University of Groningen
Galacto-oligosaccharide synthesis using immobilized -galactosidase
Benjamins, Frédéric
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2014
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Benjamins, F. (2014). Galacto-oligosaccharide synthesis using immobilized -galactosidase [S.l.]: [S.n.]
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5.
Formation of allo-lactose during GOS
synthesis in lactose slurry using high
enzyme/substrate ratio
ABSTRACT
During the conversion of lactose to GOS, catalyzed by B. circulans β-galactosidase a
significant amount of allo-lactose is formed. It was shown that the rate of allo-lactose
formation is determined by the enzyme dosage, due to the fact that the reaction is
kinetically controlled. The increase of allo-lactose is caused by the (indirect)
transglycosylation of galactose to the C-6 carbon of a free glucose molecule and
secondly, the hydrolysis of trisaccharides and higher oligosaccharides that were built up
from allo-lactose. No evidence was found for the direct synthesis of allo-lactose by this
enzyme. The preferential substrate utilization of β-galactosidase from B. circulans gives
rise to accumulation of allo-lactose.
This chapter has been submitted as: Benjamins, E., van Leeuwen, S.S., van Lente, T.,
Cao, L. & Broekhuis, A.A. Formation of allo-lactose during GOS synthesis in lactose
slurry using high enzyme/substrate ratio.
183
5.1 Introduction
Galacto-oligosaccharides (GOS) are oligomers that can be enzymatically synthesized
from lactose by the reversed action of a β-galactosidase (EC 3.2.1.23). GOS molecules
are built up from β-D-glucose and β-D-galactose, linked by β1→4 and β1→6, β1→3 and
β1→2 linkages, however non-reducing oligosaccharides (e.g. α-D-Glcp-(1↔1)-β-D-Galp)
have also been characterized (Fransen et al., 1998). The linkage types that are formed
mainly depend on the source of β-galactosidase enzyme used (Alles et al., 1999; Toba,
Yokota, & Adachi, 1985). The use of different enzymes and process parameters have
great influence on linkage types and GOS compositions in commercial GOS products
such as Vivinal GOS, Cup-Oligo and OligomateTM (Coulier et al., 2009; Otieno, 2010).
In GOS processing, the use of single or multiple β-galactosidases has been reported. The
latter usually aims at conversion of the residual lactose to glucose and galactose which in
turn, can be removed by further processing. Approaches for the latter are for instance
process chromatography or “selective fermentation” by yeast strains (Giacomelli et al.,
2010; Li, Xiao, Lu, & Li, 2008; Sheu, Li, Duan, & Chen, 1998). The composition of the
GOS mixture is likely to be altered to some extent, however, since besides the residual
lactose, the synthesized GOS structures can also be suitable substrates for a second βgalactosidase (Usui et al., 1996). Splechtna et al. have investigated the GOS synthesis of
Lactobacillus reuteri and the linkage types that were formed. As opposed to a batch
process, in a continuous mode, β-galactosidase from L. reuteri showed up to 2-fold
higher specificity toward the formation of β-(1→6)-linked GOS, including β-D-Galp(1→6)-D-Glcp (allo-lactose) and β-D-Galp-(1→6)-D-Galp. Allo-lactose is an isomer of
184
lactose (Figure 1), differing in the glycosidic linkage position of galactose to glucose. The
authors indicated this could offer possibilities to synthesize more specific GOS products
(Splechtna, Nguyen, & Haltrich, 2007). Others have also found that allo-lactose is a
component that is formed in significant amounts when lactose is converted to GOS by βgalactosidase (Martínez-Villaluenga, Cardelle-Cobas, Corzo, Olano, & Villamiel, 2008;
Splechtna et al., 2006). Little is known regarding the biological relevance of allo-lactose,
apart from its function as the inducer for the lac operon. The lac operon is an operon
required for the transport and metabolism of lactose in Escherichia coli and some other
enteric bacteria. The lac operon is regulated by several factors including the availability
of glucose and of lactose, a dual control mechanism. When lactose is the available energy
source the cells can produce β-galactosidase to hydrolyze lactose.
