying

133

P. pastoris whole-cell biocatalysts (Pp-ANGL-GCW61) were used for subsequent

134

experiments.

135

The synthetic ability of Pp-ANGL-GCW61 was analyzed with maltose as the

136

substrate26. To this end, 0.1 g of the whole-cell biocatalyst was added to 2 mL of 30%

137

(w/v) maltose dissolved in 0.04 M Britton-Robinson buffer (pH 4.0) in a 10-mL

138

stoppered glass Erlenmeyer flask. The mixture was incubated on a rotary shaker at

139

55 °C and 200 rpm for 1 h. One unit of synthesis activity corresponded to the amount

140

of biocatalyst that caused 1 µmol of maltose into IMOs (IMOs=IG2+P+IG3) per min

141

at 55 °C. A control reaction was performed using the same procedure and the control

142

whole cells but without the whole-cell biocatalyst. Using this method, the synthetic

143

ability of Pp-ANGL-GCW61 was 54.76 U/ (g dry cell).

144

Optimization of synthesis of IMOs by Pp-ANGL-GCW61

145

The IMOs synthesis by transglycosylation was carried out in 10 mL Erlenmeyer shake

146

flasks containing 2 mL of reactant. The flasks were placed in a shaking incubator at

147

200 rpm and at an appropriate temperature. The pH, temperature, and whole-cell

148

biocatalyst and substrate concentrations were optimized through univariate

149

optimization experiments. 8


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150

The effects of pH were examined at 55 °C and 200 rpm using 62.5 mg/mL

151

Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer.

152

When a single variable was changed, all of the other variables were kept constant. The

153

pH value was set at 3.0, 4.0, 5.0，6.0 or 7.0. The effect of temperature was also

154

examined by performing the reactions at 40, 45, 50, 55 and 60 °C with 50 mg/mL

155

Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer

156

(pH 4.0) at 200 rpm for 4 h. After this reaction, the whole-cell biocatalysts were kept

157

warm for 15 h at the selected pH or temperature before the tolerance of

158

Pp-ANGL-GCW61 was tested.

159

The effect of the substrate concentration on the reaction was investigated by using 200,

160

300, 400 and 500 mg/mL concentrations, while the Pp-ANGL-GCW61 concentration

161

was maintained at 50 mg/mL. The Pp-ANGL-GCW61 concentration was set at 25,

162

37.5, 50, 62.5 and 75 mg/mL, while the substrate concentration was maintained at 300

163

mg/mL.

164

To study the stability of the whole-cell catalyst under the optimal reaction conditions,

165

the Pp-ANGL-GCW61 was reused three times to investigate any changes in catalytic

166

ability. The Pp-ANGL-GCW61 was collected by centrifugation after the reaction.

167

Next, the cells were washed with 0.04 M Britton-Robinson buffer three times. After

168

centrifugation and freeze-drying, the Pp-ANGL-GCW61 was used for the next

169

reaction. Moreover, the reuse experiments were performed with a 10 mL reaction

170

system in the 50-mL Erlenmeyer shake flasks.

171

At predetermined time intervals, 50 µL of reaction liquid was removed from the 9



172

reaction mixture and centrifuged at 14,000 rpm for 1 min. Three parallel samples were

173

removed for each reaction.

174

In the present study, the conversion rate was referred to as the molar conversion,

175

which is equal to the number of moles of maltose that were completely transformed

176

into product divided by the number of moles of substrate.

177

Synthesis of IMOs and reuse of Pp-ANGL-GCW61 in a 2-L stirred reactor

178

To reduce the cost, a large quantity of Pp-ANGL-GCW61 fermentation broth of 30 L

179

fed-batch fermentation was spray-dried directly without washing after 5 days of

180

methanol induction. This whole-cell catalyst was prepared to be used in large-scale

181

reactions.

182

A 0.5-L reaction system was conducted in a 2-L stirred reactor. The reaction was

183

carried out under optimized conditions: 62.5 mg/mL Pp-ANGL-GCW61, 300 mg/mL

184

maltose, and 0.04 M Britton-Robinson buffer (pH 4.0) at 55°C and 200 rpm. Aliquots

185

of the reaction liquid were removed from the reaction mixture at predetermined time

186

intervals. In addition, the Pp-ANGL-GCW61 was reused three times to investigate

187

any changes in catalytic ability over time. The Pp-ANGL-GCW61 was reused as

188

described above.

