Enzymatic Production of Melibiose from Raffinose by the

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Enzymatic production of melibiose from raffinose by the levansucrase from Leuconostoc mesenteroides B-512 FMC Wei Xu, Shuhuai Yu, Qian Liu, Tao Zhang, Bo Jiang, and Wanmeng Mu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

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Journal of Agricultural and Food Chemistry

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Enzymatic production of melibiose from raffinose by the

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levansucrase from Leuconostoc mesenteroides B-512

3

FMC

4

Wei Xu †, Shuhuai Yu †, Qian Liu †, Tao Zhang †, Bo Jiang †,§, Wanmeng Mu* †,§

5 6 7

†

Jiangsu, 214122, China.

8 9

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

§

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.

10 11 12

*

13

Address: State Key Laboratory of Food Science and Technology, Jiangnan University,

14

Wuxi, Jiangsu, 214122, P. R. China.

15

Tel: (86) 510-85919161. Fax: (86) 510-85919161.

16

Email address: [email protected]

Corresponding author.

17

1

ACS Paragon Plus Environment


18

ABSTRACT: Melibiose, which is an important reducing disaccharide, formed by

19

α-1,6 linkage between galactose and glucose, has been proven to have beneficial

20

applications in both medicine and agriculture. In this study, a characterized

21

levansucrase from Leuconostoc mesenteroides B-512 FMC was further used to study

22

the bioproduction of melibiose from raffinose. The reaction conditions were

23

optimized for melibiose synthesis. The optimal pH, temperature, substrate

24

concentration, ratio of substrates and units of enzymes were determined as pH 6.0,

25

45 °C, 210 g/L, 1:1 (210 g/L : 210 g/L) and 5 U/mL respectively. The

26

transfructosylation product of raffinose was determined to be melibiose by FTIR and

27

NMR. A high raffinose concentration was found to strongly favor the production of

28

melibiose. When 210 g/L raffinose and 210 g/L lactose were catalyzed using 5 U/mL

29

purified levansucrase at pH 6.0 and 45 °C, the maximal yield of melibiose was 88

30

g/L.

31

KEYWORDS: Levansucrase · Leuconostoc mesenteroides · Transfructosylation ·

32

Melibiose ·

2


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INTRODUCTION

34

Melibiose (α-D-galactopyranosyl-(1→6)-α-D-glucopyranoside) is a reducing

35

disaccharide composed of galactose and glucose with α-1,6 linkage. More and more

36

attention has been paid to melibiose for its benefical attributes.1 It promotes the

37

calcium absorption in intestines,2 and helps to cure atopic dermatitis.3 It is an

38

indigestible disaccharide because humans lack α-galactosidase,4 and the appropriate

39

intake of melibiose increases the growth of bifidobacteria and improves the condition

40

of stool in healthy humans.5 Dietary melibiose effectively suppresses the Th2

41

response and improves the induction of oral tolerance.6 Particularly, melibiose can be

42

used as a high-value additive to human functional foods and pharmaceuticals to

43

maintain and promote good health because of its strong ability to function as a

44

prebiotic.7

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Difficult to obtain by other methods, melibiose is mainly produced through an

46

enzymatic transglycosylation reaction with raffinose and lactose or galactose as

47

substrates (Fig. 1). Among these enzymes, β-fructofuranosidase (also called invertase,

48

EC 3.2.1.26), a member of glycoside hydrolase family 68 (GH68) that catalyzes the

49

hydrolysis and transfructosylation of sucrose, has been reported to catalyze

50

hydrolysis

51

(α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside)

52

produce melibiose.8 A commercial lactase named Lactozyme 3000L (Novozymes,

53

produced

54

β-fructofuranosidase activity and converts raffinose to melibiose.9

of

by

Klyuveromyces

lactis)

has

the

raffinose

also

been

found

to

to

possess

55

In addition to those enzymes described above, levansucrase (EC 2.4.1.10, sucrose:

56

2,6-β-D-fructan 6-β-D-fructosyltransferase), which is also a member of GH68 that

57

catalyzed the formation of levan and fructooligosaccharides (FOS) from sucrose, 3



58

could also catalyze the synthesis of melibiose from raffinose10. The crystal structure

59

of levansucrase complexed with sucrose obtained from Bacillus subtilis was the

60

earliest crystal structure resolved and reported across the entire GH68 family,11 which

61

was followed by the resolutions of the 3D structure of levansucrase from

62

Gluconacetobacter diazotrophicus,12 Bacillus megaterium,13 and Erwinia anylovora.14

63

Up to now, more and more research on the levansucrase protein engineering has been

64

sprang up based on the crystal structures, including improving the specificity and

65

enhancing the thermostabiliy, what’s more, these 3D structures could been utilized as

66

template for the modelling of levansucrase in this study through SWISS-MODEL

67

online software (https://www.swissmodel.expasy.org/).

68

Levansucrase catalyzes three distinct reactions depending on the presence of

69

different fructosyl acceptor molecules, including polymerization, transfructosylation,

70

and hydrolysis.15 The growing fructan can be used as a fructosyl acceptor to perform

71

the synthesis, and monosaccharides, disaccharides, or oligosaccharides and water

72

serve as acceptors in transfructosylation and hydrolysis respectively.16 For example,

73

the levansucrases from Microbacterium laevaniformans

74

19, 20

75

produce levan and melibiose from raffinose through polymerization, and the Bacillus

76

subtilis levansucrase produces melibiose by transfructosylation with raffinose as

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substrate 21. That’s to say, levansucrase could played an important role in synthesizing

78

kinds of fructo-oligosaccharide according to different fructosyl acceptors, including

79

levan (β-2,6 fructan)，melibiose，raffinose，lactosucrose and so on. However, the costs

80

of enzymatically producing melibiose from raffinose through hydrolysis are too

81

high,22, 23 and while the proposed solution of using whole-cell catalytic technology

