Transferase Activity of Lactobacillal and Bifidobacterial Î²

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Transferase activity of lactobacillal and bifidobacterial #galactosidases with various sugars as galactosyl acceptors Sheryl Lozel Arreola, Montira Intanon, Pairote Wongputtisin, Paul Kosma, Dietmar Haltrich, and Thu-Ha Nguyen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06009 • Publication Date (Web): 15 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016

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Journal of Agricultural and Food Chemistry

Transferase activity of lactobacillal and bifidobacterial β-galactosidases with various sugars as galactosyl acceptors

Sheryl Lozel Arreola1,2, Montira Intanon1,3, Pairote Wongputtisin1,4, Paul Kosma5, Dietmar Haltrich1 and Thu-Ha Nguyen1* 1

Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU - University of Natural

Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria 2

Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines

3

Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang

Mai University, Chiang Mai, Thailand 4

Faculty of Science, Maejo University, Chiang Mai, Thailand

5

Division of Organic Chemistry, Department of Chemistry, BOKU - University of Natural Resources and Life

Sciences, Muthgasse 18, A-1190 Vienna, Austria

Revised for publication in:


* Corresponding author: Thu-Ha Nguyen Phone: 43-1-47654 6148. Fax: 43-1-47654 6199. E-mail: [email protected]

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

The β-galactosidases from Lactobacillus reuteri L103 (Lreuβgal), Lactobacillus

3

delbrueckii subsp. bulgaricus DSM 20081 (Lbulβgal) and Bifidobacterium breve

4

DSM 20281 (Bbreβgal-I and Bbreβgal-II) were investigated in detail with respect to

5

their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products D-

6

glucose and D-galactose, and certain sugar acceptors such as N-acetyl-D-glucosamine

7

(GlcNAc), N-acetyl-D-galactosamine (GalNAc) and L-fucose (Fuc) under defined,

8

initial-velocity conditions. The rate constants or partitioning ratios (kNu/kwater)

9

determined for these different acceptors (termed nucleophiles, Nu) were used as a

10

measure for the ability of a certain substance to act as a galactosyl acceptor of these β-

11

galactosidases. When using Lbulβgal or Bbreβgal-II, the galactosyl transfer to

12

GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and

13

GlcNAc used in equimolar substrate concentrations, Lbulβgal and Bbreβgal-II

14

catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41%

15

and 24%, respectively, as calculated from the initial GlcNAc concentration.

16 17

KEYWORDS:

β-galactosidases, transgalactosylation, galactosyl acceptors,

18

galactooligosaccharides, hetero-oligosaccharides

2


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19

INTRODUCTION

20

β-Galactosidases (β-D-galactoside galactohydrolase, EC 3.2.1.23; β-gal) have long

21

been known to catalyze the hydrolysis of lactose into glucose and galactose, as well as

22

the transfer of galactosyl moiety to suitable acceptors. If lactose is present in excess,

23

β-gal will use lactose, or its hydrolysis products, glucose and galactose, as alternative

24

galactosyl acceptors to form galacto-oligosaccharides or GOS (Scheme 1). The source

25

of β-gal, lactose concentration and working temperature influence the GOS type, GOS

26

yield and the specific linkages formed, thus creating a wide array of GOS.1

27

β-Galactosidases have also been used to form hetero-oligosaccharides (HeOS) or

28

other galactosylated compounds, with mannose, fructose, N-acetylneuraminic acid,

29

glucuronic acid and a number of aromatic compounds used as galactosyl acceptor.2-7

30

Using this approach, Sulfolobus solfataricus and Kluyveromyces lactis β-

31

galactosidases were used to produce lactulose, a commercial prebiotic disaccharide,

32

and galactosylated aromatic primary alcohols.5,

33

oligosaccharide (HMO) core structure or structurally related compounds can also be

34

accessed, albeit not in pure form, through an approach based on β-galactosidase-

35

catalyzed transglycosylation with lactose as donor (thus transferring galactose onto

36

suitable acceptors) and N-acetylglucosamine (GlcNAc) as acceptor. Thereby, lacto-N-

37

biose (LNB; Gal-β-1,3-GlcNAc) and N-acetyl-lactosamine (LacNAc; Gal-β-1,4-

38

GlcNAc) together with their regioisomers can be obtained. β-Galactosidases from

39

Bacillus circulans, K. lactis and Lactobacillus bulgaricus were proved to be suitable

40

biocatalysts for the formation of N-acetyl-oligosaccharides using lactose and GlcNAc

41

as substrates.9-11 While the feasibility of this transferase reaction has been shown, no

42

study dealt with a more detailed biochemical characterization of this reaction.

8

Part of the human milk

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43

The intermolecular transfer of galactose to acceptors other than water typically

44

presents the major pathway for the formation of GOS during lactose hydrolysis by

45

retaining β-galactosidase (Scheme 1). As the sugar species in the mixture that can act

46

as a nucleophile and hence as a galactosyl acceptor change constantly during the

47

reaction, an exact prediction of product formation and degradation cannot be made.

48

However, the partitioning of the galactosylated enzyme (which is formed as an

49

intermediate in the enzymatic reaction; E-Gal in Scheme 1) between the reaction with

50

water and hence hydrolysis, and the reaction with a galactosyl acceptor can be studied

51

under defined initial velocity conditions. When complete hydrolysis of the

52

disaccharide lactose occurs, equimolar amounts of D-glucose and D-galactose are

53

formed, and therefore the initial velocities at which the two sugars are released are

54

identical and the ratio vGlu/vGal is 1.0. In the presence of an appropriate nucleophile Nu

55

(e.g., a sugar acceptor for galactose) vGlu/vGal will increase since some of the

56

galactosyl moieties will not be released but transferred onto the acceptor. Richard et

57

al.12 derived equation (1) from Scheme 1, where the rate constant ratio kNu/kwater is

58

obtained as the slope from the linear correlation of vGlu/vGal with increasing

59

concentrations of Nu:

60

Gal

61

Glc

Nu

= 1+

water

[Nu]

(Equation 1)

When looking at the process for the formation of HeOS that mimic HMO, the

62

main

candidates

for

galactosyl

acceptors

are

N-acetylglucosamine,

N-

63

acetylgalactosamine and L-fucose (which will be added to the reaction mixture), the

64

substrate lactose, and its hydrolysis products D-glucose and D-galactose. The rate

65

constant ratios determined for the different acceptors can therefore be used as a

66

measure for the ability of a certain substance to act as a galactosyl acceptor (i.e.

