Enzymatic glucosylation of salidroside from starch by Î±-amylase

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Biotechnology and Biological Transformations

Enzymatic glucosylation of salidroside from starch by #-amylase Ke Wang, Tingting Qi, Longcheng Guo, Zhongxuan Ma, Guofeng Gu, Min Xiao, and Lili Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06618 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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Journal of Agricultural and Food Chemistry

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Enzymatic glucosylation of salidroside from starch by α-amylase

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Ke Wang,†, 1 Tingting Qi,‡, 1 Longcheng Guo,‡ Zhongxuan Ma,‡ Guofeng Gu,‡

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Min Xiao,*,‡ Lili Lu*,†,‡

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School of Pharmacy, Tongji Medical College, Huazhong University of Science and

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†

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Technology, Wuhan 430030, PR China.

9

‡

National Glycoengineering Research Center, Shandong Provincial Key Laboratory

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of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial

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Technology, Shandong University, Qingdao 266237, PR China.

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These authors contributed equally to this work

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1

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*Corresponding author.

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E-mail: [email protected] (M. Xiao); [email protected] (L. Lu)

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1

ACS Paragon Plus Environment


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ABSTRACT α-Amylases are among the most important and widely used industrial enzymes for

21

starch processing. In this work, an α-amylase from Bacillus subtilis XL8 was purified

22

and found to possess both hydrolysis and transglycosylation activities. The optimal pH

23

and temperature for starch hydrolysis were pH 5.0 and 70°C, respectively. The enzyme

24

could degrade soluble starch into beneficial malto-oligosaccharides ranging from dimer

25

to hexamer. More importantly, it was able to catalyze α-glycosyl transfer from the

26

soluble starch to salidroside, a medicinal plant-derived component with broad

27

pharmacological properties. The transglycosylation reaction catalyzed by the enzyme

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generated six derivatives in a total high yield of 73.4% when incubating with 100

29

mg/mL soluble starch and 50 mM salidroside (pH 7.5) at 50 °C for 2 h. These

30

derivatives were identified as α-1,4-glucosyl, maltosyl, maltotriosyl, maltotetraosyl,

31

maltopentaosyl, and maltohexaosyl salidrosides, respectively. They were novel

32

promising compounds that might integrate the bioactive functions of

33

malto-oligosaccharides and salidroside.

34

Key words: α-amylases; purification; glycosylation; starch; salidroside

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■ INTRODUCTION Salidroside (p-hydroxyphenethyl-β-D-glucoside or tyrosol glucoside) is an

38

important aromatic compound derived from the medical plant Rhodiola rosea, of which

39

the root extract has been popularly used as health products to increase the body’s

40

resistance to stress, exhaustion and fatigue.1,2 As one of the main effective ingredients

41

of R. rosea extract, salidroside was found to favorably affect a number of physiological

42

functions and possess broad bioactivities, such as anti-fatigue, antidepressant,

43

antioxidative, anti-inflammatory, anticancer, hepatoprotective, cardioprotective, 2


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neurotrophic and neuroprotective activities.3-13 Due to the importance of salidroside,

45

considerable efforts have been devoted to modification of its structure to develop novel

46

analogs with improved pharmacological efficacy .14.17 It is well known that

47

glycosylation of natural products is a useful method for novel drug discovery and

48

development, as the addition of sugar moiety could diversify the chemical structures

49

with altered pharmacology/pharmacokinetics and target specificities on tissue, cellular,

50

and/or molecular levels. 18, 19 However, the glycosylation of salidroside has yet not been

51

reported up to date. The traditional chemical method for glycosylation is rather sophisticated because a

52 53

sugar moiety possesses multiple hydroxyl groups with similar reactivity and thus

54

multiple protection/deprotection steps are required to control regio- and

55

stereoselectivities. Alternatively, enzyme-dependent approaches enable one-step

56

synthesis of a specific glycoside linkage due to the advantages of stereo- and

57

regioselectivity. 20 Also the enzymes catalyzed the reactions in a sustainable and

58

environment-friendly way. Glycosyltransferases (EC 2.4) and glycosidases (EC 3.2.1)

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are the two main classes of enzymes responsible for glycosylation. The former enzymes

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are effective but require costly glycosyl donors and have strict acceptor specificity,

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whereas the latter accommodate simple, inexpensive donor substrates and broad

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acceptors but usually give modest product yields. 21 α-Amylases (EC 3.2.1.1) are among the most important glycosidases that catalyze

63 64

the hydrolysis of the internal α-1,4-glucosidic bonds in starch and related α-glucans.

65

22-25

66

that have beneficial effects on human health. These oligosaccharides are considered a

67

promising energy source for athletes and some special patients. They are non-digestible

68

in the stomach and utilized by the intestinal α-glucosidases, thus providing continuous

The hydrolysis ability can be used to convert starch into malto-oligosaccharides

3



69

and steady energy. 26 Besides hydrolysis activity, some α-amylases are also able to

70

catalyze transglycosylation. For example, the α-amylases TRa2 from Trichoderma

71

viride JCM22452 was reported to catalyze glycosyl transfer from dextrins to various

72

natural flavonoids, resulting in superior α-glucosides with higher heat stability and

73

solubility and lower astringency and astringent stimulation than their aglycons. 27 Thus,

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the use of α-amylase is currently extended to novel areas like the synthesis of the

75

compounds important in food and pharmaceutical industries. 28

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In this work, an α-amylase from B. subtilis XL8 was purified and found to possess

77

both hydrolysis and transglycosylation activities. This enzyme produced

78

malto-oligosaccharides from soluble starch. Also it could glycosylate salidroside using

79

the soluble starch as glycosyl donor. It turned out to be a powerful tool for glycosylation

80

since it synthesized a series of α-glucosylated salidroside with a surprisingly high yield.