82

instead of enzymatic hydrolysis can reduce the cost, detailed knowledge of the

17, 18

and Zymomonas mobilis

not only effectively hydrolyze raffinose to melibiose and fructose but also

4


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reaction mechanism of whole-cell catalysis and effective methods to evaluate

84

production by the strain remained unclear.7

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Leuconostoc mesenteroides, belonging to lactobacillaceae, is a Gram-positive,

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facultative anaerobe, chemoorganotroph and catenation-shaped bacterium. Many

87

leuconostoc strains can produce polymers, such as dextrans and levans, which have a

88

wide variety of commercial applications, such as dextransucrase from Leuconostoc

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mesenteroides

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alternansucrases from Leuconostoc mesenteroides B-1355,26 and levansucrase from

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Leuconostoc mesenteroides B-512 FMC.11

NRRL

B-512F24 and

Leuconostoc

mesenteroides

IBT-PQ,25

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The levansucrase from Leuconostoc mesenteroides B-512 FMC has been reported

93

to catalyze the biosynthesis of levan and lactosucrose.11,27 Our recent work found the

94

recombinant enzyme showed high melibiose-producing ability. In this study, we

95

investigated the synthesis of melibiose from raffinose by the levansucrase, via two

96

methods:

97

transfructosylation by using lactose as fructosyl acceptor and sucrose as fructosyl

98

donor. Also, we investigated the optimum conditions (substrates concentration, pH,

99

temperature, enzyme amount) for melibiose biosynthesis. To our best knowledge, it

100

was the first time to report the melibiose production through transfructosylation by

101

levansucrase.

hydrolysis

and

transfructosylation.

We

mainly

focused

on

the

102 103

MATERIALS AND METHODS

104

Chemicals and Reagents. Raffinose and melibiose were purchased from Sigma (St

105

Louis, MO, USA) for high-performance liquid chromatography (HPLC) analysis. The

106

acetonitrile (HPLC grade) was obtained from Tedia Co. Inc. (Fairfield, OH, USA).

107

Yeast extract, tryptone, Isopropyl-β-D-1-thiogalactopyranosid (IPTG), ampicillin, and 5



108

other chemicals of analytical grade were purchased from Sangon Biological

109

Engineering Technology & Services Co. Ltd. (Shanghai, China). The E. coli DH5α

110

and E. coli BL 21(DE3) were used as host cells for cloning and expression of the

111

target gene, respectively.

112 113

Heterologous Expression of L. mesenteroides Levansucrase in E. coli. In this

114

article, the full length of the levansucrase-encoding gene, 2418 bp (GenBank

115

accession No. AY665464) was synthesized by Shanghai Generay Biotech Co., Ltd.

116

(Shanghai, China). The commercially synthesized gene was designed to contain a

117

C-terminal in-frame 6×histidine-tag sequence with two restriction sites NdeI and XhoI

118

at the 5’- and 3’- terminus, respectively, and the gene was subcloned into the

119

expression vector pET-22b(+), generating the recombinant plasmid, namely,

120

pET-Leme-Lev. The recombinant plasmid was transformed into the host E. coli BL21

121

(DE3) for heterologous expression. The E. coli BL21 (DE3) containing recombinant

122

plasmid was cultivated in Luria-Bertani (LB) broth (consisting of 10 g/L tryptone, 5

123

g/L yeast extract and 10 g/L sodium chloride at pH 7.0) supplemented with 50 µg/mL

124

ampicillin in a rotary shaker at 37 °C and 200 rpm. When the optical density OD600

125

reached 0.6-0.8, IPTG was added at a 1 mM concentration to induce the levansucrase

126

expression at 20 °C for 16 h.

127 128

Preparation of the Recombinant L. mesenteroides Levansucrase. The cells were

129

harvested by centrifugation (10,000 × g, 20 min, 4 °C), the pellets were washed twice

130

with 50 mM phosphate buffer containing 100 mM NaCl (pH 7.0) and subsequently

131

disrupted by sonication using a Vibra-CellTM72405 Sonicator (BioBlock Scientific,

132

Illkirch, France) for 15 min (1 s sonication with 2 s breaks). The purification steps 6


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were similar as in the previous study 27. Besides, Coomassie Brilliant Blue R250 was

134

used for protein staining. Protein concentration was determined by the method of

135

Lowry 28 using bovine serum albumin as a standard.

136 137

Measurement of Enzyme Activity. Both hydrolysis and transfructosylation activity

138

were determined. To determine the hydrolytic activity, the reactions were performed

139

in 1 L mixture containing 200 mM raffinose and 5 µg/mL purified enzyme at pH 6.0

140

and 45 °C for 20 min and terminated by boiling for 10 min. One unit of hydrolysis

141

activity was defined as the amount of enzyme that released 1 µmol of melibiose from

142

raffinose per minute. The reaction conditions to determine the rate of

143

transfructosylation were the same as those used above but in presence of 200 mM

144

lactose or galactose as fructosyl acceptor, and one unit of transfructoslyation activity

145

was defined as the amount of enzyme releasing 1 µmol melibiose by

146

transfructosylation. In this article, the quantification of enzyme activity was based

147

primarily on the raffinose hydrolysis activity27. Actually various concentrations (from

148

4 to 50 mM) of substrates (sucrose and raffinose) were used to measure the kinetic

149

parameters of L. mesenteroides levansucrase hydrolysis. The enzyme reactions were

150

performed at 45 °C in 50 mM sodium acetate buffer (pH 6.0). The kinetic parameters,

151

Michaelis-Menten constant (Km), turnover number (kcat) and catalytic efficiency

152

(kcat/Km) for substrates were determined by fitting the data to the Lineweaver-Burk

153

plot.