67

nucleophile), which in turn allows an estimation of the transgalactosylation products 4


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68

obtained for a known reaction mixture, or the suitability of a certain enzyme for the

69

efficient synthesis of HeOS.

70

In this paper, the propensity of the β-galactosidases from Lactobacillus

71

delbrueckii subsp. bulgaricus DSM 20081 (L. bulgaricus, Lbulβgal), L. reuteri L103

72

(Lreuβgal), Bifidobacterium breve DSM 20281 β-gal I (Bbreβgal-I) and β-gal II

73

(Bbreβgal-II) to transfer galactosyl moiety to different acceptors such as lactose (Lac),

74

glucose (D-Glc), galactose (D-Gal), L-fucose (Fuc), N-acetyl-D-glucosamine

75

(GlcNAc) and N-acetyl-D-galactosamine (GalNAc), was determined in order to

76

deepen the understanding of the relative extent of galactosyl transfer of a specific β-

77

galactosidase to different sugar acceptors. The four β-galactosidases belong to

78

glycoside hydrolase family 2 (GH 2) and are of the hetero-oligomeric LacLM type

79

(Lreuβgal) or the homo-oligomeric LacZ type (Lbulβgal, Bbreβgal-I, Bbreβgal-II).

80

The enzymatic synthesis of N-acetyl oligosaccharides using the two enzymes

81

Lbulβgal and Bbreβgal-II will be also presented.

82 83

MATERIALS AND METHODS

84

Chemicals. All chemicals and enzymes were purchased from Sigma (St. Louis,

85

MO, USA) unless stated otherwise, and were of the highest quality available. The test

86

kit for the determination of D-glucose was obtained from Megazyme (Wicklow,

87

Ireland). Galacto-oligosaccharide standards of β-D-Galp-(13)-D-Glc, β-D-Galp-

88

(16)-D-Glc, β-D-Galp-(13)-D-Gal, β-D-Galp-(14)-D-Gal, β-D-Galp-(16)-D-Gal,

89

β-D-Galp-(13)-β-D-Galp-(14)-D-Glc, β-D-Galp-(14)-β-D-Galp-(14)-D-Glc, β-D-

90

Galp-(16)-β-D-Galp-(14)-D-Glc were purchased from Carbosynth (Berkshire, UK)

91

while β-D-Galp-(13)-D-GlcNAc (Lacto-N-biose I, LNB I) and β-D-Galp-(14)-D-

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GlcNAc (N-acetyl-D-lactosamine, LacNAc) were purchased from Dextra Laboratories

93

(Reading, UK).

94

Enzyme preparation. β-Galactosidase from L. reuteri L103 (Lreuβgal) was

95

recombinantly produced in Escherichia coli and purified as reported previously,13

96

while the lacZ gene encoding β-gal from L. bulgaricus DSM 20081 was expressed in

97

L. plantarum WCFS1 and the corresponding protein was purified as described

98

before.14 B. breve DSM 20231 β-gal I and βgal II were prepared according to Arreola

99

et al.15

100

β-Galactosidase assays. The measurement of β-galactosidase activity using o-

101

nitrophenyl β-D-galactopyranoside (oNPG) or lactose as substrates was carried out as

102

described previously.16 Briefly, these assays were performed in 50 mM sodium

103

phosphate buffer of pH 6.5 at 30 °C, and the final substrate concentrations in the 10-

104

min assay were 22 mM for oNPG and 600 mM for lactose. Protein concentrations

105

were determined using the method of Bradford with bovine serum albumin (BSA) as a

106

standard.

107

GOS synthesis. The ability of the four recombinant β-galactosidases to

108

synthesize GOS was compared by carrying out discontinuous conversion reactions in

109

a 2-mL scale. The activities (ULac/mL) of the recombinant β-gals used were as

110

follows: L. reuteri, 0.8; L. bulgaricus, 1.5; B. breve β-gal I 1.0; B. breve β-gal II, 2.5.

111

Reaction conditions were 600 mM initial lactose concentration in sodium phosphate

112

buffer (50 mM, pH 6.5) containing 1 mM Mg2+ with the incubation temperature set at

113

30 oC and continuous agitation at 300 rpm. At certain time intervals, samples were

114

withdrawn and the reaction was stopped by heating at 95 oC for 5 minutes. The

115

composition of the GOS mixture was analyzed by HPAEC-PAD following the

6


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method described previously.15 D-Glc, D-Gal, lactose, and GOS components were

117

identified and quantified using the external standard technique.

118

Intermolecular galactosyl transfer under defined initial-velocity conditions.

119

Initial velocities of D-Glc or D-Gal release were determined using 50 mM sodium

120

phosphate buffer, pH 6.5 at 30 °C using either 10 mM oNPG or 100 mM lactose as

121

substrate. These substrate concentrations were a compromise between the practical

122

requirement to measure initial velocity of D-Gal (and/or D-Glc) formation and to

123

maximize the transfer of D-Gal to the external, added nucleophile but not to the

124

substrate. The final enzyme concentration used was ≤ 1.0 U/mL. The relationship

125

between [oNP] (or [D-Glc]) and [D-Gal] was found to be linear up to 30 min. Thus,

126

the standard reaction time of 20 min was used. νoNP, νGlc, and νGal were obtained from

127

measuring the molar concentrations of oNP, D-Glc, and D-Gal, respectively. The

128

ratios of νoNP and νGal were measured in the absence and presence of D-glucose with

129

its concentration varied between 2.5 and 20 mM.