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These newly-formed compounds were promising candidates for food and

82

pharmaceutical applications as they might combine the steady energy supply properties

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of malto-oligosaccharides and the excellent bioactivities of salidroside.

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■ MATERIALS AND METHODS

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Materials. Salidroside and soluble starch (with ~100 polymer degree) were

86

purchased from Sangon (Shanghai, China). DEAE Sepharose Fast Flow, Source 15Q and

87

Sephadex G200 were from GE Healthcare (Sweden). Bio-Gel P2 was from Bio-Rad

88

Laboratories (Hercules, USA). Silica gel 60 F254 plates coated with flourescent

89

indicator were supplied by Merck (Darmstadt, Germany). HPLC grade acetonitrile was

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purchased from Honeywell Burdick & Jackson (Muskegon, USA). Other chemicals

91

were of analytical grade.

92

Bacterial medium and cultivation. B. subtilis XL8 was inoculated in the

93

liquid medium (pH 7.0) containing 10 g/L peptone, 5 g/L yeast extract and 7 g/L NaCl 4


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and cultivated at 37 ℃ for 12 h. Then, the cell culture was transferred with a ratio of 2%

95

(v/v) into the fresh medium containing 3 g/L soluble starch, 10 g/L peptone, 5 g/L yeast

96

extract and 7 g/L NaCl and incubated at 37 ℃ for 72 h. Afterwards, the cell culture was

97

centrifuged at 18514 × g for 5 min and the resulting extracellular supernatant was used

98

as the crude enzyme for the purification of α-amylase.

99

Enzyme purification. The crude enzyme was concentrated by ammonium

100

sulfate precipitation (0-60% saturation). The resulting precipitate was dissolved in

101

phosphate buffer (pH 7.0), dialyzed and subjected to the column chromatography which

102

was subsequently performed at 4°C through the ÄKTA/FPLC machine (GE Healthcare,

103

Sweden). A DEAE Sepharose Fast Flow column (1.5×10 cm) was firstly prepared,

104

equilibrated with 50 mM phosphate buffer (pH 7.0) and loaded with the sample,

105

followed by the gradient elution with sodium chloride ranging from 0 to 500 mM. The

106

eluted fractions with the enzyme activity were detected by SDS-PAGE, combined and

107

dialyzed in 50 mM phosphate buffer. Then the sample was subjected to the Source 15Q

108

column (1.5×10 cm) according to the same procedures as those for DEAE column.

109

Afterwards, the sample was loaded on the Sephadex G200 column (1.5×15 cm) and

110

eluted by 50 mM phosphate buffer. The finally purified enzyme was concentrated by

111

ultrafiltration and stored at -80°C.

112

Enzyme and protein assays. α-Amylase activity was assayed by DNS

113

(3,5-dinitrosalycilic acid) method to detect the release of reduced sugar from starch. 29

114

The reaction mixture containing 0.1 mL enzyme and 1.0 mL soluble starch (1% w/v) in

115

0.1 M phosphate buffer (pH 7.5) was incubated at 50 ℃ for 5 minutes, followed by

116

addition of 1 mL of DNS and boiling for 10 minutes. Afterwards, the amount of the

117

reducing sugar released was measured at 540 nm. One unit of α-amylase activity was

118

defined as the amount of enzyme required to liberate 1 μmol of maltose per minute 5



119

under the assay conditions. The amount of protein was quantified by Bradford assay

120

based on the use of the dye Coomassie Brilliant Blue G-250. SDS-PAGE and Native

121

gradient PAGE for protein detection were performed in 10% (w/v) and 5 to 10% gels,

122

respectively.

123

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Biochemical studies. The optimal pH was assayed by incubating the enzyme

124

with 1% soluble starch in 50 mM buffers ranging from pH 2.0 to 8.0. The effect of pH

125

on enzyme stability was determined by incubation of the enzyme at pH from 2.0 to 10.5

126

at 4 °C for 12 h and then the residual enzyme activity was measured following the

127

standard enzyme assay conditions. The optimal temperature was determined by

128

measuring enzyme activities at different temperatures ranging from 30 to 85 °C.

129

Thermal stability was studied by assessing enzyme activity after exposing the enzyme

130

samples at the above temperatures for 1 h. To determine the effects of chemicals,

131

enzyme activities were assayed in the presence of 1 mM metal salts or 10 mM additives.

132

Kinetic constants of the enzyme were estimated by using various concentrations of

133

soluble starch (3 to 60 mg/mL) under enzyme assay conditions as described above. The

134

Km and Vmax values of the enzyme were determined with GraphPad Prism 8 software.

135

Gene cloning and heterogenous expression. A pair of degenerate primers

136

for the cloning of the entire gene were designed based on the genome sequence of B.

137

subtilis 168 (GenBank no. NC_000964.3) as well as the α-amylase gene sequences of B.

138

subtilis strains OI1085, US572, DR8806 (GenBank nos. FJ643607.1, MG264159.1,

139

KC262177.1, respectively). The nucleotide sequences were

140

5′-ATGTTTGCAAAACGATTCAAA-3′ and 5′-TCAATSGGGAAGAGAASCGCT-3′,

141

respectively. For the heterogenous expression of the gene lacking the signal-encoding

142

sequence, the forward primer (F127-22b) was designed based on the N-terminal

143

sequence of the purified enzyme and its sequence was 6


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5′-CAGCGAGCTCCACAGCGCCATCGATCAAAAGC-3′ (Sac Ⅰ site is underlined).