(1)
Figure
1
(1)
Allo-lactose
(2)
(6-O-β-D-Galactopyranosyl-D-glucose)
and
(2)
lactose
(4-O-β-D-
Galactopyranosyl-D-glucose)
However, in the absence of lactose, if glucose is readily available, the lac repressor
prevents the production of β-galactosidase (McClean & Johnson, 2013). While the
impression might appear that lactose is the inducer of the lac operon and thus of the
production of β-galactosidases, it is actually allo-lactose that induces the lac operon as
shown by Jobe and Bourgeois (Jobe & Bourgeois, 1973). Lactose is immediately
185
converted to allo-lactose by β-galactosidase internally (Hall, 1982; Huber, Kurz, &
Wallenfels, 1976; Huber, Pisko-Dubienski, & Hurlburt, 1980). This is called direct
transglycosylation; i.e. glucose does not leave the active site after cleavage of the
glycosidic linkage. Galactose is subsequently transferred to the C-6 position of glucose.
This so-called internal-transgalactosylation route is a remarkable feature of various βgalactosidases leading to the formation of disaccharides other than lactose. Evidently,
these
non-lactose
disaccharides
can
also
be
formed
through
the
indirect
transglycosylation, i.e. the transfer of the galactose moiety to a glucose molecule outside
the enzymes’ catalytic center. The nature and concentration of these disaccharide species
vary and depend largely on the enzyme and the reaction extent. A number of disaccharide
GOS structures might completely disappear due to their integration into other
oligosaccharides with a higher degree of polymerization (DP) (e.g. tri- and
tetrasaccharides), while some are more inert and remain largely in the final reaction
mixture.
In the present study the formation of specific components is studied in the enzymatic
conversion of lactose by the β-galactosidase from Bacillus circulans both in its free and
immobilized form. Under varying conditions specific attention was paid to the formation
of allo-lactose and the mechanism by which this component was formed.
5.2 Materials and Methods
Lactose (Lactochem) was a kind gift from DMV Fonterra Excipients (Goch, Germany).
Vivinal® GOS was obtained from FrieslandCampina (Amersfoort, The Netherlands). The
β-galactosidase preparation from B. circulans (Biolacta N5) was purchased from Amano
186
Enzyme Inc. (Nagoya, Japan). The protein content was 13% as determined by Quick
StartTM Bradford protein assay Kit (Bio-Rad, Veenendaal, the Netherlands). Eupergit C
250L (methacrylic carrier with epoxide functionality) was a kind gift from Evonik
(Essen, Germany). Relizyme EC-HA beads were a gift from Resindion Srl. (Milano,
Italy). Allo-lactose was from Glentham Life Science (London, UK). 4'-galactosyllactose
was purchased from Carbosynth (Berkshire, UK). Hexokinase from Saccharomyces
cerevisiae, ATP and MgCl2, were purchased from Sigma-Aldrich (Zwijndrecht, The
Netherlands).
5.2.1 Experimental
Determination of enzymatic activity – The β-galactosidase activity was determined by
measuring the glucose release in a lactose solution by using a Glucose oxidase /
peroxidase (GOPOD) kit (Megazyme; Wicklow, Ireland). To 5 mL of 12% lactose
solution, pH 6.0, 1 mL enzyme solution, appropriately diluted, was added. The mixture
was incubated at 40 °C for exactly 10 min. The reaction was terminated by addition of 1
mL 1.5 M NaOH and the mixture was left in the water bath for another 5 min. After this,
1 mL 1.5 M HCl was added and the reaction tube was placed on ice. The liberated
glucose was determined with the GOPOD kit by measurement of the absorbance at 510
nm using a Biotek PW plate reader (Biotek, Winooski, VT, USA). The enzymatic activity
was expressed in Lactase Units. One lactase unit (LU) is defined as the amount of
enzyme that liberates 1 µmol of glucose per min at the early stage of the reaction at 40 °C
and pH 6.0. For immobilized enzyme, an amount of 5 - 10 mg of immobilized enzyme
was accurately weighed into a reaction tube and 1 mL of 0.1 M sodium acetate buffer, pH
6.0 was added. The activity measurement was the same; except occasional repeated
187
swirling was applied to prevent accumulation of immobilized enzyme at the bottom of the
tube. The activity of the immobilized enzyme was expressed as LU / g immobilized
material.