154 155

HPLC Analysis. Quantitative determination of raffinose, lactose, galactose and the

156

reaction products were conducted by HPLC (Agilent 1260, CA, USA) equipped with

157

a refractive index detector and a column of Asahipak NH2P-50-4E (Shodex, Tokyo, 7



158

Japan, 4.6 mm id × 250 mm). External standard method was applied to quantify the

159

carbohydrates by calculating different peak areas of the stand reference and sample. A

160

solution of 75% (V/V) acetonitrile was used as the mobile phase with an elution rate

161

of 1 mL/min at 35 °C. The reaction products were purified and collected by

162

preparative HPLC (Waters 1525, MA, USA) with a refractive index detector and a

163

preparative XBridgeTM Prep Amide column (5 µm, 10 mm id × 250 mm, Waters, MA,

164

USA).

165 166

Fourier-Transform Infrared (FTIR) Spectroscopy. The FTIR spectroscopy analysis

167

was performed to determine the functional groups in melibiose. A small sample was

168

mixed with KBr, ground thoroughly, and then pressed into a 1 mm pellet. The FTIR

169

spectra of the dried sample film were recorded over a wavenumber range of 4000 -

170

400 cm-1 on a Thermo Nicolet NEXUS 470 FT-IR (Thermo Fisher Scientific, USA),

171

while in the internal reflectance (Attenuated Total Reflectance, ATR) mode. The

172

resolution for scanning the spectrum was set as 4 cm-1.

173 174

Nuclear Magnetic Resonance (NMR) Measurement. The sugars prepared for NMR

175

analysis were obtained by concentration and lyophilization. The products in

176

lyophilized powder form (~20 mg) were dissolved in D2O at 30 °C and then subjected

177

to NMR. The 1H NMR and

178

400 MHz Digital NMR Spectrometer (Bruker, Karlsruhe, Germany) in order to

179

determine the structures. The chemical shifts were determined with respect to the

180

signals for sodium 4,4 -dimethyl-4-silapentane-1-sulfonate (DSS) (δH = δC = 0.00

181

ppm) as the internal reference standard.

182

Production of melibiose from raffinose hydrolysis reaction. For melibiose

13

C NMR spectra were recorded by a Bruker Avance III

8


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production by hydrolysis reaction of raffinose, the reaction was carried out at pH 6.0

184

and 45 °C in 1 L solution that composed of 210 g/L raffinose and 5 U/mL purified

185

levansucrase The amount of the products was determined by high-performance liquid

186

chromatography (HPLC) at time intervals as described above.

187

Effect of pH and Temperature on the Melibiose Bioproduction. Three buffer

188

systems (50 mM), including sodium acetate buffer (pH 4.0 - 6.0), sodium phosphate

189

buffer (pH 6.0 - 7.5), and Tris-HCl buffer (pH 7.5 - 9.0), were used to study the

190

influence of pH on the melibiose production at 45 °C.

191

The effect of temperature was investigated in sodium phosphate buffer (50 mM, pH

192

6.0) by measuring the melibiose production at the temperatures ranging from 30 -

193

70 °C. All reactions were performed with 10 U/mL (hydrolysis activity) of the

194

purified recombinant levansucrase containing 210 g/L raffinose and 210 g/L lactose

195

for 1 h in 1 L reaction volume.

196 197

Effect of Substrate Concentration and Ratio on Melibiose Production. To study

198

the effect of substrate concentration, different concentrations (3%, 6%, 9%, 12%, 15%,

199

18%, 21%, 24%, 27%, and 30%, W/V) of both raffinose and lactose (equal

200

concentration) were used to investigate the melibiose production. To determine the

201

effect of substrate ratio, the ratios of raffinose (W/V) to lactose (W/V) were set as 6%:

202

21%, 12%: 21%, 18%: 21%, 21%: 21%, 24%: 21%, 21%: 24%, 21%: 18%, 21%:

203

12%, and 21%: 6%. All reactions were performed with 10 U/mL (hydrolysis activity)

204

of the recombinant levansucrase at pH 6.5 and 45 °C for 1 h.

205 206

Effect of Enzyme Amount on Melibiose Bioproduction. The reaction mixtures were

207

prepared with 210 g/L raffinose and 210 g/L lactose and enzyme amounts varying 9



208

from 2 to 10 U/mL (hydrolysis activity). All reactions were performed at pH 6.0 and

209

45 °C in 1 L volume for 1 h .

210 211

Production of Melibiose by Raffinose Transfructosylation with Galactose as

212

Acceptor.

213

For melibiose production by using galactose as fructosyl acceptor, the reaction was

214

carried out at pH 6.0 and 45 °C in 1 L solution that composed of 210 g/L raffinose and

215

210 g/L (W/V) galactose, and 5 U/mL purified levansucrase. The amount of the

216

products was determined by high-performance liquid chromatography (HPLC) at time

217

intervals.

218 219

Production of Melibiose from Raffinose by L. mesenteroides Levansucrase Under

220

Optimized Conditions. For melibiose production by transfructosylation, the reaction

221

was carried out at pH 6.0 and 45 °C in 1 L solution that composed of 210 g/L

222

raffinose and 210 g/L (W/V) lactose, and 5 U/mL purified levansucrase. The amount

223

of the products was determined by high-performance liquid chromatography (HPLC)

224

at time intervals as described above.

225 226

RESULTS AND DISCUSSIONS

227

Purification of the Recombinant L. mesenteroides Levansucrase. To the best of our

228

knowledge, there has not been any research published on the bioproduction of

229

melibiose from raffinose by the recombinant L. mesenteroides levansucrase, although

230

much attention has been focused on the ability of this levansucrase to produce

231

fructooligosaccharides and levan from sucrose. In this study, the levansucrase-

232

encoding gene was successfully cloned and expressed in E. coli, and the recombinant 10


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Leme-levansucrase was purified to homogeneity. The molecular mass of the protein

234

subunit was estimated to be 45 kDa based on SDS-PAGE analysis, which was

235

consistent with the results reported in a previous work.11

236 237

Determination of Transfructosyl Oligosaccharide Production from Raffinose and

238

Lactose. Using raffinose as asubstrate, and lactose as fructosyl acceptor, the

239

recombinant L. mesenteroides levansucrase produced melibiose as the main product in

240

addition to lactosucrose and fructose (Fig. S1), and the fructose production was

241

significantly lower than the production of lactosucrose, thus indicating that the

242

predominant reaction during the process was transfructosylation.