145

The reverse primer (R-22b) was

146

5′-GCGCAAGCTTATGGGGAAGAGAACCGCTTAA-3′ (Hind Ⅲ site is underlined).

147

The PCR reactions were performed in the presence of EasyPfu DNA Polymerase

148

(TransGen Biotech), following the procedures including 3 min at 94°C, 30 cycles of 30

149

s at 94°C, 30 s at 66°C, 4 min at 72°C, and a final 5 min at 72°C.

150

For enzyme expression, the PCR products using the primers F127-22b and R-22b

151

were purified and digested by restriction enzymes. Then they were ligated onto the

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pET-22b (+) vector and transformed into E. coli BL21 (DE3). The correct transformant

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was inoculated in LB medium containing ampicillin (50 μg/mL) at 37°C, and the

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enzyme was induced by adding isopropyl-1-thio-β-D-galactoside (IPTG) when the cell

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density reached 0.6~1.0 at 600 nm. After continuous cultivation for three hours, cells

156

were harvested and disrupted by ultrasonic treatment. The lysate was centrifuged and

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the enzyme was purified from the suspension by Ni2+ chelation chromatography.

158

Glucosylation of salidroside by α-amylase. The glucosylation reactions

159

catalyzed by the α-amylase from B. subtilis XL8 were performed by incubation of the

160

enzyme with salidroside and soluble starch. Three groups of reactions were used as

161

control: (I) salidroside and enzyme; (II) enzyme and starch; (III) inactivated enzyme,

162

starch and salidroside. The effects of the starch concentrations were tested by incubation

163

of the enzyme with starch at different concentrations (1 to 140 g/L) in the presence of

164

50 mM salidroside in 50 mM potassium phosphate buffer (pH 7.0) at 50 °C for 1 h. The

165

influences of the salidroside concentrations were tested at 10 to 700 mM. The effects of

166

pH values were investigated at pH 3.5 to 8.0 by using 100 g/L starch and 50 mM

167

salidroside as substrates. The effects of temperature were assayed by incubating the

168

enzyme with 100 g/L starch and 50 mM salidroside (pH 7.5) at 30 to 80°C, respectively. 7



169

To study the effects of reaction time, assays were performed in an enzyme reaction

170

mixture (pH 7.5) containing 100 g/L mM starch and 50 mM salidroside, which were

171

incubated at 50°C with aliquots serially analyzed at 0.5 to10 h. All reactions were

172

stopped by heating at 100 °C for 10 min, and the resulting products were detected by

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TLC and HPLC.

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Purification of salidroside derivatives. The synthesis of salidroside

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derivatives by α-amylase was carried out following the optimized reaction conditions.

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The resulting reaction mixture was first concentrated by vacuum freeze dehydration.

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Then it was applied on a Bio-Gel P2 column (1.5 × 100 cm) and eluted by distilled

178

water at a flow rate of 0.2 mL/min. 16 The eluted fractions were collected at 1.5 mL per

179

tube, which were detected through spotting on TLC plate followed by spraying

180

3,5-dihydroxytoluene/sulfuric acid and heating. The fractions containing sugars were

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loaded on TLC plate again, and developed using n-butanol/ethanol/water as the mobile

182

phase. The samples with identical sugar compositions were combined and concentrated

183

to dry powder.

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TLC and HPLC analysis. TLC was performed by loading the sugar samples on

185

the Silica gel 60 F254 plates. The loaded samples were developed by a mixture of

186

n-butanol/ethanol/water (5: 3: 2, v/v/v) and subsequently visualized by spraying with

187

0.5% (w/v) 3,5-dihydroxytoluene in 20% (v/v) sulfuric acid and heating at 120 °C for 5

188

min. Before coloration, salidroside and its derivatives could also be directly detected

189

under UV light at 254 nm. Quantitative sugar analysis was performed by HPLC using

190

Agilent 1200 series equipped with a Thermo Hypersil GOLD Amino column (4.6 × 250

191

mm) at 30 °C. Samples were eluted with 73% acetonitrile at a flow rate of 1.0 mL/min,

192

and detected through a UV detector (G1314B) at 275 nm.

193

MS and NMR analysis. The MS analysis was performed through Shimadzu 8


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LCMS-IT-TOF (Kyoto, Japan) equipped with an ESI source in positive ion mode at a

195

resolution of 10,000 full width at half-maximum. The NMR spectra were recorded on

196

Agilent DD2-600 spectrometer at 600 MHz for 1H and at 150 MHz for 13C. Chemical

197

shifts were expressed in parts per million (ppm) downfield from internal

198

tetramethylsilane of D2O. One- and two-dimensional NMR experiments, involving 1H

199

NMR, 13C NMR, correlation spectroscopy (COSY), hetero-nuclear single quantum

200

coherence (HSQC), and hetero-nuclear multiple band correlation (HMBC), were used to

201

obtain the assignments of the chemical structures.

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■ RESULTS AND DISCUSSION

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Enzyme purification, characterization and gene cloning. The α-amylase

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from B. subtilis XL8 was purified from the extracellular culture liquid with a 4.9% yield,

205

through sequential steps of ammonium sulphate precipitation, DEAE anion exchange

206

chromatography, Source 15Q anion exchange chromatography, and Sephadex G-200 gel

207

filtration chromatography (Table 1, Figure 1). The specific activity of the pure enzyme

208

was 526.5 U/mg. The molecular mass of the enzyme as determined by SDS-PAGE and

209

Native gradient PAGE was about 70.4 kDa and 162.7 kDa, respectively, indicating a

210

homodimer. The purified enzyme was subsequently electroblotted onto a

211

polyvinylidene difluoride membrane. The resulting protein band in the membrane was

212

cut out and sequenced by the method of Edman degradation. The N-terminal amino acid

213

sequence of the enzyme was determined as T-A-P-S-I-K-S-G (Supplementary Figure

214

S1), which exhibited 100% identity with the N-terminal sequence of the α-amylases

215

from Bacillus genus, such as the enzymes from Bacillus subtilis JN16 (GenBank no.