Preparation of lactose slurry - Lactose slurries were prepared by added 53 g 0.01M
KH2PO4, pH 6.3 to 70 g lactose while stirring. The slurry was heated to 58 °C and kept at
this temperature for at least 90 minutes to allow the saturation of the solution.
GOS synthesis in lactose slurry – The enzyme dosage for both free and immobilized
(Eupergit C250L and Relizyme EC-HA) enzyme was 15 LU / g lactose. The reaction
was carried out for 24 hours. After 24 hours the reaction with immobilized enzyme was
stopped by removal of immobilized enzyme from the GOS solution. The reaction with
free enzyme was stopped by heating the reaction mixture at 95 °C for 30 minutes.
GOS synthesis in lactose solution – A metastable lactose solution was prepared by
heating lactose (70 g lactose, 53 g 0.01M KH2PO4 buffer, pH 6.3) to 90 – 95 °C until a
clear solution was obtained and allowing the solution to cool off to 58 °C – 60 °C. The
(immobilized) enzyme dosage (E/S ratio) was 15LU / g lactose. The reaction was carried
out for 24 hours. After 24 hours the reaction was stopped by removal of immobilized
enzyme from the GOS solution.
GOS synthesis in liquid phase of a lactose slurry – After preparation of the lactose slurry
the stirrer was switched off to allow the crystalline lactose to precipitate. The supernatant
was decanted and brought into contact with B. circulans β-galactosidase immobilized on
188
Relizyme EC-HA. The reaction was carried out for 24 hour, samples were taken
periodically.
GOS synthesis in lactose slurry (long incubation) - A lactose slurry (70 g lactose, 53 g
0.01M KH2PO4 buffer, pH 6.3) was heated to 58 °C. The enzyme dosage (E/S ratio) was
4 LU / g lactose. The reaction was carried out for 96 hours. After 96 hours reaction was
stopped by heating the GOS solution for 45 minutes at 80 °C.
Lactose conversion with glucose removal by hexokinase - The β-galactosidase from B.
circulans (10 LU / g lactose) was incubated with lactose (10% w/w), 20 mM MgCl2, 0.1
M phosphate buffer (pH 7), 20 µl of 10 mg / ml hexokinase from Saccharomyces
cerevisiae (50 U / mg), 0.35 M ATP and dH2O up to a volume of 1 ml. The temperature
was maintained at 35 °C due to the optimum activity of hexokinase. Additional
hexokinase (2 µl of 10 mg / ml) was added at 2, 4, 6 and 22 h to make sure that all the
glucose was phosphorylated. A control experiment did not have hexokinase and ATP in
the mixture. The control was supplemented with distilled water when hexokinase was
added to the other experiment. Reaction samples of 50 µl were taken and the reaction was
stopped by adding equal amounts of 5 % citric acid (pH 3). The experiment was carried
out in an Eppendorf tube that was placed in a water bath to control the temperature.
Lactose conversion in the presence of free glucose – The β-galactosidase from B.
circulans (15 LU / g lactose) was incubated with lactose (15% w/w), glucose (3% w/w)
and 0.1 M phosphate buffer (pH 6.3) in a volume of 100 ml. The temperature was kept at
58 °C. The control was carried out under the same conditions without the presence of free
189
glucose. Reaction samples of 1 ml were taken and stopped by adding equal amounts of 5
% citric acid (pH adjusted to 3). The experiment was carried out in a 100 ml jacketed
glass reactor to maintain a constant reaction temperature.
Fractionation of GOS - Separate DP-fractions of Vivinal GOS were obtained by
preparative FPLC using a XK50 column (50x100 cm) (GE Healthcare, Diegem, Belgium)
filled with Biogel P2 resin (Bio-Rad, Veenendaal, The Netherlands) and an Äkta FPLC
system (GE Healthcare, Diegem, Belgium). GOS syrup was diluted with demineralized
water to approximately 38% solids and was applied to the column via a 2 mL sample
loop. The system was operated at 30 °C.
Fractions were obtained via the sample
collector of the Äkta system. Separate fractions of repetitive fractionations were pooled
and lyophilized.
