a r t i c l e i n f o a b s t r a c t
Article history: In this study, the combination of two b-galactosidases to synthesize prebiotic galactooligosaccharides
Received 8 October 2018 (GOS) was evaluated in terms of total GOS yield as well as GOS structures (chain length). Two different
Accepted 15 October 2018 combinations of either Aspergillus oryzae and Cryptococcus laurentii or Aspergillus oryzae and Kluyver-
Available online xxx
omyces lactis were tested to examine the influence of enzyme origin. Neither consecutive nor simulta-
neous synthesis with A. oryzae and C. laurentii led to an increased GOS yield. However, with the latter,
Keywords:
synthesis of higher GOS (3 monomer units) was enhanced from 38.5% to 40% with special emphasis of
Galactooligosaccharide
tetra- and pentasaccharides, which increased from 6.7% to 12.8% and from 0.4% to 3.3%, respectively.
GOS
Prebiotics
Additionally, due to the different preferences of the two b-galactosidases in terms of types of glycosidic
Beta-galactosidase linkages, the structural diversity of the final GOS product could be increased. Using K. lactis following the
Transgalactosylation synthesis with A. oryzae increased the yield of total GOS from 24.6% to 33.1%, which was mainly due to
Combination the formation of GOS disaccharides. On the other hand, applying A. oryzae as the second enzyme led to a
degradation of di- and trisaccharides, and thus total GOS yield was diminished, although the yield of
tetrasaccharides could be enhanced. In conclusion, with both studied enzyme combinations it was
possible to increase the percentage of higher GOS and reduce the residual lactose content of the final
mixture, which is beneficial for subsequent purification processes. Thus, using more than one b-galac-
tosidase during the synthesis of GOS represents an interesting research area, which should be explored in
more detail in the future.
© 2018 Elsevier Inc. All rights reserved.
1. Introduction With the enzyme source being one of the most important fac-
tors influencing GOS yield [9], as well as GOS structure in terms of
The research in the optimization of the synthesis of gal- b-glycosidic linkage [10], we propose that the combination of two
actooligosaccharides (GOS) is of ongoing interest as they exhibit b-galactosidases from different origins, which thus have different
prebiotic properties, and thus are beneficial compounds for infants preferences for transgalactosylation and hydrolysis, might lead to
[1e3] and adults [4e6] alike. GOS are generated in a side reaction an enhanced GOS yield. However, research on this topic is very rare
called transgalactosylation during the hydrolysis of lactose by b- with only a handful of publications during a period of more than 30
galactosidase (EC 3.2.1.23), if the acceptor molecule is another sugar years of GOS research. While Yakult Pharmaceutical Industry Co.,
instead of water [7]. As this sugar molecule can be any sugar in the Ltd uses two enzymes from Sporobolomyces singularis and Kluy-
reaction mixture, that is glucose, galactose, lactose, or GOS itself, veromyces lactis during the production of Oligomate® [11], with the
the chain length (or degree of polymerization (DP)) of the gener- second enzyme aiming mainly for hydrolysis of unreacted lactose, it
ated GOS will increase with ongoing reaction time. However, GOS is not clear whether the second enzyme also contributes to the total
are also possible substrates for the enzyme and their hydrolysis will GOS yield. Similar, Vitalus Nutrition Inc. [12] submitted a patent
exceed their synthesis after a certain degree of lactose conversion is application for a consecutive combination of b-galactosidases from
reached [8]. Aspergillus oryzae and K. lactis, which led to an increased GOS yield
from about 32% to 41%. On the other hand, Moon et al. [13] reported
a reduced synthesis of GOS tri- and tetrasaccharides (disaccharides
* Corresponding author. were unchanged), when A. oryzae and Kluyveromyces fragilis were
E-mail addresses: christin.fischer@hs-anhalt.de (C. Fischer), thomas. used simultaneously in comparison to K. fragilis alone, ultimately
kleinschmidt@hs-anhalt.de (T. Kleinschmidt).