216

AFI62032.1), Bacillus atrophaeus BA59 (GenBank no. ATO28609.1), Bacillus

217

amyloliquefaciens DL-3-4-1 (GenBank no. ADH93703.1), and Bacillus sp. KR-8104

218

(GenBank no. ACD93218.3). 9



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The enzyme was stable between pH 5.5 and 7.5, and the optimal pH for enzyme

220

activity was 5.0 (Figure 2a). It was highly active at a high temperature of 70°C, but kept

221

stable at lower temperatures and the residual enzyme activity remained above 60% after

222

incubation below 50°C for 1 h (Figure 2b). Most known α-amylases from different

223

strains of B. subtilis showed optimal activity at temperatures ranging from 37°C to

224

60 °C and in the pH from 5 to 9. 22, 30 Also there existed exceptional examples. For

225

instance, the α-amylases from a strain of B. subtilis isolated from fresh sheep’s milk

226

exhibited maximal activities at 135℃ .31 The Km and Vmax values of the enzyme for

227

soluble starch were calculated as 4.9 mg/mL and 1188 μmol/mL/mg, respectively.

228

Ca2+, Mg2+ and Mn2+ significantly increased the enzyme activity with 19.4%, 28.6%

229

and 40%, respectively. Hg2+ completely inhibited the enzyme activity, while EDTA

230

exhibited partial inhibition (Figure 2c). Various metal ions had been previously reported

231

to influence the activity of α-amylases, among which Ca2+ was found to enhance the

232

enzyme activity in most cases. However, there still exist a few enzymes that were

233

Ca2+-independent or even could be inhibited by Ca2+ ions, such as the enzymes from B.

234

amyloliquefaciens and Geobacillus thermoleovorans. 30 On the other hand, the

235

inhibition of Hg2+ on the enzyme activity was a common phenomenon among

236

α-amylases. The enzymes from Lactobacillus manihotivorans LMG 18010T,

237

Aspergillus niger UO-1, and Cryptococcus flavus could all be inactivated after addition

238

of Hg2+ . 22

239

The gene of the α-amylase from B. subtilis XL8 was subsequently obtained by

240

PCR. It contained an open reading frame (ORF) of 1980 nucleotides, encoding a 559

241

amino-acid protein with a predicted molecular mass of ~72.3 kDa. The bioinformatics

242

analysis result of the deduced protein revealed it had a putative signal peptide in the

243

N-terminal region. Based on the N-terminal amino acid sequence of the purified enzyme 10


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as described above, the cleavage site of the signal peptide was determined to locate

245

between the 42th and 43th amino acids. The nucleotide sequence encoding the mature

246

gene without the signal peptide was obtained by PCR, ligated into pET-22b (+), and

247

successfully expressed in E. coli BL21 (DE3). The resulting recombinant mature

248

α-amylase was purified and verified to have similar molecular mass and enzyme activity

249

to the pure enzyme prepared from B. subtilis XL8. Thus the gene sequence of the

250

enzyme was submitted to GenBank with the accession number MK234875.

251

Glucosylation of salidroside. The transglycosylation activity of the α-amylase

252

from B. subtilis XL8 was detected by incubation with soluble starch as glycosyl donor

253

and salidroside as glycosyl acceptor.

254

As shown in Figure 3a, no products were found in I and III control reactions,

255

whereas new sugar spots appeared above starch in the control reaction II catalyzed by

256

the enzyme. The hydrolysis ability of the enzyme seemed excellent since the starch spot

257

became very pale after conversion by the enzyme. The resulting malto-oligosaccharide

258

products were purified and identified to be mainly composed of di- to hexasaccharide

259

by MS analysis (Supplementary Figure S2). When the salidroside was added to the

260

reaction mixture containing the enzyme and starch, the product distribution changed

261

greatly. The hydrolysis of starch was inhibited while the transglycosylation reactions

262

occurred. There existed a series of novel spots below the spot of salidroside in the TLC

263

plate. These spots showed signals under UV light (Supplementary Figure S3), indicating

264

that they were salidroside derivatives as the presence of tyrosol in the salidroside was

265

related to the UV absorption.

266

The result of the HPLC spectrum from a UV detector showed that there were six

267

novel visible peaks existent in the reaction mixture besides salidroside (Figure 3b). Each

268

peak of the sample was subsequently collected, concentrated and analyzed by TLC 11



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269

under UV light (Supplementary Figure S3). The result revealed that the peaks at 6.0

270

min, 7.5 min, 9.7 min, 13.2 min, 18.5 min, and 25.8 min corresponded to P1 to P6

271

products in Figure 3a, respectively, which also confirmed the production of salidroside

272

derivatives by the α-amylase. The impacts of the reaction conditions such as substrate

273

concentration, pH, temperature, and reaction time on the product yield were further

274

investigated and optimized.

275

As for the effect of donor substrate, the total product yield continued to rise when

276

the starch concentration was increased from 1 g/L to 100 g/L (Figure 4a). It reached the

277

maximum of 70.1% at 100 g/L starch and then decreased with continuous increment of

278

the donor substrate. The yield dropped to 57.9% when the starch was used at 140 g/L.