130

The intermolecular transgalactosylation to lactose was performed with varying

131

initial lactose concentrations (9–600 mM), while galactosyl transfer to either GlcNAc,

132

GalNAc and L-fucose were determined using 100 mM lactose with acceptor

133

concentrations varying from 12.5 to 200 mM. After incubation of the reaction mixture

134

for 20 min at 30 °C, the reaction was stopped by heating for 5 min at 95 °C. The rate

135

of formation of oNP (νoNP) was measured using the standard β-galactosidase assay,

136

while D-galactose (νGal) or D-glucose (νGlc) measurement was carried out by HPLC

137

(Dionex; MA, USA) using an Aminex HPX-87K column (300 x 7.8 mm; Bio-Rad,

138

Hercules, CA, USA) equipped with a refractive index detector. Water was used as

139

mobile phase at a flow rate of 0.80 mL min-1 and the column temperature was 80 °C.

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140

N-acetyl oligosaccharide formation. N-acetyl-oligosaccharide synthesis was

141

carried out using lactose and GlcNAc (or GalNAc) as substrates with either Lbulβgal

142

or Bbreβgal-II. The effects of temperature (30 and 50 °C), substrate concentration (0.6

143

M and 1 M), molar ratio of donor to acceptor (1:2, 1:1 and 2:1), and enzyme

144

concentration (2.5 and 5.0 U/mL) on the synthesis were also investigated. Substrates

145

were dissolved in 50 mM sodium phosphate buffer, pH 6.5 containing 1 mM Mg2+.

146

The enzyme was added, and the incubation was done at the required temperature at

147

300 rpm with a thermomixer (Eppendorf, Hamburg, Germany). Aliquots of samples

148

were withdrawn at certain time intervals to determine the residual activities and

149

carbohydrate content using either HPAEC-PAD as described in17 or an HPLC system

150

consisting of a UV detector and the Hypercarb column (0.32 × 150 mm, inner

151

diameter 5 µm; Thermo Scientific). Ammonium formate buffer (0.3% formic acid, pH

152

9.0) was used as Buffer A, and a gradient was performed from 0 to 35% acetonitrile

153

within 35 min using a Dionex Ultimate 3000 pump (cap flow, 1 mL/min). The

154

GlcNAc transgalactosylation yield was determined based on the starting GlcNAc

155

concentration and was calculated using Equation 2.

156

GlcNAc transgalactosylation yield % =

GlcNAcinitial -GlcNAcremaining GlcNAcinitial

157

× 100

(Equation 2)

158

Purification of N-acetyl-oligosaccharides. For purification and identification of

159

GlcNAc transfer products, a 10-mL discontinuous batch reaction using Bbreβgal-II (5

160

ULac/mL) was carried out at 30 °C using initial equimolar concentrations of lactose

161

and GlcNAc (600 mM each) dissolved in 50 mM sodium phosphate buffer (pH 6.5)

162

with 1 mM Mg2+. Agitation was at 300 rpm on a rotary shaker. After 4 h, the reaction

163

was stopped by heating at 95 °C. GlcNAc-containing oligosaccharides were prepared

8


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also with Lbulβgal in here in a similar way, and here the reaction conditions were 2.5

165

ULac/mL with 1 M lactose and 1 M GlcNAc at 50 oC. Due to the complex course of

166

transgalactosylation reactions, the reaction mixture was partially purified by gel

167

permeation chromatography on Bio-Gel P2 (2.0 x 100 cm) equilibrated in water

168

containing 5% (v/v) ethanol and 0.0015 % (w/v) NaCl. The elution was followed by

169

UV reading at 210 nm to detect the presence of GlcNAc and transgalactosylation

170

products. The fractions containing the desired transgalactosylation products were

171

pooled, freeze-dried, and re-dissolved in acetonitrile. The complete purification of the

172

transgalactosylation products was obtained by using an HPLC system and the

173

Hypercarb column as described above.

174

NMR Measurements. NMR spectra were recorded at 27 oC in 99.9% D2O on a

175

Bruker Avance IIITM 600 spectrometer (1H at 600.13 MHz and

13

176

equipped with a BBFO broad-band inverse probe head and z-gradients using standard

177

Bruker NMR software. COSY experiments were recorded using the program cosygpqf

178

with 2048 x 256 data points, respectively, per t1-increment. Multiplicity-edited HSQC

179

spectra were recorded using hsqcedetgp with 1024 x 128 data points and 16 scans,

180

respectively, per t1-increment. 1H NMR spectra were referenced to internal DSS (δ=

181

0); 13C NMR spectra were referenced to external 1,4-dioxane (δ= 67.4).

C at 150 MHz)

182 183

RESULTS AND DISCUSSION

184

We studied in detail different β-galactosidases from probiotic strains of lactic acid

185

bacteria (Lreuβgal and Lbulβgal) and bifidobacteria (Bbreβgal-I and Bbreβgal-II) with

186

respect to their propensity to transfer galactosyl moieties onto certain sugar acceptors.

187

While biochemical properties of these enzymes have been investigated in earlier

9



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188

works,14-17 detailed kinetic analyses of the formation of oligosaccharides based on the

189

transfer constants (kNu/kwater) (Scheme 1) have not been reported.

190

Hydrolysis versus transgalactosylation using lactose as substrate. The

191

measurement of the D-Glc/D-Gal ratio as a function of the reaction time provides a

192

good estimate, as to which extent transgalactosylation (onto lactose, D-Glc or D-Gal

193

as acceptors) competes with hydrolysis during lactose conversion. This ratio,

194

however, does not accurately reflect the true extent of lactose conversion because a

195

transfer of the galactosyl moiety can occur either via intramolecular or intermolecular

196

reaction.

197

The formation of D-Glc and D-Gal was monitored over the entire course of the

198

conversion using an initial lactose concentration of 600 mM (Figure 1A). At all times,

199

the ratio of D-Glc/D-Gal was higher for Lbulβgal when compared with that of

200

Lreuβgal, Bbreβgal-I and Bbreβgal-II, indicating that this enzyme shows a more

201

pronounced transferase activity. The maximum value of the D-Glc/D-Gal ratio for

202

Lbulβgal was 3.0 at 16% lactose conversion where trisaccharides form predominantly.