Incubation of separate DP fraction with B. circulans β-galactosidase – The lyophilized
DP3 fraction of the commercial GOS mixture, were separately dissolved in a 0.1M
sodium acetate buffer, pH 6.0. Since the β-galactosidase preparation contains a
considerable amount of lactose, incubations were carried out with the enzyme
immobilized on Eupergit C250L to ensure the absence of lactose as a substrate at the
beginning of the incubation. The re-dissolved DP fractions were transferred in a HPLC
vial with a screw cap and the vial was placed in a container and the whole was placed in a
shaker / incubator (VWR, Amsterdam, the Netherlands). The shaking speed was 100 rpm
and the incubation temperature was 58 °C. Aliquots of 100 µL were withdrawn
190
periodically and the enzyme was inactivated by addition of 100 µL of a 5% citric acid
solution. The samples were stored in the refrigerator or freezer until further analysis.
5.2.2 Analytical
NMR spectroscopy – Lyophilised samples were exchanged twice with 99.9%atom D2O
(99.9%atom, Cambridge Isotope Laboratories Inc.) with intermediate lyophilisation.
Samples were dissolved in 550 µL D2O, containing acetone (δ 2.225) as internal standard.
One-dimensional 1H NMR spectra were recorded on a Varian Inova 500 spectrometer
(GBB, NMR Center, University of Groningen) at probe temperatures of 300K, with a
spectral width of 5000 Hz and zero filled to 32k. A pre-saturation pulse was applied to
suppress the HOD signal. Spectra were processed with MestReNova 5.3 (Mestrelabs
Research SL, Santiago de Compostella, Spain).
HPAEC-PAD isolation – Incubated samples were separated on a Carbopac PA-1 (250 x 9
mm; Dionex BV, Amsterdam, The Netherlands) column, using a Dionex DX500
instrument (Dionex BV, Amsterdam, The Netherlands), with a ED40 pulsed
amperometric detector. Components were eluted with a gradient of NaOAc and NaOH.
Isolated peaks were desalted on Carbograph SPE (300 mg, Grace) columns, eluted with
40 % acetonitrile, containing 0.05% TFA. Samples were dried under N2 stream, followed
by lyophilisation.
HPAEC-PAD analysis - GOS samples were analyzed using a Dionex ICS-3000 HPAECPAD instrument (Dionex BV, part of Thermo Fisher Scientific, Amsterdam, The
Netherlands) equipped with a CarboPac PA1 column (250 x 4 mm; Dionex BV, part of
191
Thermo Fisher Scientific, Amsterdam, The Netherlands), with a DC-1 pulsed
amperometric detector. Components were eluted with gradients of sodium hydroxide and
sodium acetate at a flow rate of 1.0 mL/min and a column temperature of 30 °C.
HPLC-RI analysis – The DP-distribution of GOS was obtained by GPC using an
Ultimate 3000 HPLC system (Dionex BV, part of Thermo Fisher Scientific, Amsterdam,
The Netherlands) equipped with a Phenomenex Rezex RSO-Oligosaccharide AG column
(200 x 10 mm; Phenomenex, Torrance, USA) and a Shodex R-101 Refractive Index
detector (Showa Denko Europe GmbH, Munich, Germany). Water was used as the
mobile phase. The column temperature was 75 °C.
5.3 Results and discussion
GOS synthesis in lactose slurry
The synthesis of GOS from lactose slurry was carried out with the immobilized βgalactosidase from B. circulans immobilized on Eupergit C250L and Relizyme ECHA. A notable increase in allo-lactose was observed when using immobilized enzyme
with an E/S ratio of 15 LU / g of lactose. Figure 21 shows the overlay of the HPAECPAD chromatograms for the products obtained in time during 24 hour incubation with B.
circulans β-galactosidase immobilized on Eupergit C250L. Incubation of lactose slurry
with B. circulans β-galactosidase immobilized on Relizyme EC-HA gave a similar
result. The observed increase of allo-lactose however was not a result of the
immobilization of the enzyme, since a similar increase was observed under the same
conditions with the soluble enzyme (results not shown). The quantification of allo-lactose
192
was performed using HPAEC-PAD, based on the description by Coulier et al. (Coulier et
al., 2009). The commercially available product Vivinal GOS, which is also produced
with the β-galactosidase from B. circulans, contains typically 6% allo-lactose expressed
on the total solids content.