279

The existence of such a high concentration of starch might occupy the active site of the

280

enzyme and thus slow down the transglycosylation reaction. As shown in Figure 4b, the

281

total product yields were at high levels when the salidroside concentration rose from 10

282

mM to 200 mM. The yield achieved the peak value at 50 mM salidroside and decreased

283

to 59.0% when the salidroside was used at 300 mM. Continuing to increase the

284

salidroside from 300 mM to 700 mM, the yield kept stable without obvious fluctuations.

285

It seemed that high concentrations of substrates in a certain range facilitated the reaction

286

equilibrium toward the transglycosylation over the hydrolysis by the enzyme, a

287

common phenomenon that had been found in reactions catalyzed by glycosidases. 32, 33

288

In Figure 4c, the product formation was slightly affected by the pH value ranging

289

from pH 3.5 to 8.0. The yield gradually increased with the increased pH values. It

290

reached the maximum at pH 7.5, and dropped outside this pH value. In Figure 4d, the

291

reaction temperature displayed remarkable influences on the glycoside formation by the

292

enzyme. The product yields were keeping increased from 30 °C and reached the peak

293

value at 50 °C. When the reaction temperatures were continuously elevated to 80 °C, 12


Page 13 of 27


294

there appeared sharp decreases of the yields. As the enzyme was stable below 50°C,

295

higher temperatures might inactivate it and reduced the glycoside production. Notably,

296

the optimal pH and temperature of the enzyme for transglycosylation were quite

297

different from those for hydrolysis (pH 5.0 and 70°C). This property might contribute to

298

accumulate glycoside products in a high yield as the hydrolysis activity could be

299

partially inhibited under the optimal transglcosylation conditions.

300

The influences of reaction time on the product yield by α-amylase were

301

investigated by tracing time curves within 10 h (Figure 4e). The reaction proceeded

302

quickly and the products were accumulated in a relatively high amount at 30 min and

303

increased to the peak value at 2 h. When the reaction time was prolonged to 10 h, the

304

yield gradually reduced. The reduction of the product might be related to the hydrolysis

305

activity of glycosidases as they can utilize glycoside products as substrates for

306

hydrolysis. Generally, there exists competition between the transglycosylation and the

307

hydrolysis processes catalyzed by glycosidases, and the final product yield depends on

308

the relative kinetics of synthesis and degradation. The α-amylase from B. subtilis XL8

309

catalyzed the synthesis in a quite rapid speed, and high-yield formation of glycosides

310

was achieved within a short time (2 h). The reported α-amylases used for the synthesis

311

reaction generally required more than 10 hours, such as 18 to 24 h for the enzyme TRa2

312

from T. viride JCM22452 to glycosylate catechin and epigallocatechin 27 and 16 h for

313

the enzyme HGE from B. subtilis X-23 to modify caffeic acid. 34

314

In summary, the optimal conditions for glucosylation of salidrosides by the

315

α-amylase from B. subtilis XL8 were 100 g/L starch and 50 mM salidroside at pH 7.5

316

and 2 h incubation at 50 °C. Under these conditions, the yield of total salidroside

317

derivatives reached a maximum of 73.4%. It is surprising that the enzyme could directly

318

utilize inexpensive, large molecule of soluble starch with polymerization degree of ~100 13



319

as the glycosyl donor to achieve such a high yield of glycosides. The reported

320

α-amylases mostly employed small molecules like maltopentaose or dextrin as a

321

glucosyl donor, since small donors are generally easier for glycosidase to utilize than

322

large ones. 33 Due to high efficiency and low cost, the α-amylase from B. subtilis XL8

323

might be extended to glucosylate more valuable compounds at a large scale in the

324

future.

325

Page 14 of 27

Isolation and identification of salidroside derivatives. The salidroside

326

derivatives produced by α-amylase were purified and analyzed by MS and NMR

327

spectroscopy (Supplementary Figure S4-S5). The positive-ion ESI-mass spectrum of the

328

product P1 showed peaks of [M+NH4]+ at m/z 480.2100, [M+Na]+ ion at m/z 485.1655,

329

and [M+K]+ ion at m/z 501.1376, consistent with the molecular mass of

330

monoglucosyated salidroside (462). In the 1 H NMR spectrum of P1, there appeared

331

two characteristic double peaks of sugar H-1s at 5.21 (J = 3.9 Hz) and 4.29 ppm

332

(J = 7.9 Hz), implying an α-configuration and a β-configuration of sugar

333

moieties, respectively. The H-1 signal at 4.29 ppm was considered to belong to

334

the β-glucose residue of salidroside, while the H-1 signal at 5.21 ppm was predicted

335

to come from the newly added α-glucose residue by the enzyme. In HMBC, the cross

336

peak clearly existed between C-4′ (δ 76.53) of the sugar ring from the salidroside and

337

the H-1′′ (δ 5.21) of the newly added sugar moiety, conforming an α-1, 4 linkage

338

between the two sugar residues. Therefore, the structure of P1 was determined to be

339

α-1,4-glucosyl salidroside (Figure 5). The relevant NMR data were listed in Table S1.

340

The MS result of the product P2 showed peaks of [M+Na]+ ion at m/z 647.2164,

341

consistent with the molecular mass of diglucosyated salidroside (624). Three

342

characteristic double peaks of sugar H-1s were presented at 5.20 ppm (J = 3.9 Hz),

343

5.19 ppm (J = 3.9 Hz), and 4.27 ppm (J = 8.0 Hz), respectively, indicating that two 14


Page 15 of 27


344

α-linked glucose residues have been added to the β-glucose residue of salidroside.