243 244

FTIR result. To identify the melibiose, the structure of melibiose was determined by

245

both FTIR and NMR measurement. The FTIR spectrum (Fig. S2A) displayed two

246

typical bands at approximately 3,346 and 2,933 cm-1 that represent O-H and C-H

247

stretching, respectively.29 Typically, the O-H stretching vibration was observed within

248

the wavenumber range of 3,600 - 3,200 cm-1, and the broad and pure peak at 3,346

249

cm-1 resulted from the intermolecular hydrogen bonding.30 A shoulder peak at 2,933

250

cm−1 was attributed to alkane C-H stretch. In addition, the spectrum displayed a peak

251

at 1,645 cm−1, which was due to C-O stretching31. Furthermore, the FTIR spectrum of

252

the product featured a strong absorption at 840 cm−1 indicating the presence of α-type

253

glycosidic linkages of melibiose.

254 13

C NMR (Fig. S2B) and 1H NMR

255

NMR result. In addition to FTIR spectrum,

256

spectra (Fig. S2C) of the melibiose were also measured. The chemical shifts of

257

NMR spectra for the melibiose was listed in Table. 1. The chemical shifts of melibiose 11


13

C


Page 12 of 40

258

were compared to the results of melibiose reported in previous studies.32 As we

259

predicted, the glucose moiety of the melibiose included α- and β- configurations, and

260

there was a little difference in the chemical shifts of these two kinds of glucose.33 In

261

this study, the

262

groups of anomeric carbons (ߜC, 98.24, 92.24 ppm and ߜC, 98.24, 96.12 ppm) with a

263

large downfield (Fig. S2B) and two groups of protons on anomeric sugar carbons (ߜH,

264

5.01, 5.27 ppm and ߜH, 5.01, 4.68 ppm) (Fig. S2C). The chemical shift comparison

265

between the reaction product and the reported melibiose showed a very high similarity.

266

In addition, the 13C and 1H NMR analyses of standard melibiose were also performed

267

and showed the same spectra as those produced by recombinant L. mesenteroides

268

levansucrase from raffinose. Thus, the transfructosyl oligosaccharide produced by

269

levansucrase

270

(α-D-galactopyranosyl-(1→6)-α-D-glucopyranoside).

13

C NMR spectrum indeed revealed 18 carbon molecules with two

from

raffinose

was

identified

as

melibiose

271 272

In addition, lactosucrose could also be formed during the transfructoslyation when

273

raffinose and lactose were used as substrates. Compared to previous reports about the

274

lactosucrose chemical shift,

275

produced by levansucrase from raffinose and lactose was identified as lactosucrose.

18,27, 34

an additional transfructosyl oligosaccharide

276 277

Effect of pH on Melibiose Production. The effect of pH on melibiose production

278

was investigated at 40 °C and pH at values ranging from 4.0 to 9.0. (Fig. 2A).

279

Maximal amounts of melibiose were achieved at pH 6.0, and the purified recombinant

280

L. mesenteroides levansucrase could effectively catalyze the bioproduction of

281

melibiose at a pH ranging from 4.5 to 6.5, but the production was dramatically

282

reduced at pH values of 7 - 9. By comparison, the enzyme showed relatively high 12


Page 13 of 40


283

hydrolysis activity over a wide range of pHs from 5.5 to 9.0. The enzyme exhibited a

284

more dominant transfructosylation at pH 5.5- 6.0 and a more dominant hydrolyzation

285

when the pH was less than 5.5 or more than 6.0. As reported previously, many

286

levansucrases displayed optimal activity at slightly pHs values 5.0 to 6.5, for example,

287

the levansucrase from Bacillus licheniformis RN-01 (pH 6.5),35 Bacillus sp. TH4-2

288

(pH 6.0),36 E. amylovora ATCC 49946 (pH 6.5),18 Pseudomonas syringae pv.

289

phaseolicola (pH 5.0-7.0),37 Bacillus megaterium (pH 6.6)

290

sanfranciscensis TMW 1.392 (pH 5.4).39

38

and Lactobacillus

291 292

Effect of Temperature on Melibiose Production. The effect of temperature on the

293

production of melibiose was another important factor. As a general rule, increased

294

temperature is favorable to increase the substrate solubility and improves the reaction

295

rate,40 but a lower temperature was reported to be more suitable for polymerization of

296

levansucrase.41 The optimal temperature for melibiose production was determined to

297

be 45 °C (Fig. 2B), and the bioproduction of melibiose steadily increase at

298

temperature ranging from 30 - 45 °C. However, the production of lactosucrose

299

decreased more dramatically than that of melibiose when the temperature was greater

300

than 50 °C, owing to the fact that lactosucrose was produced exclusively from the

301

transfructosylation, while melibiose was produced not only by transfructosylation but

302

also by hydrolysis of raffinose. The concentration of fructose, which was only

303

produced by hydrolysis, showed a steady increase when the temperature ranged from

304

30 to 55 °C and dropped when the temperature was higher than 55 °C, indicating the

305

optimal temperature for hydrolysis was 55 °C. These results were consistent with the

306

finding from other sources of levansucrase that indicated that lower temperature

307

favors the polymerization while higher temperature favors the hydrolysis reaction. By 13



Page 14 of 40

308

comparison, the Z. mobilis levansucrase showed the greatest amounts of sucrose

309

hydrolysis and levan formation at 50 and 30 °C,17 and the optimal temperature of

310

Bacillus

311

transfructosylation were 30 and 4 °C,42 and the enzyme from M. laevaniformans

312

ATCC 15953 exhibited the highest activites of hydrolysis and transfructosylation at 45