Glc
Allo-lac
Gal
Lac
24 h
23 h
8h
4h
1h
0
5
10
15
20
25
Figure 2. Overlay of HPAEC-PAD chromatograms of samples taken during the incubation of lactose slurry
with immobilized Biolacta N5 on Eupergit C250L.
In the commercial sample used in this study a similar concentration was found (results
not shown). For the samples obtained with B. circulans β-galactosidase immobilized on
Eupergit C250L en Relizyme EC-HA, the allo-lactose values were 18.9% and 11.3%
(w / w), respectively after 24 h of incubation. Since the E/S ratio was based on the total
amount of substrate present in the system, but not all substrate is available due to the fact
that it is partially undissolved, the initial E/S ratio is even higher. The solubility of lactose
at 58 °C is approximately 37% (Machadoa, Coutinho. J.A., & Macedo, 2000) and
therefore the initial E/S ratio is 22 LU / g of lactose.
193
In Table 1 the composition of the GOS mixture during the 24-hour incubation is
displayed. Allo-lactose is displayed separately from the other GOS, thus providing a
better insight in the formation of allo-lactose in time during the reaction.
Table 1. Composition of GOS synthesized from lactose slurry using Biolacta immobilized on Eupergit
C250L.
% on total solids
Time
Galactose
Glucose
Lactose
Lactulose
GOS*
[h]
Allolactose
0.5
0.7
16.2
38.8
1.2
46.0
1.1
1
1.0
18.4
24.9
1.1
52.5
2.1
4
2.0
21.4
8.6
1.1
59.0
7.9
8
2.9
21.7
5.2
1.2
55.4
13.5
22
6.6
26.1
5.0
1.5
41.6
19.1
24
6.6
25.4
2.7
1.4
44.9
18.9
* Ex. allo-lactose
Figure 3 shows an overlay of HPLC-SEC chromatograms the DP distribution. Where
initially the distribution is comparable to that of the commercial product, after 24 hours
especially the higher DP fractions have been hydrolyzed indicated by the disappearing
peaks in the chromatogram and the increase in galactose.
194
24h
18h
DP2
2h
DP3
Glc
DP4
DP6
DP5
Gal
1h
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
RT [min]
Figure 3. Overlay of DP-distribution HPLC-SEC chromatograms of incubation of lactose with BN5
immobilized on Eupergit C250L. Conditions: 39% lactose, T = 58 °C, pH 6.3, E/S = 15 LU / g, t = 24
hours.
GOS synthesis in the liquid phase of lactose slurry
In order to investigate whether the presence of lactose crystals had an influence on the
formation of allo-lactose the reaction was carried out in a 37% lactose solution obtained
by decanting the supernatant after sedimentation of lactose slurry. During incubation of
enzyme in the supernatant of the lactose slurry, 19.9 % (w/w) allo-lactose was found.
Identification of allo-lactose by HPAEC-PAD
In order to confirm the increase of allo-lactose the sample containing the peak assigned as
allo-lactose was spiked with commercially available allo-lactose. The HPAEC-PAD
195
chromatogram of the sample is shown in Figure 4. The increase in area of the peak with
retention time 13.2 minutes is clearly visible.
Figure 4. Identification of allo-lactose by HPAEC-PAD – Solid line: GOS sample from synthesis using
immobilized enzyme.
Dashed line: Same sample spiked with commercially available reference allo-
lactose (Carbosynth, UK)
This provided already a strong indication that the detected compound was indeed allolactose. However, for definite characterization NMR spectroscopy was applied on the
peak isolated with preparative HPAEC-PAD.
196
Identification of allo-lactose using NMR
The GOS mixture obtained by incubation of lactose slurry with BN5 immobilized on
Relizyme EC-HA was used for isolation by HPAEC of the peak at 7.183 min, matching
elution of reference allo-lactose (Carbosynth). The retention time differs from the earlier
mentioned retention time for allo-lactose (Figure 2) because of a different gradient.