189

HPLC analysis

190

The quantitative analyses of maltose (M), isomaltose (IG2), panose (P) and

191

isomaltotriose (IG3) were performed using an HPLC (Waters model 2695) equipped

192

with an evaporative light-scattering detector (Waters model 2424 ELSD) and a Waters

193

SPHERISORB NH2 column (particle size, 5 µm; dimensions, 250 mm × 4.6 mm). 10


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194

The reaction liquid was centrifuged at 13,000 rpm for 2 min. The supernatant (20 µL)

195

was collected, diluted 50-fold with water to a total volume of 1 mL and filtered

196

through a cellulose nitrate membrane (0.25 µm). Next, the samples were characterized

197

by HPLC. A standard calibration curve was prepared using IG2, P and IG3 standards

198

that were purchased from Sigma–Aldrich. The analysis was carried out at a column

199

temperature of 30 °C using an acetonitrile-water (80:20, v/v HPLC GRADE) solvent

200

as the mobile phase, at a flow rate of 1.0 mL/min. Using the ELSD detector, the drift

201

tube was set at 65 °C using a nebulizer gas as the carrier gas at a flow rate of 30 psi.

202

RESULTS AND DISCUSSION

203

Display of ANGL on the yeast cell surface

204

The constructed plasmid pPIC9K-ANGL-GCW61 is shown in Figure 1a. Gel

205

electrophoresis of an EcoRI/MluI digestion of the recombinant vector revealed that

206

this plasmid was constructed successfully. The isolated transformants were cultured in

207

BMGY/BMMY medium. A growth curve of the transformants is shown in Figure 1b.

208

As seen from the growth curves, the growth of the recombinant yeast strain is

209

consistent with the growth of GS115/9K, showing that the expression of the fusion

210

protein has no obvious influence on the growth of this strain. With the increase of the

211

induction time, the enzyme activity and OD600 increased substantially, reaching the

212

highest levels (Figure 1c).

213

According

214

transglycosylation activity of the Aspergillus niger α-glucosidase that was obtained

215

via secretory expression in Aspergillus niger or other microorganisms. This report is

to

previous

studies,

researchers

studied

11


the

hydrolysis

and


216

the first in which the Aspergillus niger α-glucosidase was displayed on the yeast cell

217

surface with a higher activity, and there are little activity existed in the free-form

218

(Figure 1d). As a cost-effective whole-cell catalyst, its preparation and storage were

219

convenient. Moreover, its operation stability was suitable for large-scale reactions.

220

Effect of various reaction parameters on the IMOs yield by Pp-ANGL-GCW61

221

The products were quantified using HPLC. Glucose (G), maltose (M), isomaltose

222

(IG2), panose (P) and isomaltotriose (IG3) were well separated by a Waters amino

223

propyl column (2). The retention times were 5.820 min for G, 7.575 min for M, 8.430

224

for IG2, 11.142 min for P and12.607 min for IG3. The conversion rate was greatly

225

affected by the reaction conditions, such as the pH, temperature, whole-cell

226

biocatalyst concentration, substrate concentration and the properties of the enzyme.

227

Effect of pH on IMOs synthesis

228

A tiny deviation from the optimal pH can change the ionization of groups in the

229

enzyme active site and reduce the activity of the enzyme. In addition, large deviations,

230

which disturb many non-covalent bonds that maintain the enzyme’s three-dimensional

231

structure, cause the enzyme to denature. As shown in Figure 3a, the effect of pH was

232

obvious. The highest molar conversion was reached at pH 4.0 and reaction duration

233

for 2 h. The initial synthesis activity was increased when the pH value decreased. The

234

molar conversion of all reaction mixtures was approximately 30% under different pH

235

conditions when the reaction was performed for 4 h.

236

The pH-activity curves of IMOs synthesis for the Amano enzyme are shown in Figure

237

3b. The optimal pH of the activity of Pp-ANGL-GCW61 is from 3.0~4.0, while the 12


Page 12 of 35

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238

optimal pH is from 5.0~6.0 for transglucosidase L (Amano Enzymes Inc, Japan).

239

These changes in the optimal pH range have rarely been reported by other

240

researchers18-20. The essential ionizable groups of the α-glucosidase are two kinds of

241

carboxyl groups: one is charged, and the other is a protonated 27. One possible reason

242

for this behavior was that the GPI-modified cell-wall protein (GCW), which was

243

co-expressed with ANGL, influenced the three-dimensional structure and the charge

244

of the enzyme. We also studied the relationship between pH and stability of

245

Pp-ANGL-GCW61. The activity was still high when the pH value was 3 and 4. We

246

chose pH 4.0 as the optimal pH of the transglucosylation reaction.