https://doi.org/10.1016/j.bbrc.2018.10.091
0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
2 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
resulting in a decreased total GOS yield (37% compared to 27%). A the consecutive (with inactivation of the first enzyme by heat
consecutive application of A. oryzae followed by Bacillus circulans treatment for 5 min at 95 C) or simultaneous combination of
could increase GOS yield (DP3-DP6) from 26% to 34% [14]. However, A. oryzae and K. lactis as well as A. oryzae and C. laurentii. Lactose
this was considerably lower compared to using only the B. circulans was dissolved in PEM buffer pH 6.5 for the former two enzymes and
enzyme (40% GOS yield). Investigating the consecutive (but McIlvaine buffer pH 4.5 for the latter two enzymes at a concen-
without inactivation of the first enzyme) and simultaneous tration of 200 g/L for all experiments. Enzyme/substrate ratios were
coupling of b-galactosidases from B. circulans with those from based on the initial lactose concentration and set to 50 UONPG/
A. oryzae or K. lactis, also showed no increase in GOS yield (exem- gLactose for A. oryzae and K. lactis and to 1 UONPG/gLactose for
plified by the two main trisaccharides) compared to B. circulans C. laurentii. In the simultaneous approach, both enzymes were used
alone [15]. This study aims to investigate the consecutive and in a ratio of 1:1. Reaction temperature was set to 45 C for K. lactis
simultaneous combination of different b-galactosidase enzymes and the simultaneous A. oryzae/K. lactis experiments. All other re-
and its effect on total GOS yield, as well as potential compositional actions were carried out at 55 C. Samples were withdrawn at
changes. different time intervals and enzymes were inactivated by heating at
95 C for 5 min in a thermo shaker (model 5436 from Eppendorf
2. Materials and methods Vertrieb Deutschland GmbH, Germany).
The b-galactosidase enzymes were from Aspergillus oryzae GOS were analyzed on a LaChrom Elite HPLC system (VWR In-
(Maxilact A4 from DSM Food Specialties B.V., The Netherlands) and ternational GmbH, Germany). For the determination of the sugar
Kluyveromyces lactis (optilactase LX2 from optiferm GmbH, Ger- classes according to chain length, a Hi-Plex Na column
many). Additionally, whole cells of Cryptococcus laurentii (strain (300 mm 7.7 mm, Agilent Technologies Sales & Services GmbH &
DSM 27153 from Leibniz Institut DSMZ - Deutsche Sammlung von Co. KG, Germany) was used at 80 C with 0.2% sodium azide in
Mikroorganismen und Zellkulturen GmbH, Germany) were used. water as an eluent and a flow rate of 0.3 mL/min. GOS-disaccharides
Cells were cultivated in an enzyme production medium proposed were separated from lactose either on a Zorbax Carbohydrate col-
by Ohtsuka et al. [16]. A preculture was grown in 15 mL medium umn (250 mm 4.6 mm, Agilent Technologies Sales & Services
(100 mL Erlenmeyer flask) at 30 C and 160 rpm in a rotary shaker GmbH & Co. KG, Germany; eluent 75:25 acetonitrile:water, 1.4 mL/
(model KS 4000 i control, IKA®-Werke GmbH & Co. KG, Germany) min, 35 C) or a Microsorb-MV 100 NH2 column (250 mm 4.6 mm
for 24 h. The main culture (100 mL in a 500 mL Erlenmeyer flask) with guard column Polaris 5 NH2 MetaGuard, Agilent Technologies
was inoculated with 2% of the preculture and incubated (30 C, Sales & Services GmbH & Co. KG, Germany; eluent 70:30 acetoni-
160 rpm) for 78e96 h until the stationary growth phase was trile:water, 1.3 mL/min, 40 C). Both columns had a very similar
reached (data not shown). Cells were harvested by centrifugation peak pattern and could therefore be used equally (see
(25,000 g, 10 min, 4 C, Sigma 3K30 from Sigma Laborzentrifugen supplemental Fig. S1). Sugars were detected by refractive index and
GmbH, Germany), washed once with buffer (50 mM sodium concentrations were determined using external standard calibra-
phosphate pH 5.0), resuspended in the same buffer, and stored tions. For the GOS structures, lactose was used for disaccharides,
at 20 C until use. raffinose for trisaccharides and maltotetraose for tetrasaccharides
and higher oligosaccharides. Samples were subjected to protein
2.2. Enzyme activity assay precipitation using Carrez reagent, diluted appropriately and
filtered through a PES 0.22 mm syringe filter (Carl Roth GmbH,
b-Galactosidase activity was measured using o-nitrophenyl-b-D- Germany).