Isolated fractions were analyzed by 1D 1H NMR spectroscopy and compared to the 1D
1
H NMR spectrum of reference allo-lactose (Figure 5). The 1D 1H NMR profiles match
exactly, showing peaks at δ 5.226 (J1,2 4.0 Hz) and 4.654 (J1,2 8.1 Hz), corresponding with
α-D-Glcp and β-D-Glcp H-1, respectively, and δ 4.426 (J1,2 7.9) and 4.444 (J1,2 8.0),
corresponding with β -D-Gal-(1-6)- H-1, linked to α-D-Glcp and β -D-Glcp, respectively.
This typical splitting of the non-reducing anomeric proton is a common occurrence, also
observed in α-glucans linked by (1-2), (1-3) or (1-6) linkage to the reducing residue (van
Leeuwen et al., 2008). Further peaks at 4.162 (J5,6 1.5; J6,6’ -11.9) and 4.217 (J5,6 1.5; J6,6’
-11.6) corresponding to 6-substituted α-D-Glcp and β-D-Glcp, respectively and α-D-Glcp
H-5 at δ 3.989 ppm, are shifted downfield significantly and are important 1D 1H NMR
markers for allo-lactose. These data indicate that the peak at 7.183 min is indeed allolactose.
197
Figure 5. 1D 1H NMR spectra of A. reference allo-lactose (Carbosynth) and B. isolated peak at 7.183 min
from GOS sample produced with B. circulans β-galactosidase immobilized on Relizyme EC-HA. Distinct
peaks outside of the bulk-area are marked in the allo-lactose reference spectrum.
GOS synthesis in lactose slurry (long incubation)
Allo-lactose is formed through a kinetically controlled reaction, which determines its
yield, just like the yield of other transglycosylation products. The yield of products is
independent of the enzyme concentration. Therefore the E/S ratio in itself does not
determine the final yield. The time to reach this yield, however, is inversely proportional
to the enzyme concentration (Kasche, Haufler, & Riechmann, 1984). The results of an
experiment with a lower E/S ratio combined with an extended incubation time support
this. During a 96-hour incubation with a E/S ratio of 4 LU / g of lactose (initially 5.9 LU /
198
g of lactose due to the slurry system), the final product obtained also shows a significant
increase in allo-lactose (Figure 6).
Glc
Lac
Allo-lac
Gal
96 h
20 h
4.5 h
1h
0
5
10
15
20
25
Figure 6. HPAEC-PAD chromatograms overlay of incubation of lactose slurry with free B. circulans βgalactosidase. Conditions: Lactose 55% (w/w/), T = 58 °C, pH 6.3, t = 96 h.
Although the increase is lower than observed earlier during the 24-hour / high E/S
experiments, the concentration of allo-lactose is still 33% higher than the levels found in
the commercial sample. The lower concentration of allo-lactose found in this experiment
compared to the previously mentioned experiments, can be attributed to the inactivation
of the enzyme due to the long incubation time at a temperature of 58 °C. The composition
after 96 hours was 2.3% galactose, 29.0% glucose, 12.4% lactose, 0.9% lactulose, 10.0%
allo-lactose and 46.3% GOS (ex. allo-lactose).
These results raise two questions. Firstly, what is the explanation for the increase of allolactose at the cost of other oligosaccharides? And secondly, how is allo-lactose formed?
199
Whereas Huber et al. (Huber et al., 1976; Huber et al., 1980) indicated that 50% of the
allo-lactose is formed initially in the beginning of the reaction with E. coli βgalactosidase, remarkably, results from our experiments show that especially in a later
stage of the reaction relatively large amounts of allo-lactose are formed. The reason for
this apparent contradiction could be that allo-lactose is an excellent acceptor for a
galactose moiety and therefore reacts very quickly to form trisaccharides and thus only
limited concentrations can be detected. On the other hand, allo-lactose is possibly a poor
substrate for the β-galactosidase from B. circulans. This was observed by Warmerdam et
al. (Warmerdam, Paudel, Jia, Boom, & Janssen, 2013) who used allo-lactose as substrate
in Isothermal Titration Calorimetry (ITC) experiments. The "poor substrate" properties
were derived from the incomplete (13% and 21%) degradation of allo-lactose by β galactosidase A and β-galactosidase D, respectively. In contrast, 4'-galactosyllactose was
broken down for more than 90% (applies to both β -galactosidases). This is likely the
explanation for the accumulation during the conversion of lactose to GOS. When given
sufficient time, the hydrolysis reaction will eventually yield glucose and galactose
(Gosling et al., 2009; Nakanishi, Matsuno, Torii, Yamamoto, & Kamikubo, 1983) after
breaking down previously formed structures in a certain order of substrate preference.