247

Effect of temperature on IMOs synthesis

248

The influence of temperature on the enzymatic reaction includes two aspects; on the

249

one hand, when the temperature rises, the reaction rate is accelerated, as in a general

250

chemical reaction. On the other hand, the enzyme gradually degrades as the

251

temperature rises, thus slowing the enzyme’s reaction by reducing the activity of the

252

enzyme. Just below the optimal temperature, the former effect is dominant; but above

253

the optimal temperature, the latter effect is dominant, resulting in the loss of enzyme

254

activity and a decrease in the reaction rate.

255

As shown in Figure 4a, the Pp-ANGL-GCW61 activity gradually increases with an

256

increase in temperature; the high temperature resulted in a high initial rate because the

257

high temperature accelerated molecular diffusion and increased the solubility of the

258

substrate. When the temperature reached 60 °C, the molar conversion was highest.

259

When the temperature continued to increase, the enzyme would be inactivated 13



260

irreversibly, so the molar conversion sharply reduced. At 50, 55 and 60 °C, the molar

261

conversion was higher and above 35%. Its properties are similar to the properties of

262

the transglucosidase L (Amano Enzymes Inc, Japan)16.

263

The 50 mg/mL whole-cell biocatalyst was kept warm for 15 h to test its thermal

264

tolerance at 50, 55 and 60 °C. As shown in Figure 4b, the Pp-ANGL-GCW61 has

265

good thermal stability at 50 and 55 °C, and the molar conversion still can reach

266

approximately 25% after the heat treatment, although the conversion only reaches 10%

267

at 60°C. Therefore, 55 °C was considered to be the most suitable temperature for

268

further optimization research and biocatalyst reuse.

269

Effect of the Pp-ANGL-GCW61 concentration on IMOs synthesis

270

As the Pp-ANGL-GCW61 concentration increases, the initial reaction rate increases.

271

The larger the dose of enzyme was added, the shorter reaction time was required to

272

get to the molar conversion peak. As shown in Figure 5a, when 75 mg/mL

273

Pp-ANGL-GCW61 was added, the conversion rate peaked at 32% in 3 h of reaction.

274

When the added amount of catalyst was 25 mg/mL, the maximum conversion rate

275

(30%) required the reaction to continue for 9 h. Optimizing the enzyme dose is

276

important to reduce the reaction time, as well as to control costs.

277

The results showed that the ultimate conversion rate that the different enzyme doses

278

achieved was consistent, approximately 30%, when the reaction continues for a

279

sufficient period. When the enzyme concentration reaches a certain value, restricted

280

by the amount of substrate, the enzymatic reaction rate will not increase. When 62.5

281

and 75 mg/mL Pp-ANGL-GCW61 were added, the conversion rate curves were 14


Page 14 of 35

Page 15 of 35


282

similar (Figure 5a). Therefore, considering the reaction time and enzyme cost, 62.5

283

mg/mL Pp-ANGL-GCW61 was found to be the optimal enzyme concentration.

284

Effect of maltose concentration on IMOs synthesis

285

ANGL has exhibited typical substrate inhibition kinetics10. The rate increased linearly

286

with increasing substrate concentration (up to 100 mg/mL maltose) and then exhibited

287

a lower, incrementally changing slope in a non-linear phase between 100 and 200

288

mg/mL of substrate. Once the substrate concentration increased above 200 mg/mL,

289

the reaction velocity entered a non-linear deceleration phase, indicating strong

290

substrate inhibition by the maltose10.

291

As shown in Figure 5b , when the maltose concentration increased from 200 mg/mL,

292

the IMOs concentrations showed a corresponding increase, in contrast, the

293

conversation rates decreased. The reaction mixtures containing 200, 300, 400 and 500

294

mg/mL maltose reached the highest conversion rate (approximately 35%) in 3, 5, 7

295

and 9 h, respectively. After enough reaction time, the reaction system is a mixture of

296

IMOs, glucose and small amounts of maltose. Since the maximum conversion rates of

297

the reaction system of different substrate concentrations are similar, when the initial

298

substrate concentration is higher, more maltose is not converted into IMOs.