galactopyranoside (oNPG) as the substrate. For the enzymes Max-
ilact A4 and optilactase LX2, 480 mL buffered substrate solution 3. Results and discussion
(30 mM oNPG) was tempered at least 10 min in a thermo shaker
(model TS-100C from Biosan, Lativa), then 20 mL of appropriately 3.1. Combination of A. oryzae and C. laurentii
diluted enzyme solution were added. The reaction was stopped
after 10 min by addition of 0.4 M Na2CO3 and absorbance was Fig. 1 displays the total GOS yield as a function of lactose con-
measured at 420 nm. For the C. laurentii cell solution, 700 mL of a version obtained by various combinations of b-galactosidases from
65 mM oNPG solution was used, 50 mL of the cell solution was A. oryzae and C. laurentii. In general, C. laurentii exhibits a much
added and the reaction was stopped after 10 min by adding 750 mL higher transfer activity compared to A. oryzae with the maximum
of 1 M Na2CO3. Samples were centrifuged for 10 min (model VR-1 GOS yield being about twice as high (50% compared to 24%). At the
from Heraeus Instruments GmbH, Germany) to remove the cells. same time, the former has a very low hydrolytic activity with a GOS/
The supernatant was transferred into cuvettes and then measured galactose ratio of 55.6 compared to 2.5 for A. oryzae (see also
at 420 nm. Assay buffers and temperatures were selected according supplemental Table S1). In the consecutive combination with
to the GOS synthesis parameters, that is PEM buffer pH 6.5 (50 mM A. oryzae, the enzyme from C. laurentii was able to continue GOS
potassium phosphate containing 1 mM MgSO4 * 7 H2O and 0.056 mM synthesis, and thus increased GOS yield by 20%. This was mainly
EDTA * 2 H2O according to the FCC method [17]) at 45 C for opti- because of the formation of new disaccharide structures (increase
lactase LX2, 50 mM McIlvaine buffer pH 4.5 and PEM buffer pH from 2.6% to 12%), but the concentration of tri- and tetrasaccharides
6.5 at 55 C and 45 C for Maxilact A4, and 50 mM McIlvaine buffer was also enhanced by 5.9% and 3.9%, respectively (see
pH 4.5 at 55 C for C. laurentii. One unit was defined as the amount supplemental Fig. S2). Probably because of an inhibiting effect of
of enzyme that releases 1 mmol of o-nitrophenol per minute under glucose and/or galactose, which were present at the start of the
the respective assay conditions. reaction due to the hydrolysis activity of the first enzyme
(A. oryzae), the total GOS yield (44%) compared to the single
2.3. Galactooligosaccharide synthesis enzyme approach was diminished. Despite this inhibiting effect,
the fact that the free galactose concentration was decreased from
Two experimental approaches were carried out studying either 9.2% (3 h with A. oryzae) to 8.1% (another 168 h with C. laurentii,
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5 3
Fig. 2. GOS composition expressed as percent of total sugars as a function of lactose conversion. A: Consecutive coupling of C. laurentii (96 h, 64% hydrolysis) and A. oryzae. B:
Simultaneous combination of C. laurentii and A. oryzae (open symbols) and synthesis with C. laurentii only (closed symbols). Symbols: C/B disaccharides other than lactose, :/D
⋄
trisaccharides, A/ tetrasaccharides, -/, pentasaccharides, þ hexasaccharides (not detected if C. laurentii was used alone).
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
4 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Fig. 4. GOS composition expressed as percent of total sugars as a function of lactose conversion. A: Consecutive coupling of K. lactis (0.5 h, 90% hydrolysis) and A. oryzae. B:
Consecutive coupling of A. oryzae (3 h, 45% hydrolysis) and K. lactis (closed symbols) and synthesis with K. lactis only (open symbols). Symbols: C/B disaccharides other than
⋄
lactose, :/D trisaccharides, A/ tetrasaccharides, -/, pentasaccharides (not detected if K. lactis was used alone).
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5 5
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Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091