Preferential substrate utilization causes also the observed increase of allo-lactose during
long incubations of B. circulans β-galactosidase. Clearly the breakdown of larger
structures is visible and an accumulation of allo-lactose takes place instead. (See also
GOS synthesis in lactose slurry (long incubation). These results also confirm the poor
substrate properties of allo-lactose for β -galactosidases from B. circulans.
200
Lactose conversion in the presence of free glucose
The mechanism for allo-lactose formation by E. coli cells was already described by
Wallenfels and Malhotra in 1961 (Wallenfels & Malhotra, 1961). The authors
distinguished between direct transglycosylation and indirect transglycosylation. The
former is considered to be the mechanism for intracellular allo-lactose formation. It is,
however, unclear if the formation of allo-lactose in the previously described experiments
follows the same mechanism, since allo-lactose formation seems to occur mainly in a
later stage of the reaction. If the formation of allo-lactose occurs solely via intramolecular
rearrangement, addition of free glucose would not influence the allo-lactose
concentration. During incubation in the presence however, a faster increase in allo-lactose
is observed than during incubation with only lactose present (Figure 7). These results
indicate that allo-lactose synthesis by B. circulans β-galactosidase occurs in any case
through the indirect transglycosylation mechanism.
4000
3500
Allo-lactose [ppm]
3000
2500
2000
1500
1000
500
0
0
1
2
3
4
5
6
7
Time [h]
Figure 7. Formation of allo-lactose during incubation of lactose with B. circulans β-galactosidase. Closed
triangles: 15% lactose, open squares: lactose + glucose (3%). T = 58 °C, pH 6.3. Lines to guide eye.
201
Lactose conversion with glucose removal by hexokinase
In order to investigate the occurrence of allo-lactose formation by direct
transglycosylation, free D-glucose, released during the conversion of lactose, was
removed by simultaneous incubation with a hexokinase (Figure 8). After 6 h of
incubation allo-lactose becomes detectable. These results indicate that in case the
formation of allo-lactose through direct transglycosylation occurs, it is only minimal. The
increase after 6 h is likely caused by a decrease in activity of the hexokinase and a
subsequent increase in glucose, which in turn leads to indirect transglycosylation.In E.
coli both mechanisms were shown to occur (Huber, R. E., Kurz, G., & Wallenfels, K.,
1976). The allo-lactose formation rate of β-galactosidase from E. coli seems to be much
higher with than that of B. circulans β-galactosidase. This can be explained by the fact
that two mechanism simultaneously take place in E. coli. Moreover, E. coli β galactosidase shows a strong preference for the formation of β(1-6) linkages during
transglycosylation (Reuter S, Rusborg Nygaard A. & Zimmermann W., 1999) and thus is
more likely to form allo-lactose.
202
3000
2500
Allo-lactose [ppm]
2000
1500
1000
500
0
0
5
10
15
Time [h]
20
25
30
35
Figure 8. Allo-lactose formation during lactose (10%) incubation with Biolacta N5. Glucose removal with
hexokinase (closed triangles) No glucose removal (open squares). Lines to guide the eye. Conditions: pH 7,
T = 35 °C.
Incubation of separate DP fraction with B. circulans β-galactosidase
Next to the synthesis of allo-lactose by transglycosylation, its increase can also be caused
by the hydrolysis of trisaccharides that were built up from allo-lactose initially. In order
to investigate the latter possibility, the DP3 fraction of a GOS produced with B. circulans
β-galactosidase was isolated and incubated with Biolacta N5 immobilized on Eupergit
C250L. Immobilized enzyme was chosen in order to prevent the presence of lactose in
the initial reaction mixture (the commercial enzyme preparation is standardized with
lactose). Figure 9 is an overlay of the samples that were taken during the incubation. The
decrease of a number of DP3 peaks is clearly visible as well as the formation of allolactose, lactose, and galactose.