313

and 30 °C,17 respectively. Moreover, the levansucrase from G. stearothermophilus

314

and Bacillus sp. TH4-236 display higher levels of thermostability with their maximal

315

transfructosylation activities at 57 °C and 60 °C, respectively, which were the two

316

thermostable levansucrase enzymes reported up to this date.

amyloliquefaciens

levansucrase

for

sucrose

hydrolysis

and

43

317 318

Production of Melibiose from Raffinose Hydrolysis. Enzymatic production of

319

melibiose by raffinose hydrolysis was studied in 1 L solutions. The results clearly

320

demonstrated that the production of melibiose steadily increased with longer reaction

321

times, and the molar ratio of melibiose to fructose produced consistently remained at

322

approximately 1 : 1 (Fig. 3), and the chromatography trace did not show any other

323

obvious peaks besides the melibiose and fructose (Fig. 3), indicating that the

324

hydrolysis reaction primarily occurred in the presence of raffinose as the sole

325

substrate. Except for fructose, the only hydrolysis product identified by NMR was

326

α-D-galactopyranosyl-(1→6)-α-D-glucopyranoside, i.e. melibiose (Table 1). After a

327

reaction time of 150 min, 138 ± 6.17 mM melibiose was produced from 300 mM

328

raffinose.

329 330

Production of Melibiose by Raffinose Transfructosylation with Lactose as

331

Acceptor. It was previously reported that L. mesenteroides levansucrase displayed

332

high transfructosylation activity using sucrose in the presence of a high concentration 14


Page 15 of 40


333

of lactose and that the specific transfructosylation activity was much higher than the

334

sucrose hydrolysis activity.28 Thus, this study also determined the transfructosylation

335

of L. mesenteroides levansucrase with raffinose. Melibiose production by

336

transfructosylation was studied in 1 L solution containing 210 g/L raffinose and 210

337

g/L lactose at pH 6.0 and 45 °C, and the results suggested that the enzyme could

338

utilize raffinose as an effective fructosyl donor in addition to its ability to utilize

339

sucrose. When raffinose and lactose were used as the fructosyl donor and acceptor, the

340

transfructosylation activity was 285 ± 14 U/mg at pH 6.0 and 45 °C, which was much

341

higher than the specific raffinose hydrolysis activity (195 ± 16 U/mg) described above,

342

indicating that the enzyme preferred to catalyze the transfructosylation in the presence

343

of a fructosyl acceptor.

344 345

Effect of Substrate Concentration and Ratio on Melibiose Production. Several

346

concentrations of raffinose were used to investigate the optimal production of

347

melibiose, and the total substrate concentration varied from 3% to 30%. As shown in

348

Fig. 4A, when each substrate was added at 3% (W/V) up to 21%, the melibiose yield

349

reached 78.2 g/L, which was the highest yield observed, and the production of

350

lactosucrose was 67.7 g/L. With the increase of total substrate concentration, the

351

content of melibiose produced out of the total sugar increased from 1.4 g/L to 78.2

352

g/L, and the concentration remained at a relative stable level up to the substrate

353

concentration of 30%. In contrast, the greatest amount of fructose production occurred

354

with a substrate concentration of 18% and decreased to an almost undetectable level

355

when the total substrate concentration was more than 30%. Thus, a high concentration

356

of substrate favored the fructosyl transfer reaction to produce melibiose, and a similar

357

phenomenon was reported in the production of raffinose production from melibiose 15



358

and sucrose, which was due to the fact that a high sugar concentration resulted a low

359

activity of water, may indirectly inhibit di- or oligosaccharide hydrolysis and promote

360

the fructosyl transfer activity 34. By comparison, the effect of substrate concentration

361

on the yield and rate of the transfer and hydrolysis reactions were also explored in a

362

study using levansucrase from Paenibacillus polymyxa, which proposed that the large

363

excesses of both the acceptor and the donor glycosides at high concentrations could

364

promote an efficient transglycosylation reaction by levansucrase by competing with

365

water for the fructosyl-enzyme intermediate, similar to the transgalactosylation

366

reaction by α-galactosidase.40, 44

367

The effect of the ratio of raffinose and lactose on the melibiose production was also

368

studied, and it was found that the highest melibiose production was realized by using

369

210 g/L raffinose and 210 g/L lactose (Fig. 4B). We kept lactose concentration

370

unchanged at 21% (W/V), and varied raffinose concentration from 6% to 24% (W/V),

371

founding the increase of raffinose concentration resulted in increased melibiose

372

production, and the maximal melibiose production was observed at 210 g/L raffinose.

373

Lactose concentration had a similar influence on melibiose production when the

374

raffinose concentration was fixed at 21% (W/V). Therefore, both fructosyl donor and

375

acceptor concentrations had significant effects on melibiose production, and in all the

376

tests the highest melibiose production (approximately 75 g/L) was gained at 21% / 21%

377

(W/V) of raffinose / lactose. This result was similar to the lactulose production

378

reported by β-galactosidase in permeabilized cells of Kluyveromyces lactis, which

379

suggested that with equimolar concentrations of lactose and fructose, i.e., 40:20

380

(W/V, %), could result in maximal lactulose production.45

381 382

Effect of Enzyme Amount on Melibiose Production. The effect of enzyme amount 16


Page 16 of 40

Page 17 of 40


383

on the melibiose production was studied by the recombinant levansucrase from 210

384

g/L raffinose and 210 g/L lactose at pH 6.0 and 45 °C (Fig. 5). The production of

385

melibiose was steadily increased when increasing the enzyme dosage from 2 U/mL to

386

5 U/mL, but the fructose production did not increase, meaning that the improvement

387

of transfructosylation activity was higher than that of sucrose hydrolysis activity using

388

this amount of enzyme. In addition, the highest yield of melibiose occurred at 5 U/mL,

389

but the melibiose production dropped more slowly than the lactosucorse production

390

when more than 5 U/mL of enzyme was added. However, the change in the release of

391

fructose was negligible during the whole reaction, indicating that the sucrose

392

hydrolysis activity consistently increased.