203

This had also been confirmed in our previous work showing that β-D-Galp-(16)-β-D-

204

Galp-(14)-D-Glc

205

transgalactosylation products at the beginning of the reaction when using this

206

enzyme.14 This ratio decreased to 2.71 at ~30% lactose conversion, then remained

207

constant until lactose conversion was ~70%, and decreased dramatically with more

208

than 90% lactose conversion. The same trend was observed for Bbreβgal-I where the

209

highest D-Glc/D-Gal ratio was observed at the initial stage of the reaction and further

210

decreased as the reaction progressed. For Lreuβgal and Bbreβgal-II, maximum values

211

of the D-Glc/D-Gal ratio were found over a rather broad range of lactose conversion

212

(20–80%). At about 98–99% lactose conversion, the D-Glc/D-Gal ratio was close to

and

β-D-Galp-(13)-β-D-Galp-(14)-D-Glc

are

the

main

10


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213

1.0 for Lreuβgal and Bbreβgal-I suggesting that lactose and all GOS formed were

214

extensively hydrolyzed. In contrast, the D-Glc/D-Gal ratio with Lbulβgal and

215

Bbreβgal-II was still 1.76 and 1.42, respectively, at almost complete lactose

216

conversion, implying that a significant amount of GOS resisted hydrolysis even when

217

lactose conversion was almost 100%.

218

The ratio of D-Glc/D-Gal measured can be related directly to the level of GOS

219

formation when these enzymes are used for the reaction with lactose. Lbulβgal, which

220

exhibited the highest D-Glc/D-Gal ratio, showed the highest GOS yield at all lactose

221

conversion levels compared to that of the other three β-gals (Figure 1B). The yields as

222

well as the type of GOS formed differ significantly among the β-gals studied. Overall,

223

Lreuβgal and Lbulβgal yielded the same mixture of GOS, which is different from that

224

of Bbreβgal-I and Bbreβgal-II. Typical HPLC chromatograms of GOS formed by

225

Lbulβgal and Bbreβgal-II are depicted in Figure S1.

226

Partitioning analysis. The transfer constant kNu/kwater, which is obtained from the

227

velocity ratios of νGlc/νGal or νoNP/νGal, provides a useful tool to measure the ability of a

228

certain substance to act as a galactosyl acceptor (i.e. a nucleophile, Nu), which in turn

229

allows an estimation of the level of transgalactosylation products obtained from a

230

known reaction mixture. Initial velocities were measured at 30 °C in 50 mM sodium

231

phosphate buffer, pH 6.5. Plots of (νoNP/νGal) or (νGlc/νGal) against [Nu] were linear for

232

a specific range of the acceptor concentration. Deviation from linearity, which

233

occurred mainly at low and high concentrations of the nucleophile, may be due to

234

competition for binding to the nucleophile-binding site of the galactosyl-enzyme

235

intermediate [E-Gal].18 The F-test at 95% probability level confirmed the validity of

236

the linear fit for the range of [Nu] as shown in Figure S2. Moreover, the fit of the lines

237

as represented by r2 was usually greater than 0.98. When kGlc/kwater was determined,

11



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238

Bbreβgal-II showed the lowest partitioning ratio (3.91 ± 0.44 M-1) while Lbulβgal

239

gave the highest (9.36 ± 0.56 M-1) as shown in Table 1. Likewise, when lactose alone

240

was used as the substrate, where the only possible galactosyl acceptors are lactose and

241

its hydrolysis products, D-Gal and D-Glc, Bbreβgal-II showed the lowest kLac/kwater

242

ratio (0.53 M-1) while that of Lbulβgal was the highest (2.79 M-1), again confirming

243

the high transferase activity of this latter enzyme.

244

When kGlc/kLac was determined (obtained from the ratio of kGlc/kwater to kLac/kwater),

245

Bbreβgal-II showed the highest ratio of 7.4 and Bbreβgal-I a ratio of 4.2, while

246

Lreuβgal and Lbulβgal showed lower ratios of 3.4 and 3.3, respectively. These values

247

suggest that D-Glc is a far better galactosyl acceptor for the two bifidobacterial β-

248

galactosidases than lactose when compared to the two lactobacillal β-galactosidase.

249

Hence, disaccharides other than lactose will make up a large proportion of GOS

250

mixtures formed with the two bifidobacterial enzymes. Figure 2A confirms that D-

251

glucose is in fact a better galactosyl acceptor than D-lactose when looking at the ratio

252

of total GalGlc disaccharides to Galβ-D-Galp-(14)-D-Glc trisaccharides formed. This

253

was especially pronounced at the beginning of the reaction, where this ratio was ~5

254

for Bbreβgal-I or ~4 for Bbreβgal-II at 20% lactose conversion while it was 0.44 for

255

both Lbulβgal and Lreuβgal. We reported in our recent study that the predominant

256

oligosaccharide product of both bifidobacterial enzymes was β-D-Galp-(16)-D-Glc

257

(allolactose), accounting for approximately 45% and 50% of the GOS formed by

258

transgalactosylation by Bbreβgal-I and Bbreβgal-II, respectively, at maximum total

259

GOS yield 15, confirming the results predicted by the partionining coefficients.

260

Furthermore, the propensity of the four β-galactosidases to transfer the galactose

261

moiety to either GlcNAc, GalNAc or Fuc was determined using a fixed lactose

262

concentration (100 mM) as galactosyl donor and the respective nucleophile in varying

12


Page 13 of 31


263

concentrations as galactosyl acceptor. In the presence of 100 mM lactose and the

264

absence of any external galactosyl acceptor, νGlc/νGal was found to be ~1.3 for

265

Lbulβgal suggesting GOS formation even at this low lactose concentration while

266

νGlc/νGal of the other three β-gals was nearly 1.0 indicating that hydrolysis of lactose is

267

the main reaction.