345

Similarly, the MS result of P3 revealed the signal of [M+Na]+ ion at m/z 809.2716, in

346

agreement with the molecular mass of triglucosyated salidroside (786). In the 1 H NMR

347

spectrum, H-1s of the newly added α-glucose moieties showed characteristic

348

double peaks at 5.21 (J = 3.9 Hz), 5.20 ppm (J = 2.6 Hz), 5.19 ppm (J = 3.9 Hz),

349

while the signal of H-1 of the β-glucose residue of salidroside appeared at 4.27 ppm

350

(J = 8.0 Hz ). In the case of P4，MS analysis showed the signal of [M+Na]+ ion at m/z

351

971.3267, suggesting a tetraglucosyated salidroside (molecular mass: 948). In 1 H NMR

352

spectrum, there were five characteristic double peaks which located at 5.22 ppm (J

353

= 2.7 Hz), 5.21 ppm (J = 2.6 Hz), 5.20 ppm (J = 3.9 Hz), 5.19 ppm (J = 2.8 Hz), and

354

4.28 ppm (J = 8.0 Hz), respectively, indicating four α-glucose moieties added to the

355

β-glucose residue of salidroside. The MS analysis of P5 and P6 revealed signals of

356

[M+Na]+ ions at m/z 1133.3766 and 1295.4304, and [M+K]+ ions at m/z 1149.3529 and

357

1311.4082, respectively, conforming they were pentaglucosyated (molecular mass:

358

1110) and hexaglucosyated salidrosides (molecular mass: 1272). Based on all these

359

results, P2 to P6 were considered to be maltosyl, maltotriosyl, maltotetraosyl,

360

maltopentaosyl, and maltohexaosyl salidrosides that contained two to six α-1,4 glucose

361

residues, respectively (Figure 5).

362

Obviously, this enzyme was proved to be a powerful tool for one-pot, one-step

363

glycosylation of highly valuable compounds. It sequentially added glucose to the

364

salidroside and salidroside derivatives, exhibited strict regioselectivity and produced a

365

single glycoside product in each molecular mass, without isomer production. This

366

property was of great importance for the glycosylation as the isolation of the products

367

from the reaction mixture would become quite easy when without the isomer

368

interference. Additionally, the enzyme showed flexible acceptor specificity and could 15



Page 16 of 27

369

accommodate salidroside and various salidroside derivatives for glycosylation. The

370

monoglucosylated to hexaglucosyated salidrosides accounted for 40.3%, 25.2%, 17.4%,

371

8.5%, 5.4%, and 3.2%, respectively, in the total products. These attracting

372

α-glucosylated derivatives might combine functions of salidroside and

373

malto-oligosaccharides, and thus have promising application in food and

374

pharmaceutical industries in the future. Further work is currently underway to

375

investigate the bioactivity of these newly-synthesized compounds.

376

■ ASSOCIATED CONTENT

377

Supporting Information

378

Table S1 and Figure S1−S5, as noted in the text

379

■ AUTHOR INFORMATION

380

Corresponding Author

381

Email: [email protected]; [email protected]

382

ORCID

383

Lili Lu: 0000-0002-0191-1792

384

Funding

385

This work was supported by National Natural Science Foundation of China (No.21877044,

386

31872626, 31670062), Fundamental Research Funds for the Central Universities

387

(2018KFYYXJJ020).

388

Notes

389

The authors declare no competing financial interest.

390

REFERENCES

391

(1) Panossian, A.; Wikman, G.; Sarris, J. Rosen root (Rhodiola rosea): traditional use, chemical

392

composition, pharmacology and clinical efficacy. Phytomedicine. 2010, 17, 481-493.

393

(2) Anghelescu, I. G.; Edwards, D.; Seifritz, E.; Kasper, S. Stress management and the role of

394

Rhodiola rosea: a review. Int. J. Psychiatry Clin. Pract. 2018, doi:

395

10.1080/13651501.2017.1417442. [Epub ahead of print]

396 397 398

(3) Ma, L.; Cai, D.; Li, H.; Tong, B.; Song, L.; Wang, Y. Anti-fatigue effects of salidroside in mice. Journal of Medical Colleges of PLA. 2008, 23, 288-293. (4) Yang, S. J.; Yu, H. Y.; Kang, D. Y.; Ma, Z. Q.; Qu, R.; Fu, Q.; Ma, S. P. Antidepressant-like 16


Page 17 of 27


399

effects of salidroside on olfactory bulbectomy-induced pro-inflammatory cytokine production and

400

hyperactivity of HPA axis in rats. Pharmacol. Biochem. Behav. 2014, 124, 451-457.

401

(5) Yang, D. W.; Kang, O. H.; Lee, Y. S.; Han, S. H.; Lee, S. W.; Cha, S. W.; Seo, Y. S.; Mun, S. H.;

402

Gong, R.; Shin, D. W.; Kwon, D. Y. Anti-inflammatory effect of salidroside on

403

phorbol-12-myristate-13-acetate plus A23187-mediated inflammation in HMC-1 cells. Int. J. Mol.

404

Med. 2016, 38, 1864-1870.

405

(6) Ju, L.; Wen, X.; Wang, C.; Wei, Y.; Peng, Y.; Ding, Y.; Feng, L.; Shu, L. Salidroside, a natural

406

antioxidant, improves β-cell survival and function via activating AMPK pathway. Front.

407

Pharmacol. 2017, 8, 749.

408

(7) Kong, Y. H.; Xu, S. P. Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a

409

mouse model through suppression of inflammation and promotion of apoptosis. Oncol. Rep. 2018,

410

39, 2513-2526.