393 394

Biological Production of Melibiose from Raffionse and Lactose Under Optimal

395

Conditions. Melibiose biosynthesis under optimized conditions was investigated

396

where the pH, temperature, ratio of raffinose and lactose, and the amount of enzyme

397

were pH 6.0, 45 °C, 210 g/L raffinose, 210 g/L lactose, and 5 U/mL, respectively (Fig.

398

6). The production of melibiose and lactocucrose increased quickly in the first 30 min,

399

and the highest production level of melibiose 88 g/L was obtained when the reaction

400

reached an equilibrium with a ratio of melibiose to raffinose of 49.0%. During the

401

whole reaction process, there was no obvious change in the fructose production,

402

which demonstrated the consistent occurrence of hydrolysis. The synthesis of

403

raffinose by levansucrase from Clostridium arbusti SL20646 was reported to have a

404

50 % conversion ratio from 240 g/L sucrose and 240 g/L lactose, and B. subtilis

405

KCCM 32835 levansucrase could produce 183 g/L lacotsucrose from 225 g/L sucrose

406

and 225 g/L lactose in whole cell form.16 Herein, the purified recombinant L.

407

mesenteroides levansucrase produced 88 g/L melibiose from 210 g/L raffinose and 17



408

210 g/L lactose, showing a significantly competitive productivity. Therefore, it was

409

suggested that L. mesenteroides levansucrase could be used as a good producer of

410

melibiose.

411 412

Kinetic Comparison of Raffinose Hydrolysis and Sucrose Hydrolysis. At pH 6.0

413

and 45 °C, the specific activities of L. mesenteroides levansucrase were calculated to

414

be 469 ± 23 and 195 ± 16 U/mg for the hydrolysis of sucrose and raffinose,

415

respectively (Table 2). The kinetic parameters toward both raffinose and sucrose

416

hydrolysis were measured and compared (Table 2). The Michaelis-Menten constant

417

(Km) for sucrose and raffinose were measured to be 25.66 ± 1.21 and 56.82 ± 1.56

418

mM, respectively. The catalytic efficiency (kcat/Km) for sucrose was measured to be

419

2,901 ± 26 mM-1 min-1, which was much higher that of raffinose (112 ± 8 mM-1

420

min-1).

421 422 423 424 425

Production of Melibiose by Raffinose Transfructosylation with Galactose as

426

Acceptor. Melibiose production by transfructosylation from raffinose was also

427

studied using galactose as acceptor in 1 L solution containing 210 g/L raffinose and

428

210 g/L galactose (Fig. 7). The transfructosyl product of galactose was determined to

429

be O-α-D-galactopyranosyl-(1→2)-β-D-fructofuranoside (Gal-α-1,2-Fru) by NMR

430

(Table 1). At pH 6.0 and 45 °C, the transfructosylation activity with galactose as

431

acceptor was measured to be 298 ± 12 U/mg, slightly higher than that with lactose as

432

acceptor, indicating that galactose was a better acceptor than lactose. The 18


Page 18 of 40

Page 19 of 40


433

concentration of melibiose and Gal-α-1,2-Fru reached 188 ± 5.04 and 136 ± 7.1 mM,

434

respectively, after reacting for 150 min. The biocatalysis process exhibited a relatively

435

high rate of hydrolysis reaction because the production of fructose was much higher

436

than that during transfructosylation with lactose as acceptor. Gal-α-1,2-Fru is a isomer

437

of

438

O-α-D-galactopyranosyl-(1→1)-β-D-fructofuranoside (1-lactulose). The final two

439

isomers exhibit a high potential to serve as prebiotics in food industry. This study

440

provides the first report of the efficient production of Gal-α-1,2-Fru using

441

transfructosylation of raffinose and galactose.

O-β-D-galactopyranosyl-(1→4)-β-D-

fructofuranoside

(lactulose)

and

442

In this study, the recombinant L. mesenteroides levansucrase was purified and

443

studied to examine its production of melibiose from raffinose. Based on the FTIR and

444

NMR measurements, the transfructosylation product by the recombinant L.

445

mesenteroides levansucrase was determined to be melibiose. Both lactose and

446

galactose could be used as effective fructosyl acceptors for melibiose production by

447

transfructosylation from raffinose, and the byproducts were determined to be

448

lactosucrose and Gal-α-1,2-Fru (a lactulose isomer) respectively. A high concentration

449

of raffinose significantly favored the transfructoslyation, and the enzyme produced 88

450

g/L melibiose and 104 g/L lactosucrose from 210 g/L raffinose and 210 g/L lactose

451

after reaction at pH 6.0 and 45 °C for 6 h. Therefore, L. mesenteroides levansucrase

452

could be used as a potential biocatalyst for production of melibiose or functional

453

syrup containing melibiose.

454 455

Funding

456

This work was supported by the NSFC Project (No. 21276001), the 863 Project

457

(No. 2013AA102102), the Fundamental Research Funds for the Central Universities 19



458

(No. JUSRP51304A), and the Support Project of Jiangsu Province (No.

459

BK20130001).

460 461

ASSCOIATED CONTENT

462

Supporting Information

463

(A) FTIR, (B) 13C NMR and (C) 1H NMR spectra of the carbohydrate produced from

464

raffinose and lactose from the purified recombinant L. mesenteroides levansucrase

465

(Figure S1, S2). This material is available free of charge via the Internet at

466

http://pubs.acs.org.

467 468

AUTHOR INFORMATION

469

Corresponding Authors

470

*

471

[email protected].

(W. Mu) Phone: +86 510 85919161. Fax: +86 510 85919161. E-mail:

472 473 474 475 476 477

ABBREVIATIONS USED NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography; IPTG, isopropyl-β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DSS, 4,4-di-methyl-4-silapentane-1-sulfonate.