268

Both Lbulβgal and Bbreβgal-II effectively transferred the galactosyl moiety to

269

GlcNAc rather than to water as indicated by the rate constant ratio kGlcNAc/kwater of

270

16.8 M-1 and 5.42 M-1 (Table 1), respectively. Bbreβgal-II and Lbulβgal also showed

271

high preference to transfer galactosyl moiety to GlcNAc rather than to lactose, and

272

GlcNAc is a ~10- and 6-fold better galactosyl acceptor than lactose for these two

273

enzymes. The kGlcNAc/kGlc ratios of Lbulβgal and Bbreβgal-II (1.8 and 1.4,

274

respectively) indicate furthermore that GlcNAc is also the preferred galactosyl

275

acceptor over glucose. This altogether suggests that GlcNAc is an excellent acceptor

276

for Lbulβgal and Bbreβgal-II, and that the disaccharide Gal-GlcNAc (positional

277

isomers thereof) will be the main products rather than tri-GOS or other GOS when

278

using a mixture of GlcNAc and lactose as substrate.

279

To confirm this assumption, discontinuous conversion reactions using either

280

Bbreβgal-II or Lbulβgal (2.5ULac/mL) were carried out with 600 mM lactose in the

281

absence and presence of 600 mM GlcNAc. The formation of D-Glc, D-Gal, and

282

oligosaccharides was determined at different time intervals. Figure 2B shows that

283

when using lactose alone as the substrate, the maximum D-Glc/D-Gal ratios with

284

Lbulβgal and Bbreβgal-II were 3.0 and 1.5, respectively, while the presence of

285

GlcNAc with lactose as substrates resulted in significantly higher D-Glc/D-Gal ratios

286

of 11.0 and 4.6, respectively. The 3-fold increase in the D-Glc/D-Gal ratio suggests

287

that the transferase activity of the enzymes was significantly increased over

13



Page 14 of 31

288

hydrolysis, and that the galactosyl moiety was transferred preferentially to GlcNAc

289

rather than onto lactose or glucose. For example, the presence of GlcNAc resulted in a

290

decrease of GalGal formation by Bbreβgal-II, particularly of 6’-galactobiose (β-D-

291

Galp-(16)-D-Gal), as is shown in Figure 3A. Moreover, the synthesis of GOS

292

trisaccharides by Bbreβgal-II decreased significantly from 24 to 13 g/L when GlcNAc

293

was present together with lactose as substrates (Figure 3B). In addition, HPLC

294

analysis showed that presence of a prominent, novel oligosaccharide peak when

295

adding GlcNAc to the reaction mixture (see below). Overall, this indicates that

296

GlcNAc can be an excellent galactosyl acceptor for certain β-galactosidases such as

297

Lbulβgal and Bbreβgal-II.

298

N-acetyl-D-galactosamine can also serve as galactosyl acceptor when using

299

Lbulβgal as judged by the measured kGalNAc/kwater ratio (3.21 M-1). Bbreβgal-I,

300

Bbreβgal-II and Lreuβgal showed kGalNAc/kwater ≤ 1.0 M-1 suggesting that hydrolysis is

301

the preferred reaction in the presence of GalNAc. L-fucose, on the other hand, was

302

shown to be a weak nucleophilic acceptor for all four enzymes based on the kFuc/kwater

303

ratio (≤ 1.27 M-1).

304

The ratio of kGal/kwater would also be an essential kinetic parameter to measure the

305

propensity to transfer the galactosyl moiety to another galactose unit. Unfortunately,

306

kGal/kwater could not be determined because the amount of galactose released cannot be

307

measured accurately in the presence of an excess of free galactose.

308

Formation of GalNAc-containing transgalactosylation products. It was shown

309

earlier that GalNAc can also serve as galactosyl acceptor when using Lbulβgal based

310

on the measured kGalNAc/kwater ratio (3.21 M-1). The formation of N-acetyl-

311

oligosaccharides with lactose and N-acetyl-D-galactosamine (GalNAc) using Lbulβgal

312

as biocatalyst was hence investigated. The maximum GalNAc transgalactosylation

14


Page 15 of 31


313

yield (29.2%) was obtained after 1 h with a donor/acceptor molar ratio of 2:1 and

314

initial concentrations of 600 mM lactose and 300 mM GalNAc (Figure 4A). The

315

HPLC profile showed that both di- and trisaccharides containing GalNAc were

316

formed, however, the individual components were not structurally identified or

317

quantified (Figure 4B).

318

Structural characterization of GlcNAc transgalactosylation products. The

319

effects of enzyme concentrations and the molar ratios of the donor (lactose) to the

320

acceptor (GlcNAc) were studied to obtain the maximum yield of GlcNAc-containing

321

transgalactosylation products. The molar ratio of the donor (lactose) to the acceptor

322

(GlcNAc) of 1:1 (0.6 M lactose and 0.6 M GlcNAc) was found to be optimal for both

323

enzymes, β-gal from L. bulgaricus and β-gal II from B. breve (data not shown).

324

Hence, the optimal conditions for the transgalactosylation reactions for β-gal from L.

325

bulgaricus (2.5 ULac/mL with 1 M lactose and 1 M GlcNAc at 50 °C) and for β-gal II

326

from B. breve (5.0 ULac/mL with 0.6 M lactose and 0.6 M GlcNAc at 30 °C) were

327

employed for the formation of N-acetyl oligosaccharides. The reaction at 50 °C was

328

carried out only with Lbulβgal since Bbreβgal-II is not stable at higher temperature.

329

The products formed in these reactions were then separated using a Hypercarb

330

column. This chromatographic column can also separate the anomeric forms of

331

reducing sugars, and thus each oligosaccharide is represented by two peaks

332

constituting the anomeric isomers.19 The chromatographic patterns and the

333

compounds synthesized by Lbulβgal and Bbreβgal-II were found to be similar as

334

judged by HPLC analysis (Figure S3).