203
Glc
Gal
Allo-lac
Lac
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Figure 9. Incubation of GOS DP3 fraction with immobilized B. circulans β-galactosidase. Conditions: T
58 °C, pH 6.3, t = 24h. Black line is t = 0 min, red line is t = 24 h (other lines are intermediate times).
This indicates that due to hydrolysis of trisaccharides allo-lactose can be formed. For
instance when the galactose at the non-reducing end of the trisaccharide, β-D-Galp(1→4/6)-β -D-Galp(1→6)-β-D-Glcp is hydrolyzed, allo-lactose (β-D-Galp(1→6)β-D-Glcp)
is formed. This obviously requires initial formation of allo-lactose as a scaffold for the
formation of trisaccharides.
Both mechanisms may have occurred during the described experiments; as a consequence
of the enzymatic action of a β -galactosidase, both during hydrolysis as well as
transglycosylation, glucose is released. The HPAEC-PAD analysis of the samples that
were obtained during the incubation of the separate DP3 fraction of Vivinal GOS with
immobilized B. circulans β-galactosidase demonstrate that allo-lactose is indeed used a
building block for the construction of higher DP species. Historically, allo-lactose is
considered as one of the primary trans-galactosylation products of E. coli β-galactosidase
204
(Huber et al., 1976). These new DP2 GOS can be further used for building blocks to
construct higher DP GOS species together with lactose, as confirmed by Pazur et al.
already in the 1950s (Pazur, Marsh, & Tipton, 1958). For the B. circulans β-galactosidase
as well as β -galactosidases from other sources the formation of allo-lactose was
described. For instance, 3 new DP2 species, namely β-D-Galp-(1→3)-β-D-Glcp, β-DGalp-(1→2)-D-β-Glcp and β-D-Galp-(1→6)-β-D-Glcp i.e. allo-lactose were found to be
present in the GOS product formed by β-galactosidase from B. circulans. Two new DP2
species, namely β-D-Galp-(1→6)-β-D-Galp, β-D-Galp-(1→6)-β-D-Glcp were found in the
GOS product formed with β-galactosidase from K. lactis (Park & Oh, 2010). Similarly,
the presence of β-4,6-di-galactosylglucose - a branched DP3 GOS (two galactosyl groups
are linked to one glucose moiety) species and β-D-Galp-(1→4)-[β -D-Galp-(1→6)]-D-Glcp
in the GOS product formed by β-galactosidase from A. oryzae also suggested that allolactose is one of the DP2 intermediates (Alles et al., 1999; Neri et al., 2011). The fact that
β-4,6-di-galactosylglucose was formed as the main product even at the beginning of the
GOS synthesis strongly suggest that allo-lactose might be directly used by the B.
circulans β-galactosidase to form higher DP GOS without releasing into the reaction
mixture (Yanahira et al., 1995). Remarkably, the diversity of DP2 formed by a βgalactosidase also reflects, to some extent, the flexibility of the enzyme active center.
Hence, more DP2 often means that more higher DP GOS species can be formed on the
basis of these DP2 intermediates.
205
5.4 Conclusion
During the conversion of lactose to GOS, catalyzed by B. circulans β-galactosidase a
significant amount of allo-lactose is formed. It was shown that the rate of allo-lactose
formation is indeed determined by the enzyme dosage, due to the fact that the reaction is
kinetically controlled. The increase of allo-lactose is caused by two factors, namely the
(indirect) transglycosylation of galactose to the C-6 carbon of a free glucose molecule
and secondly, the hydrolysis of trisaccharides and higher oligosaccharides that were built
up from allo-lactose.
No evidence for the direct synthesis of allo-lactose by this enzyme was found in this
study, but if this mechanism occurs it is only minimal. The preferential substrate
utilization of β-galactosidase from B. circulans gives rise to accumulation of allo-lactose.
Acknowledgement
This project is jointly financed by the European Union, European Regional Development
Fund and The Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the
Delta, the Municipality of Groningen, the Provinces of Groningen, Fryslân and Drenthe
as well as the Dutch Carbohydrate Competence Center (CCC WP9).
206
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