411

(8) Feng, J.; Niu, P.; Chen, K.; Wu, L.; Liu, T.; Xu, S.; Li, J.; Li, S.; Wang, W.; Lu, X.; Yu, Q.;Liu,

412

N.; Xu, L.; Wang, F.; Dai, W.; Xia, Y.; Fan, X.; Guo, C. Salidroside mediates apoptosis and

413

autophagy inhibition in concanavalin A-induced liver injury. Exp. Ther. Med. 2018, 15,

414

4599-4614.

415

(9) Chang, X.; Zhang, K.; Zhou, R.; Luo, F.; Zhu, L.; Gao, J.; He, H.; Wei, T.; Yan, T.; Ma, C.

416

Cardioprotective effects of salidroside on myocardial ischemia-reperfusion injury in coronary

417

artery occlusion-induced rats and Langendorff-perfused rat hearts. Int. J. Cardiol. 2016, 215,

418

532-544.

419

(10) Liu, H.; Lv, P.; Zhu, Y.; Wu, H.; Zhang, X. F.; Zheng, L.; Zhao, J. Salidroside promotes

420

peripheral nerve regeneration based on tissue engineering strategy using Schwann cells and

421

PLGA: in vitro and in vivo. Sci. Rep. 2017, 7, 39869.

422

(11) Biswal, S.; Barhwal, K. K.; Das, D.; Dhingra, R.; Dhingra, N.; Nag, T. C.; Hota, S. K. Salidroside

423

mediated stabilization of Bcl -xL prevents mitophagy in CA3 hippocampal neurons during

424

hypoxia. Neurobiol. Dis. 2018, 116, 39-52.

425

(12) Jiang, J.; Yin, H.; Wang, S.; Zhuang, Y.; Liu, S.; Liu, T.; Ma, Y. Metabolic engineering of

426

Saccharomyces cerevisiae for high-Level production of Salidroside from glucose. J. Agric. Food

427

Chem. 2018, 66, 4431-4438.

428

(13) Hu, Z.; Wang, Z.; Liu, Y.; Wu, Y.; Han, X.; Zheng, J.; Yan, X.; Wang, Y. Metabolite profile of

429

Salidroside in rats by ultraperformance liquid chromatography coupled with quadrupole

430

time-of-flight mass spectrometry and high-performance liquid chromatography coupled with

431

quadrupole-linear ion trap mass spectrometry. J. Agric. Food Chem. 2015, 63, 8999-9005.

432

(14) Shi, T. Y.; Feng, S. F.; Xing, J. H.; Wu, Y. M.; Li, X. Q.; Zhang, N.; Tian, Z.; Liu, S. B.; Zhao, M.

433

G. Neuroprotective effects of Salidroside and its analogue tyrosol galactoside against focal

434

cerebral ischemia in vivo and H2O2-induced neurotoxicity in vitro. Neurotox. Res. 2012, 21,

435

358-367.

436

(15) Yu, S.; Cheng, Q.; Li, L.; Liu, M.; Yang, Y.; Ding, F.

437

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell

438

and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of 17



439

neuronal glucose transporter 3. Toxicol. Appl. Pharmacol. 2014, 277, 259-269.

440

(16) Qi, T.; Gu, G.; Xu, L.; Xiao, M.; Lu, L. Efficient synthesis of tyrosol galactosides by the

441

β-galactosidase from Enterobacter cloacae B5. Appl. Microbiol. Biotechnol. 2017, 101,

442

4995-5003.

443

Page 18 of 27

(17) Yu, S.; Wei, L.; Chi, X.; Xu, H.; Ding, F.

444

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside, an analog of salidroside,

445

contributes to neuroprotection in cerebral ischemic injury in vitro and in vivo. Neuroreport. 2018,

446

29, 426-431.

447 448 449 450 451 452 453 454 455 456 457 458

(18) Thibodeaux, C. J.; Melançon, C. E.; Liu, H. W. Unusual sugar biosynthesis and natural product glycodiversification. Nature. 2007, 446, 1008-1016. (19) Williams, G. J.; Gantt, R. W.; Thorson, J. S. The impact of enzyme engineering upon natural product glycodiversification. Curr. Opin. Chem. Biol. 2008, 12, 556-564. (20) Hayes, M.R.; Pietruszka, J. Synthesis of glycosides by glycosynthases. Molecules. 2017, 22, pii: E1434. (21) Bojarová, P.; Kren, V. Glycosidases in carbohydrate synthesis: when organic chemistry falls short. Chimia (Aarau). 2011, 65, 65-70. (22) de Souza, P. M.; de Oliveira Magalhães, P. Application of microbial α-amylase in industry - A review. Braz. J. Microbiol. 2010, 41, 850-861. (23) Janeček, Š.; Svensson, B.; MacGregor, E. A. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases. Cellular and Molecular Life Sciences. 2014, 71, 1149-1170.

459

(24) Zhang, D.; Tu, T.; Wang, Y.; Li, Y.; Luo, X.; Zheng, F.; Wang, X.; Bai, Y.; Huang, H.; Su, X.;

460

Yao, B.; Zhang, T.; Luo, H. Improving the catalytic performance of a Talaromyces leycettanus

461

α-amylase by changing the linker Length. J. Agric. Food Chem. 2017, 65, 5041-5048.

462

(25) Jeon, H. Y.; Kim, N. R.; Lee, H. W.; Choi, H. J.; Choung, W .J.; Koo, Y. S.; Ko, D. S.; Shim, J. H.

463

Characterization of a novel maltose-forming α-amylase from Lactobacillus plantarum subsp.

464

plantarum ST-III. J. Agric. Food Chem. 2016, 64, 2307-2314.

465

(26) Pan, S.; Ding, N.; Ren, J.; Gu, Z.; Li, C.; Hong, Y.; Cheng, L.; Holler, T. P.; Li, Z.