335

The major product was purified as described in Materials and Methods. This

336

major disaccharide product was found to be β-D-Galp-(16)-D-GlcNAc (N-acetyl-

337

allolactosamine) as identified by the NMR data. Despite the presence of the anomeric

15



Page 16 of 31

338

forms of the reducing glucosamine unit (α/β ratio ~1.4:1), which led to two sets of

339

spin-coupled systems, a full assignment could be achieved based on COSY and edited

340

HSQC-spectra (Figure S4, Table S1) and showed a low-field shift of carbon 6 of the

341

reducing GlcNAc to 69.4 ppm. The data -when corrected for different referencing of

342

chemical shifts- were in full agreement with published

343

allolactosamine.20-22 Peaks 2, 4, 6, 7 (Figure S3) were not identified, however, based

344

on the elution rate, peaks 6 and 7 might be assigned to the trisaccharides

345

GalGalGlcNAc, however, the exact identity of these compounds have not been

346

determined.

13

C NMR data of N-acetyl-

347

Under the optimal conditions, the maximum yields of N-acetyl-allolactosamine,

348

calculated as the percentage of initial GlcNAc, were 41% and 24% with Lbulβgal and

349

Bbreβgal-II, respectively (data not shown). The formation of disaccharides as product

350

of transgalactosylation of GlcNAc using β-galactosidases from different organisms

351

has been reported, and the linkage preference of the transgalactosylation products

352

varies for different enzymes. β-gals from Kluyveromyces lactis, L. bulgaricus and L.

353

plantarum synthesized N-acetyl-allolactosamine as the major product and N-acetyl-

354

lactosamine (LacNAc) as minor product.9, 11 β-gals from Bifidobacterium bifidum and

355

Bacillus circulans favored formation of LacNAc over N-acetyl-allolactosamine,22-24

356

whereas N-acetyl-allolactosamine was exclusively synthesized with β-gals from

357

Penicillum multicolor, Aspergillus oryzae and Bifidobacterium longum.22 The

358

presence of higher DP N-acetyl oligosaccharides in the reaction mixtures of

359

transgalactosylation by using β-gals from Sulfolobus solfataricus, A. oryzae or E. coli

360

was also observed, however were not identified.25

361

In conclusion, kinetic analyses of β-galactosidases from L. reuteri, L. bulgaricus,

362

B. breve (β-gal I and β-gal II) with various sugars as nucleophile provided an insight

16


Page 17 of 31


363

into the specificities of the given enzymes for transgalactosylation and formation of

364

hetero-oligosaccharides. The transgalactosylation reaction with GlcNAc using

365

Lbulβgal and Bbreβgal-II showed high yields of N-acetyl oligosaccharides, of which

366

N-acetyl-allolactosamine β-D-Galp-(16)-D-GlcNAc is dominant. Although the major

367

product formed is not similar to the core structures of human milk oligosaccharides

368

(HMO), which are lacto-N-biose (LNB, β-D-Galp-(13)-D-GlcNA, type I) or N-

369

acetyllactosamine (LacNAc, β-D-Galp-(14)-D-GlcNAc, type II), this hetero-

370

oligosaccharide might be of interest because of its potentially extended functionality

371

in addition to galacto-oligosaccharides.

372 373

ABBREVIATIONS USED

374

β-gal, β-galactosidase; Lreuβgal, β-galactosidase from L. reuteri L103; Lbulβgal, β-

375

galactosidase from L. delbrueckii subsp. bulgaricus DSM 20081; Bbreβgal-I, β-

376

galactosidase from B. breve DSM 20281 (β-gal I); Bbreβgal-II, β-galactosidase from

377

B. breve DSM 20281 (β-gal II); Lac, lactose; Glc, glucose; Gal, galactose; Fuc, L-

378

fucose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine;

379

LacNAc, N-acetyllactosamine.

380 381

ACKNOWLEDGEMENTS

382

The authors thank Dr. Andreas Hofinger (Department of Chemistry, BOKU) for

383

recording the NMR spectra.

384 385

FUNDING SOURCES

386

This work was supported by the Austrian Science Fund FWF (Grant P24868-B22 to

387

THN). SLA and MI are thankful for a Technology Grant Southeast Asia, and PW

17



Page 18 of 31

388

acknowledges receipt of an Ernst Mach Grant, both jointly supported by the ASEAN-

389

European Academic University Network (ASEA-UNINET), the Austrian Federal

390

Ministry of Science, Research and Economy and the Austrian Agency for

391

International Cooperation in Education and Research (OeAD-GmbH).

392 393

SUPPORTING INFORMATION

394

Figure S1. Separation and quantification by HPAEC-PAD of individual GOS

395

produced during lactose conversion catalyzed by (A) L. bulgaricus β-gal and (B) B.

396

breve β-gal I. The identified compounds are (1) D-Gal, (2) D-Glc, (3) β-D-Galp-(16)-

397

D-Gal,

398

(6) β-D-Galp-(13)-D-Gal, (7) β-D-Galp-(16)-β-D-Galp-(14)-D-Glc, (9) β-D-Galp-

399

(13)-D-Glc, (15) β-D-Galp-(13)-β-D-Galp-(14)-D-Glc, (17) β-D-Galp-(14)-β-D-

400

Galp-(14)-D-Glc. Peaks 8, 10-14, 16, 18-22 were not identified.

401

Figure S2. Ratio of initial velocities vGlu/vGal in the presence of different exogenous

402

nucleophiles, which are lactose (A), N-acetyl-D-glucosamine (B), N-acetyl-D-

403

galactosamine (C), L- fucose (D); and voNP/vGal in the presence of D-Glucose (E). The

404

enzymes are β-galactosidases from L. reuteri (●), L. bulgaricus (○), B. breve β-gal I

405

(▲), and B. breve β-gal II (∆).

