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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:
Journal of Agricultural and Food Chemistry
* Corresponding author: Thu-Ha Nguyen Phone: 43-1-47654 6148. Fax: 43-1-47654 6199. E-mail:
[email protected] 1
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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
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INTRODUCTION
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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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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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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
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method described previously.15 D-Glc, D-Gal, lactose, and GOS components were
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identified and quantified using the external standard technique.
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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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N-acetyl oligosaccharide formation. N-acetyl-oligosaccharide synthesis was
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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
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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
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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
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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,
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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
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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
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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
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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
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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
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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
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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),
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This material is available free of charge via the Internet at http://pubs.acs.org
414 415
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enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme Microb.
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Technol. 2000, 26, 271-281.
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Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans
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β-galactosidase. Biosci. Biotech. Biochem. 1998, 62, 1791-1794.
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permeabilized cells of Kluyveromyces lactis. Appl. Microbiol. Biotechnol. 2004, 64,
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787-793.
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transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose. Biosci.
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Biotech. Biochem. 2004, 68, 2086-2090.
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galactosidase catalyzed selective galactosylation of aromatic compounds. Biotechnol.
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Prog. 2006, 22, 326-330.
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value-added derivatives. Int. Dairy J. 2008, 18, 685-694.
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lactose derivatives (hetero-oligosaccharides) from lactose. Int. Dairy J. 2012, 22, 116-
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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
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fructose by a thermostable β-galactosidase from Sulfolobus solfataricus. Enzyme
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Microb. Technol. 2006, 39, 903-908.
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Structural identification of novel oligosaccharides produced by Lactobacillus
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bulgaricus and Lactobacillus plantarum. J. Agric. Food Chem. 2012, 60, 4886-94.
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and N-acetylglucosamine as substrates by using β-D-galactosidase from Bacillus
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circulans. Eur. Food Res. Technol. 2010, 231, 55-63.
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beta-galactosidases from Kluyveromyces lactis and Bacillus circulans in the
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enzymatic synthesis of N-Acetyl-lactosamine in aqueous media. Biotechnol. Prog.
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2011, 27, 386-94.
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relationships for β-galactosidase (Escherichia coli, lac Z). 2. Reactions of the
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galactosyl-enzyme intermediate with alcohols and azide ion. Biochemistry 1995, 34,
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11713-24.
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Cloning and expression of the β-galactosidase genes from Lactobacillus reuteri in
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Escherichia coli. J. Biotechnol. 2007, 129, 581-591.
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K.; Mathiesen, G.; Nguyen, T.-H.; Haltrich, D. Homodimeric β-galactosidase from
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Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus
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plantarum and biochemical characterization. J. Agric. Food Chem. 2012, 60, 1713-
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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.;
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Haltrich, D.; Nguyen, T.-H. Two β-galactosidases from the human isolate
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Bifidobacterium breve DSM 20213: Molecular cloning and expression, biochemical
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characterization and synthesis of galacto-oligosaccharides. PLoS ONE 2014, 9,
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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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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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thermostable β-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus:
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kinetic studies of the reactions of galactosylated enzyme intermediates with a range of
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nucleophiles. J. Biochem. 2001, 130, 341-9.
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oligosaccharides by graphitic carbon high-performance liquid chromatography with
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tandem mass spectrometry. Anal. Biochem. 2013, 433, 28-35.
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galactose disaccharides using β-galactosidases. Carbohydr. Res. 1997, 303, 339-345.
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N-acetyllactosamine and N-acetylallolactosamine by the use of β-D-galactosidases. J.
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Carbohyd. Chem. 1992, 11, 553-565.
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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
(24)
489
yield and enzyme stability in the β-galactosidase catalysed synthesis of N-
490
acetyllactosamine. Org. Process Res. Dev. 2002, 6, 553–557.
491
(25)
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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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
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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.
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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.
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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
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FIGURES
Figure 1. 27
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Figure 2.
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Figure 3.
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Figure 4.
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