478 479

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480

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481

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Biomolecular characterization of the levansucrase of Erwinia amylovora, a promising

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biocatalyst for the synthesis of fructooligosaccharides. J. Agr. Food Chem. 2013, 61,

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Identification of functionally important amino acid residues in Zymomonas mobilis

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levansucrase. J. Biochem. 2002, 132, 565-572.

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557

B-512F. FEMS Microbiol. Lett. 2006, 261, 203-210. 23



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Production and properties of a dextransucrase from Leuconostoc mesenteroides

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IBT-PQ isolated from 'pulque', a traditional Aztec alcoholic beverage. J. Ind.

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Environ. Microbiol. 1997, 63, 581-586.

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biosynthesis of lactosucrose from sucrose and lactose by the purified recombinant

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levansucrase from Leuconostoc mesenteroides B-512 FMC. J. Agr. Food Chem. 2015,

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63, 9755-9763.

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590

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Bacillus sp. TH4-2 capable of producing high molecular weight levan at high

594

temperature. J. Biotechnol. 2002, 99, 111-119.

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596

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597

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599

polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a

600

novel levansucrase from Bacillus megaterium. Biochem. J. 2007, 407, 189-198.

601

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602

functional characterization of a levansucrase from the sourdough isolate Lactobacillus

603

sanfranciscensis TMW 1.392. Appl. Microbiol. Biotechnol. 2005, 66, 655-663.

604

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605

bioconversion from lactose and sucrose by whole cells of Paenibacillus polymyxa

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harboring levansucrase activity. Biotechnol. Prog. 2004, 20, 1876-1879.

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(41) W. Li.; S. Yu.; T. Zhang.; B. Jiang.; W. Mu., Recent novel applications of

Enzymatic

Synthesis

of

Lactosucrose

25


and

Its

Analogues

by


Page 26 of 40

608

levansucrases. Appl. Microbiol. Biotechnol. 2015, 99, 6959-6969.

609

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610

Gene cloning, characterization, and heterologous expression of levansucrase from

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Bacillus amyloliquefaciens. J. Ind. Microbiol. Biotechnol. 2010, 37, 195-204.

612

(43) L. Inthanavong.; F. Tian.; M. Khodadadi.; S. Karboune., Properties of

613

Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis

614

of levan and fructooligosaccharides. Biotechnol. Prog. 2013, 29, 1405-1415.

615

(44) C. S. Kim.; E. S. Ji.; D. K. Oh., A new kinetic model of recombinant

616

beta-galactosidase

617

transgalactosylation reactions. Biochem. Biophys. Res. Commun. 2004, 316, 738-743.

618

(45) Y. J. Lee.; C. S. Kim.; D. K. Oh.; Lactulose production by beta-galactosidase in

619

permeabilized cells of Kluyveromyces lactis. Appl. Microbiol. Biotechnol. 2004, 64,

620

787-793.

621

(46) W. Li.; S. Yu.; T. Zhang.; B. Jiang.; W. Mu., Synthesis of raffinose by

622

transfructosylation using recombinant levansucrase from Clostridium arbusti

623

SL206[J]. J Sci Food Agr. 2017, 97, 43-49.

from

Kluyveromyces

lactis

for

26


both

hydrolysis

and

Page 27 of 40


624

Figure Legend

625

Fig. 1 (A) and (B), Schematic digram of melibiose production by L. mesenteroides

626

levansucrase from raffionse by using lactose (A) and galactose (B) as fructosly

627

acceptor, respectively. (A’) and (B’), the chemical structure of melibiose and the

628

reaction from lactose and galactose, respectively.

629 630

Fig. 2 Effect of pH on melibiose synthesis (A). All reactions were performed using 10

631

U/mL purified levansucrase at 45 °C for 1 h in a 1 L reaction mixture with different

632

50 mM solutions (sodium acetate, pH 4.0 - 6.0; sodium phosphate buffer, pH 6.0 - 7.5;

633

Tris-HCl buffer, pH 8.0 - 9.0) containing 210 g/L raffionse and 210 g/L lactose.

634

Values are means of three replications ± standard deviation.

635

Effect of temperature on melibiose synthesis (B). All reactions were performed

636

using 10 U/mL purified levansucrase at pH 6.0 for 1 h in a 1 L reaction mixture with

637

containing 210 g/L raffionse and 210 g/ L lactose, and different temperature ranging

638

from 30 to 70 °C. (Values are means of three replications ± standard deviation)

639 640

Fig. 3 Bioconversion of raffinose to melibiose through hydrolysis. HPLC profile of

641

the enzymatic reaction products from raffinose hydrolysis by the recombinant purified

642

L. mesenteroides levansucrase. The change of product concentration during the

643

hydrolysis. The enzymatic reaction was performed in 1 L solution containing 10

644

U/mL purified enzyme and 300 mM raffinose at pH 6.0 and 45 °C. Values are the

645

means of three replications ± standard deviation.

646 647

Fig. 4 Effect of substrate concentration on melibiose synthesis (A). The reactions were

648

performed using 10 U/mL purified levansucrase at pH 6.0 (sodium phosphate buffer,

649

50 mM) and 45 °C for 1 h, and different substrate concentrations ranging from 3% to

650

30%. 27



651

Effect of ratio of raffinose to lactose on melibiose synthesis (B). The reactions

652

were performed using 10 U/mL purified levansucrase at pH 6.0 (sodium phosphate

653

buffer, 50 mM) and 45 °C for 1 h, and different ratios of substrate concentrations were

654

set as: 6%: 21%, 12%: 21%, 18%: 21%, 21%: 21%, 24%: 21%, 21%: 24%, 21%: 18%,

655

21%: 12%, and 21%: 6%.

656 657

Fig. 5 Effect of enzyme amount on melibiose synthesis. The reactions were performed

658

in a reaction mixture containing 210 g/L raffionse and 210 g/L lactose for 6 h at 45 °C,

659

by varying the purified enzyme amount from 2 to 10 U/mL. Values are the means of

660

three replicates ± standard deviation.