406

Figure S3. HPLC-UV chromatogram of N-acetyl oligosaccharides produced by (A)

407

L. bulgaricus and (B) B. breve β-galactosidases II using equimolar concentration of

408

lactose and GlcNAc dissolved in 50 mM sodium phosphate buffer (pH 6.5) containing

409

1 mM MgCl2. Peak identification: (1) the anomeric isomers of GlcNAc, (3) and (5) the

410

anomeric isomers of Galp-(16)-D-GlcNAc. Peaks 2, 4, 6 and 7 were not identified.

411

Figure S4. Multiplicity edited HSQC spectrum of β-D-Galp-(16)-D-GlcNAc.

412

Table S1. NMR assignments for N-acetyl-allolactosamine.

(4) β-D-Galp-(16)-D-Glc (allolactose), (5) β-D-Galp-(14)-D-Glc (lactose),

18


Page 19 of 31


413

This material is available free of charge via the Internet at http://pubs.acs.org

414 415

REFERENCES

416

(1)

417

enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb.

418

Technol. 2000, 26, 271-281.

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(2)

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Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans

421

β-galactosidase. Biosci. Biotech. Biochem. 1998, 62, 1791-1794.

422

(3)

423

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

424

787-793.

425

(4)

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transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose. Biosci.

427

Biotech. Biochem. 2004, 68, 2086-2090.

428

(5)

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galactosidase catalyzed selective galactosylation of aromatic compounds. Biotechnol.

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Prog. 2006, 22, 326-330.

431

(6)

432

value-added derivatives. Int. Dairy J. 2008, 18, 685-694.

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(7)

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lactose derivatives (hetero-oligosaccharides) from lactose. Int. Dairy J. 2012, 22, 116-

435

122.

Boon, M. A.; Janssen, A. E. M.; van ‘t Riet, K. Effect of temperature and

Yanahira, S.; Yabe, Y.; Nakakoshi, M.; Miura, S.; Matsubara, N.; Ishikawa, H.

Lee, Y. J.; Kim, C. S.; Oh, D. K. Lactulose production by β-galactosidase in

Miyasato, M.; Ajisaka, K. Regioselectivity in β-galactosidase-catalyzed

Bridiau, N.; Taboubi, S.; Marzouki, N.; Legoy, M. D.; Maugard, T. β-

Gänzle, M. G.; Haase, G.; Jelen, P. Lactose: Crystallization, hydrolysis and

Gänzle, M. G. Enzymatic synthesis of galacto-oligosaccharides and other

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Kim, Y. S.; Park, C. S.; Oh, D. K. Lactulose production from lactose and

437

fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme

438

Microb. Technol. 2006, 39, 903-908.

439

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440

Structural identification of novel oligosaccharides produced by Lactobacillus

441

bulgaricus and Lactobacillus plantarum. J. Agric. Food Chem. 2012, 60, 4886-94.

442

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443

and N-acetylglucosamine as substrates by using β-D-galactosidase from Bacillus

444

circulans. Eur. Food Res. Technol. 2010, 231, 55-63.

445

(11)

446

beta-galactosidases from Kluyveromyces lactis and Bacillus circulans in the

447

enzymatic synthesis of N-Acetyl-lactosamine in aqueous media. Biotechnol. Prog.

448

2011, 27, 386-94.

449

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450

relationships for β-galactosidase (Escherichia coli, lac Z). 2. Reactions of the

451

galactosyl-enzyme intermediate with alcohols and azide ion. Biochemistry 1995, 34,

452

11713-24.

453

(13)

454

Cloning and expression of the β-galactosidase genes from Lactobacillus reuteri in

455

Escherichia coli. J. Biotechnol. 2007, 129, 581-591.

456

(14)

457

K.; Mathiesen, G.; Nguyen, T.-H.; Haltrich, D. Homodimeric β-galactosidase from

458

Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus

459

plantarum and biochemical characterization. J. Agric. Food Chem. 2012, 60, 1713-

460

1721.

Black, B. A.; Lee, V. S.; Zhao, Y. Y.; Hu, Y.; Curtis, J. M.; Gänzle, M. G.

Li, W.; Sun, Y.; Ye, H.; Zeng, X. Synthesis of oligosaccharides with lactose

Bridiau, N.; Maugard, T. A comparative study of the regioselectivity of the

Richard, J. P.; Westerfeld, J. G.; Lin, S.; Beard, J. Structure-reactivity

Nguyen, T.-H.; Splechtna, B.; Yamabhai, M.; Haltrich, D.; Peterbauer, C.

Nguyen, T. T.; Nguyen, H. A.; Arreola, S. L.; Mlynek, G.; Djinovic-Carugo,

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Arreola, S. L.; Intanon, M.; Suljic, J.; Kittl, R.; Pham, N. H.; Kosma, P.;

462

Haltrich, D.; Nguyen, T.-H. Two β-galactosidases from the human isolate

463

Bifidobacterium breve DSM 20213: Molecular cloning and expression, biochemical

464

characterization and synthesis of galacto-oligosaccharides. PLoS ONE 2014, 9,

465

doi:10.1371/journal.pone.0104056.

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Kulbe, K. D.; Haltrich, D. Purification and characterization of two novel beta-

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galactosidases from Lactobacillus reuteri. J. Agric. Food Chem. 2006, 54, 4989-4998.

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470

Haltrich, D. Production of prebiotic galacto-oligosaccharides from lactose using β-

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galactosidases from Lactobacillus reuteri. J. Agric. Food Chem. 2006, 54, 4999-5006.

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473

thermostable β-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus:

474

kinetic studies of the reactions of galactosylated enzyme intermediates with a range of

475

nucleophiles. J. Biochem. 2001, 130, 341-9.

476

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477

oligosaccharides by graphitic carbon high-performance liquid chromatography with

478

tandem mass spectrometry. Anal. Biochem. 2013, 433, 28-35.

479

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galactose disaccharides using β-galactosidases. Carbohydr. Res. 1997, 303, 339-345.

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482

N-acetyllactosamine and N-acetylallolactosamine by the use of β-D-galactosidases. J.

483

Carbohyd. Chem. 1992, 11, 553-565.