299

Considering the reaction time and substrate cost, 300 mg/mL maltose was used for

300

further research

301

Reuse of Pp-ANGL-GCW61 in 10-mL and 50-mL Erlenmeyer shake flasks under

302

optimum conditions

303

After single-factor optimization of the factors influencing IMOs synthesis by 15



the

optimum

reaction

conditions

304

Pp-ANGL-GCW61,

305

Pp-ANGL-GCW61, 300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer

306

(pH 4.0) at 55 °C and 200 rpm for 4 h.

307

In the initial stage of the reaction, a mass of maltose was hydrolyzed into glucose.

308

Because of the high concentrations of maltose in the mixture, free glucose residues are

309

preferred to participate in the synthesis reaction of maltose to panose; As shown in

310

Figure 6a, the synthesis rate of panose was highest28. When the reaction reached a

311

certain time, the maltose concentration was greatly reduced, and the panose

312

concentration increased; the effect of panose hydrolysis was remarkable, as shown in

313

the conversion rate drop. The glucose content in the reaction system continued to

314

increase. The reaction reached equilibrium when the glucose content was high enough

315

to inhibit the hydrolysis activity of Pp-ANGL-GCW61.

316

To study the reuse ability of the whole-cell catalyst, the Pp-ANGL-GCW61 was

317

reused three times firstly in the 2-mL reaction system. Batches 1- 3 indicated that

318

Pp-ANGL-GCW61 was reused once, twice and three times. As shown in Figure 7a,

319

the conversion rate in the 2-mL reaction system achieved approximately 35% in 4 h

320

when Pp-ANGL-GCW61 was reused once. There are two reasons: the loss of added

321

enzyme and the loss of enzyme activity, which decreased the conversion rate. Then

322

we enlarged the reaction system to 10-mL, while the conversion rate was a little

323

higher in 10-mL reaction system, as shown in Figure 7b. The conversion rate of

324

batches 2, 3 still reached more than 30% in 5 h both in 2-mL and 10-mL reaction

325

system. Besides, the curves of batches 1, 2 and 3 were similar, indicating that 16


were

Page 16 of 35

62.5

mg/mL

Page 17 of 35


326

Pp-ANGL-GCW61 was stable and reusable in a 2-mL and 10-mL reaction system.

327

Synthesis of IMOs in a 2-L stirred reactor and the reuse of Pp-ANGL-GCW61

328

IMOs were synthesized in 500-mL reaction system under the optimized conditions in

329

a 2-L stirred reactor. Batches 1- 3 indicated that Pp-ANGL-GCW61 was reused once,

330

twice and three times, respectively. Compared with the 2-mL and 10-mL reaction

331

system, the initial conversion rate of the 500-mL reaction system was a little decrease

332

in the reuse study (Table 1). The reaction in the small shake flask system may have

333

been more uniform than that in the stirred reactor. Similar to the 2-mL and 10-mL

334

reaction system, batch 1 achieved the highest conversion rate in 4 h, and the

335

conversion rate was still increasing after reacting for 5 h in batches 2 and 3 (Figure

336

7c).

337

In general, Pp-ANGL-GCW61, which has a high operational stability and the

338

advantages of being inexpensive and conveniently prepared, is very suitable for the

339

large-scale synthesis of IMOs through the transglycosylation reaction. This study

340

provided a reference for enzymatic industrial applications.

341

In conclusions, the P. pastoris strain that displayed the Aspergillus niger

342

α-glucosidase was successfully constructed. The hydrolysis and synthetic activities of

343

Pp-ANGL-GCW61 were 2.88 U/(g dry cell) and 54.76 U/(g dry cell), respectively.

344

The ANGL-displaying P. pastoris whole-cell biocatalyst (Pp-ANGL-GCW61) was

345

prepared by vacuum lyophilization. After a single-factor optimization test, we

346

determined that the optimum reaction conditions are 62.5 mg/mL Pp-ANGL-GCW61,

347

300 mg/mL maltose, and 2 mL 0.04 M Britton-Robinson buffer (pH 4.0) at 55 °C and 17



Page 18 of 35

348

200 rpm for 4 h. In a 2-mL reaction system, IMOs were synthesized with

349

approximately

350

Pp-ANGL-GCW61.

351

The whole-cell biocatalyst was reused in a 500-mL reaction system under the

352

optimized conditions in a 2-L stirred reactor. Furthermore, the reusability of the

353

Pp-ANGL-GCW61 indicated that Pp-ANGL-GCW61 was suitable for large-scale

354

synthesis because of its high stability, low cost and moderate conversation rate.