661 662

Fig. 6 Bioconversion of raffinose to melibiose under the optimized conditions. The

663

enzymatic reaction was performed with 5 U/mL (hydrolysis activity) of the purified

664

recombinant levansucrase from 210 g/L raffinose and 210 g/L lactose at pH 6.0 and

665

45 °C. All values are the means of three replications ± standard deviation.

666 667

Fig. 7 Bioconversion of raffinose to melibiose under the optimized conditions. The

668

enzymatic reaction was performed with 5 U/mL (hydrolysis activity) of the purified

669

recombinant levansucrase from 210 g/L raffinose and 210 g/L galactose at pH 6.0 and

670

45 °C. All values are the means of three replications ± standard deviation.

28


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Table 1. Chemical shifts in

1

H and

13

C spectra of reaction products by L.

mesenteroides levansucrase with raffinose as substrate.a Group

C-atom

Galactose

1 2 3 4 5 6

Melibiose δC δH 98.24 5.01 68.54 3.84 69.54 3.92 69.28 4.01 71.00 3.98 61.15 3.75

α-Glucose

1′ 2′ 3′ 4′ 5′ 6′

92.24 71.49 73.00 69.67 70.16 66.05

5.27 3.56 3.72 3.50 4.02 3.97/3.76

β-Glucose

1′ 2′ 3′ 4′ 5′ 6′

96.12 74.95 75.95 69.52 74.41 65.95

4.68 3.26 3.51 3.54 3.67 3.97/3.76

a

Group

C-atom

Galactose

1 2 3 4 5 6

Gal-α-1,2-Fru δC δH 92.33 5.43 68.02 3.82 69.18 3.89 69.14 4.01 71.449 4.12 60.90 3.71

Fructose

1’ 2’ 3’ 4’ 5’ 6’

61.58 103.64 76.62 74.12 81.33 62.39

3.67 -4.20/4.18 4.05 3.72 3.84

Chemical shifts (δ) in ppm were determined relative to the internal standard sodium

4,4-di-methyl-4-silapentane-1-sulfonate (δH = δC = 0.00 ppm).

29



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Table 2. Comparison of specific activities and kinetic parameters of L. mesenteroides levansucrase for raffinose and sucrose hydrolysis. Substrate

Sucrose Raffinose

Specific activity (U/mg) 469 ± 23 195 ± 16

Vmax (mM min-1)

Km (mM)

kcat (min-1)

5.21 ± 23 2.18 ± 23

25.66 ± 1.21 56.82 ± 1.56

15,125 ± 563 6,338 ± 126

30


kcat/Km (mM-1 min-1) 589.44 ± 6.1 112 ± 8

Page 31 of 40


Fig. 1 (A) and (B), Schematic digram of melibiose production by L. mesenteroides levansucrase from raffionse by using lactose (A) and galactose (B) as fructosly acceptor, respectively. (A’) and (B’), the chemical structure of melibiose and the reaction from lactose and galactose, respectively.

31



Fig. 2 Effect of pH on melibiose synthesis (A). All reactions were performed using 10 U/mL purified levansucrase at 45 °C for 1 h in a 1 L reaction mixture with different 50 mM solutions (sodium acetate, pH 4.0 - 6.0; sodium phosphate buffer, pH 6.0 - 7.5; Tris-HCl buffer, pH 8.0 - 9.0) containing 210 g/L raffionse and 210 g/L lactose. Values are means of three replications ± standard deviation. 32


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Effect of temperature on melibiose synthesis (B). All reactions were performed using 10 U/mL purified levansucrase at pH 6.0 for 1 h in a 1 L reaction mixture with containing 210 g/L raffionse and 210 g/ L lactose, and different temperature ranging from 30 to 70 °C. (Values are means of three replications ± standard deviation)

33



Fig. 3 Bioconversion of raffinose to melibiose through hydrolysis. HPLC profile of the enzymatic reaction products from raffinose hydrolysis by the recombinant purified L. mesenteroides levansucrase. The change of product concentration during the hydrolysis. The enzymatic reaction was performed in 1 L solution containing 10 U/mL purified enzyme and 300 mM raffinose at pH 6.0 and 45 °C. Values are the means of three replications ± standard deviation.

34


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Fig. 4 Effect of substrate concentration on melibiose synthesis (A). The reactions were performed using 10 U/mL purified levansucrase at pH 6.0 (sodium phosphate buffer, 50 mM) and 45 °C for 1 h, and different substrate concentrations ranging from 3% to 30%. Effect of ratio of raffinose to lactose on melibiose synthesis (B). The reactions were performed using 10 U/mL purified levansucrase at pH 6.0 (sodium phosphate buffer, 50 mM) and 45 °C for 1 h, and different ratios of substrate concentrations were set as: 6%: 21%, 12%: 21%, 18%: 21%, 21%: 21%, 24%: 21%, 21%: 24%, 21%: 18%, 35



21%: 12%, and 21%: 6%.

36


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Fig. 5 Effect of enzyme amount on melibiose synthesis. The reactions were performed in a reaction mixture containing 210 g/L raffionse and 210 g/L lactose for 6 h at 45 °C, by varying the purified enzyme amount from 2 to 10 U/mL. Values are the means of three replicates ± standard deviation

37



Fig. 6 Bioconversion of raffinose to melibiose under the optimized conditions. The enzymatic reaction was performed with 5 U/mL (hydrolysis activity) of the purified recombinant levansucrase from 210 g/L raffinose and 210 g/L lactose at pH 6.0 and 45 °C. All values are the means of three replications ± standard deviation.

38


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Fig. 7 Bioconversion of raffinose to melibiose under the optimized conditions. The enzymatic reaction was performed with 5 U/mL (hydrolysis activity) of the purified recombinant levansucrase from 210 g/L raffinose and 210 g/L galactose at pH 6.0 and 45 °C. All values are the means of three replications ± standard deviation.

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TOC

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Enzymatic Production of Melibiose from Raffinose by the

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