466

Maltooligosaccharide-forming amylase: Characteristics, preparation, and application. Biotechnol.

467

Adv. 2017, 35, 619-632.

468

(27) Noguchi, A.; Inohara-Ochiai, M.; Ishibashi, N.; Fukami, H.; Nakayama, T.; Nakao, M. A novel

469

glucosylation enzyme: molecular cloning, expression, and characterization of Trichoderma viride

470

JCM22452 alpha-amylase and enzymatic synthesis of some flavonoid monoglucosides and

471

oligoglucosides. J. Agric. Food Chem. 2008, 56, 12016-12024.

472

(28) Sindhu, R.; Binod, P.; Madhavan, A.; Beevi, U. S.; Mathew, A. K.; Abraham, A.; Pandey, A.;

473

Kumar, V. Molecular improvements in microbial α-amylases for enhanced stability and catalytic

474

efficiency. Bioresour. Technol. 2017, 245, 1740-1748.

475

(29) Bano, S. U.; Qader, S. A.; Aman, A.; Syed, M. N.; Azhar, A. Purification and characterization of

476

novel α-amylase from Bacillus subtilis KIBGE HAS. AAPS PharmSciTech. 2011, 12, 255-261.

477

(30) Mehta, D.; Satyanarayana, T. Bacterial and archaeal α-Amylases: diversity and amelioration of

478

the desirable characteristics for industrial applications. Front. Microbiol. 2016, 7, 1129. 18


Page 19 of 27

479


(31) Konsoula, Z.; Liakopoulou-Kyriakides, M. Co-production of alpha-amylase and

480

beta-galactosidase by Bacillus subtilis in complex organic substrates. Bioresour. Technol. 2007,

481

98, 150-157.

482

(32) Lu, L.; Xu, X.; Gu, G.; Jin, L.; Xiao, M.; Wang, F. Synthesis of novel galactose containing

483

chemicals by beta-galactosidase from Enterobacter cloacae B5. Bioresour Technol. 2010, 101,

484

6868-6872.

485

(33) Guo, L.; Chen, X.; Xu, L.; Xiao, M.; Lu, L. Enzymatic synthesis of 6'-sialyllactose, a dominant

486

sialylated human milk oligosaccharide, by a novel exo-α-sialidase from Bacteroides fragilis

487

NCTC9343. Appl. Environ. Microbiol. 2018, 84, e00071-18.

488

(34) Nishimura, T.; Kometani, T.; Takii, H.; Terada, Y.; Okada, S. Glucosylation of caffeic acid with

489

Bacillus subtilis X-23 α-amylase and a description of the glucosides. J. Ferment. Bioeng. 1995, 80,

490

18-23.

491

19



492 493

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Figure captions Figure 1. SDS-PAGE analysis of the purification of α-amylase from B. subtilis

494

XL8. Lane 1, crude enzyme from extracellular supernatant; lane 2, 60%(NH4)2SO4

495

precipitation followed by desalting; lane 3, DEAE Fast Flow chromatography; lane 4,

496

Source 15Q chromatography; lane 5, Sephadex G 200 chromatography; M, marker

497

proteins.

498

Figure 2. Biochemical properties of the α-amylase from B. subtilis XL8. (a) Effect

499

of pH on the activity (red triangle) and stability (blue cycle) of the enzyme; (b) Effect of

500

temperature on the activity (red triangle) and stability (blue cycle) of the enzyme; (c)

501

Effect of metal ions and chemical reagents on the activity of the enzyme. Data points

502

represent the means ± S.D. of three replicates.

503

Figure 3. TLC (a) and HPLC (b) analysis of the transglycosylation reaction

504

catalyzed by the α-amylase from B. subtilis XL8. (a) Lane 1, salidroside; lane 2, control

505

reaction containing salidroside and α-amylase; lane 3, control reaction containing starch

506

and α-amylase; lane 4, control reaction containing starch, salidroside and inactivated

507

α-amylase; lane 5, reaction containing salidroside, starch and α-amylase. (b) The

508

remaining time of salidroside is 5.0 min. The peaks at 6.0 min, 7.5 min, 9.7 min, 13.2

509

min, 18.5 min, and 25.8 min are signals of salidroside derivatives.

510

Figure 4. The effects of reaction conditions on the production of salidroside

511

derivatives by the α-amylase from B. subtilis XL8. (a) Soluble starch concentration; (b)

512

Salidroside concentration; (c) Reaction pH; (d) Reaction temperature; (e) Reaction time.

513

The total yield of the product was defined as the ratio of the concentration of the

514

synthesized glycoside product (mM) to the initial concentration of salidroside (mM).

515

Data points represent the means ± S.D. of three replicates.

516 517

Figure 5. Outlines of the salidroside glycosylation by α-amylase as well as the chemical structures of the salidroside derivative products.

518

20


Page 21 of 27


Table 1 Purification of the α-amylase from B. subtilis XL8 Purification step

Total protein

Total activity

Specific activity

Yield

(mg)

(U)

(U/mg)

(%)

Crude enzyme

6611.6

16002.5

2.4

100

60%(NH4)2SO4

862.4

10276.9

11.9

64.2

DEAE FF

28.7

1621.0

56.5

10.1

Source 15Q

11.8

1049.5

88.9

6.6

Sephadex G200

1.5

789.7

526.5

4.9

21



Page 22 of 27

Figure 1

22


Page 23 of 27


Figure 2

23



Page 24 of 27

Figure 3

24


Page 25 of 27


Figure 4

25



Page 26 of 27

Figure 5

26


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TOC Graphic


Enzymatic glucosylation of salidroside from starch by Î±-amylase

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