355

Because of its cost-effectiveness, high stability and catalytic activity, the whole-cell

356

catalyst Pp-ANGL-GCW61 provides a reference for the enzymatic synthesis of IMOS

357

on an industrial scale.

358

Abbreviations Used

359

GPI,

360

pNPG, 4-nitrophenyl-α-D-glucopyranoside; GCW61, GPI-modified cell wall protein

361

from Pichia pastoris; M, maltose; IMOs, isomalto-oligosaccharides; IG2, isomaltose;

362

P,

363

ACKNOWLEDGMENT

364

All the authors are thankful for the financial support of the Science and Technology

365

Planning Project of Guangzhou City (No.201607010307), the National Natural

366

Science Foundation of China (No.31671840), the Recruitment Program of Leading

367

Talents in Innovation and Entrepreneurship of Guangzhou (LCY201322) to Suiping

368

Zheng.

369

NOTES

49%

conversion

in

2

h

under

optimum

conditions

by

glycosylphosphatidylinositol; ANGL, the Aspergillus niger α-glucosidase;

panose; IG3, isomaltotriose; rpm, revolutions per minute.

18


Page 19 of 35


370

The authors declare no competing financial interest.

371

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25



524

Table titles

525

Table 1 Initial conversion rate of Pp-ANGL-GCW61 in reuse study in 0.5 h.

526 527

Figure captions

528

Figure 1. (a) Construction of the pPIC9K-ANGL-GCW61 plasmid used for

529

displaying ANGL on the yeast cell surface. (b) Growth-related curves of P. pastoris

530

GS115/ANGL–GCW61 and GS115/9K in 250-mL shake flasks. (c) Fermentation time

531

curves of P. pastoris GS115/ANGL–GCW61 and GS115/9K in 250-mL shake flasks.

532

(d) Determination of Pp-ANGL-GCW61 fermentations upermatant hydrolytic activity.

533

Error bars represent standard deviations, and three replicates were performed.

534 535

Figure 2. HPLC-ELSD analyses of reaction products from maltose by enzymatic

536

activity of Pp-ANGL-GCW61; G, glucose; M, maltose; IG2, isomaltose; P, panose ;

537

IG3, isomaltotriose.

538 539

Figure 3. (a) Effect of pH on IMOs synthesis by Pp-ANGL-GCW61, (b) pH-activity

540

curves of Amano and Pp-ANGL-GCW61 for IMOs synthesis, and (c) pH-stability

541

relationship of Pp-ANGL-GCW61 for IMOs synthesis. Error bars represent standard

542

deviations, and three replicates were performed.

543

544

Figure 4. (a) Effect of temperature on IMOs synthesis by Pp-ANGL-GCW61 (b)

545

Temperature-stability relationship of Pp-ANGL-GCW61 for IMOs synthesis. Error 26


Page 26 of 35

Page 27 of 35


546

bars represent standard deviations, and three replicates were performed.

547

548

Figure 5. (a) Effect of the whole-cell biocatalyst Pp-ANGL-GCW61 concentration on

549

IMOs synthesis. (b) Effect of the maltose concentration on IMOs synthesis. Error bars

550

represent standard deviations, and three replicates were performed.

551

Figure 6. Production of IMOs under optimum conditions. Error bars represent

552

standard deviations, and three replicates were performed.

553

554

Figure 7. (a) Reuse of Pp-ANGL-GCW61 in the 2-mL reaction system. (b) Reuse of

555

Pp-ANGL-GCW61 in the 10-mL reaction system. (c) Reuse of Pp-ANGL-GCW61 in

556

the 500-mL reaction system. Error bars represent standard deviations, and three

557

replicates were performed.

558

27



Page 28 of 35

Tables Table 1 times reused

2-mL system

10-mL system

500-mL system

batch 1

9.84%

9.97%

9.68%

batch 2

9.26%

9.67%

7.42%

batch 3

6.74%

8.94%

5.40%

28


Page 29 of 35


Figures Figure 1

29



Figure 2

30


Page 30 of 35

Page 31 of 35


Figure 3

31



Figure 4

32


Page 32 of 35

Page 33 of 35


Figure 5

Figure 6

33



Figure 7

34


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For Table of Contents Only

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Synthesis of Isomalto-Oligosaccharides by Pichia pastoris Displaying

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