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485

acetyllactosamine in an organic co-solvent. Carbohydr. Res. 2000, 327, 377-83.

Nguyen, T.-H.; Splechtna, B.; Steinböck, M.; Kneifel, W.; Lettner, H. P.;

Splechtna, B.; Nguyen, T.-H.; Steinböck, M.; Kulbe, K. D.; Lorenz, W.;

Petzelbauer, I.; Splechtna, B.; Nidetzky, B. Galactosyl transfer catalyzed by

Bao, Y.; Chen, C.; Newburg, D. S. Quantification of neutral human milk

Finch, P.; Yoon, J. H. The effects of organic solvents on the synthesis of

Sakai, K.; Katsumi, R.; Ohi, H.; Usui, T.; Ishido, Y. Enzymatic syntheses of

Yoon, J. H.; Rhee, J. S. The efficient enzymatic synthesis of N-

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Vetere, A.; Paoletti, S. High-yield synthesis of N-acetyllactosamine by

487

regioselective transglycosylation. Biochem. Biophys. Res. Commun. 1996, 219, 6-13.

488

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489

yield and enzyme stability in the β-galactosidase catalysed synthesis of N-

490

acetyllactosamine. Org. Process Res. Dev. 2002, 6, 553–557.

491

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492

synthesis with β-galactosidases from Sulfolobus solfataricus, Aspergillus oryzae, and

493

Escherichia coli. Enzyme Microb. Technol. 1999, 25, 509-516.

Kaftzik , N.; Wasserscheid, P.; Kragl, U. Use of ionic liquids to increase the

Reuter, S.; Nygaard, A. R.; Zimmermann, W. β-Galactooligosaccharide

494

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495

FIGURE LEGENDS

496

Figure 1. D-Glucose/D-Galactose ratio (A) and formation and degradation of galacto-

497

oligosaccharide (B) during lactose conversion by β-galactosidases from L. reuteri (●),

498

L. bulgaricus (○), B. breve β-gal I (▲), and B. breve β-gal II (∆). The reactions were

499

performed at 30 °C at an initial lactose concentration of 200 g L-1 in sodium

500

phosphate buffer (pH 6.5) and 1 mM MgCl2.

501

Figure 2. GalGlc/Galβ-D-Galp-(14)-D-Glc (or GalGlc/GalLac) ratio during lactose

502

conversion (A) and D-Glc/D-Gal ratio during galacto-oligosaccharides (solid line) and

503

N-acetyl-oligosaccharides (dashed line) formation (B). The reactions for galacto-

504

oligosaccharides production were performed at 30 °C at an initial lactose

505

concentration of 200 g L-1 in sodium phosphate buffer (pH 6.5) and 1 mM MgCl2. The

506

reactions for N-acetyl-oligosaccharides production were at 30 °C with equimolar of

507

lactose and GlcNAc (600 mM each) in sodium phosphate buffer (pH 6.5) and 1 mM

508

MgCl2. The enzymes are β-galactosidases from L. reuteri (●), L. bulgaricus (○), B.

509

breve β-gal I (▲), and B. breve β-gal II (∆).

510

Figure 3. Formation of 6’-galactobiose (β-D-Galp-(16)-D-Gal) (A) and tri-galacto-

511

oligosaccharides (B) during lactose conversion catalyzed by B. breve β-gal II in the

512

absence (solid line) and presence (broken line) of N-acetyl-D-glucosamine. The

513

reactions were performed at 30 °C at an initial lactose concentration of 600 mM in

514

sodium phosphate buffer (pH 6.5) and 1 mM MgCl2. Initial GlcNAc concentration

515

used was 600 mM.

516

Figure 4. (A) GalNAc transgalactosylation yield at different initial lactose and

517

GlcNAc molar ratios (solid line: 600 mM lactose and 600 mM GalNAc; dotted line:

518

600 mM lactose and 300 mM GalNAc; short dash: 300 mM lactose and 600 mM

519

GalNAc); and (B) HPLC-UV profile of GalNAc-containing oligosaccharides

23



Page 24 of 31

520

catalyzed by β-galactosidase from L. bulgaricus. The reaction was done in 50 mM

521

sodium phosphate buffer (pH 6.5) containing 1 mM Mg2+ incubated at 50 °C. Peaks I

522

and II are the tri- and di-GalNAc containing oligosaccharides, respectively and Peak

523

III is the free GalNAc.

24


Page 25 of 31


SCHEME

Scheme 1. Hydrolysis and galactosyl transfer reactions during the β-galactosidase catalyzed conversion of lactose. E, enzyme; Lac, lactose; Gal, galactose; Glc, glucose; Nu, nucleophile.

25



Page 26 of 31

TABLES

Table 1. Partitioning ratios (kNu/kwater, M-1) for reaction of β-galactosidases with exogenous nucleophiles and with water. Nucleophile β-galactosidase source

D-Glca

Lactose

GlcNAc

GalNAc

L-fucose

Bifidobacterium breve β-galI β-galII

6.73 ± 0.62 3.91 ± 0.44

1.61 ± 0.05 0.53 ± 0.02

1.01 ± 0.03 5.42 ± 0.05

0.36 ± 0.03 0.39 ± 0.02

1.27 ± 0.12 1.16 ± 0.05

Lactobacillus bulgaricus

9.36 ± 0.56

2.79 ± 0.15

16.8 ± 0.7

3.21 ± 0.26

0.54 ± 0.05

6.7 ± 0.3* 1.91 ± 0.12* 0.27 ± 0.01 1.07 ± 0.09 Lactobacillus reuteri a Measured with 10 mM oNPGal as substrate and calculated from the νoNP/νGal *Ref. 17

0.67 ± 0.06

26


Page 27 of 31


FIGURES

Figure 1. 27



Page 28 of 31

Figure 2.

28


Page 29 of 31


Figure 3.

29



Page 30 of 31

Figure 4.

30


Page 31 of 31


FOR TABLE OF CONTENTS ONLY

31


Transferase Activity of Lactobacillal and Bifidobacterial Î²

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