Assessment of repetitive batch-wise synthesis of galacto-oligosaccharides from lactose slurry using immobilized βgalactosidase from Bacillus circulans

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Publication date: 2014
Citation for published version (APA): Benjamins, F. (2014). Galacto-oligosaccharide synthesis using immobilized β-galactosidase. [Thesis fully internal (DIV), University of Groningen]. [S.n.].
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galactosidase from Bacillus circulans
ABSTRACT
The synthesis of galacto-oligosaccharides using covalently immobilized β-galactosidase from Bacillus circulans was carried out in lactose slurry rather than in solution. Repeated batch-wise synthesis at 58 °C and 55% lactose (w/w) could be carried out for at least 15 successive runs. All 15 runs were completed within 4 h of reaction time. The reduction of heat exposure due to these short incubation times, contributed to the retention of 60% of the initial activity. The product properties of the galacto-oligosaccharide mixture obtained with the immobilized biocatalyst were compared to the free enzyme product. Oligosaccharide yields and product pattern were highly similar as was the degree of polymerization. The productivity of the immobilized enzyme system was compared to the free enzyme. The enzymatic productivity (i.e. g GOS / g enzyme) after 15 consecutive runs was 165% higher for the immobilized enzyme as opposed to the free enzyme.
This chapter has been published as: Eric Benjamins, Laura Boxem, Janke KleinJan-
Noeverman & Ton A. Broekhuis (2014). Assessment of repetitive batch-wise synthesis of
galacto-oligosaccharides from lactose slurry using immobilised β-galactosidase from
Bacillus circulans. International Dairy Journal, Volume 38, Issue 2, Pages 160–168.
124
galactose to an acceptor molecule, thus synthesizing galacto-oligosaccharides (GOS)
(Torres, do Pilar, Goncalves, Teixeira, & Rodrigues, 2010). Due to their prebiotic
properties GOS are widely applied in infant nutrition and food in general (Gibson &
Rastall, 2006; Rastall, 2010; Tzortzis & Vulevic, 2009). Generally, production processes
for GOS are executed as robust and simple batch processes in which the reaction is
allowed sufficient time to reach the desired degree of lactose conversion. Further
downstream processing yields either GOS syrup or powder (Friesland Foods Domo,
2007; FrieslandCampina Domo, 2007; GTC Nutrition, 2009a; GTC Nutrition, 2009b;
Yakult Pharmaceutical Industry Co., Ltd., 2010a; Yakult Pharmaceutical Industry Co.,
Ltd., 2010b). Undesirably, the enzyme is used only once, as it will be lost during
downstream processing. Furthermore, the volumetric productivity of batch processes is in
general much lower than that of continuous processes. Continuous systems for GOS
synthesis have been described in the literature (Albayrak & Yang, 2002 a,b; Li, Xiao, Lu,
& Li, 2008; Mozaffar, Nakanishi, & Matsuno, 1986; Shin, Park, & Yang, 1998), but thus
far they are not commonly used on industrial scale.
The production of high fructose corn syrup (HFCS) is a good example of a comparable
process that has been successfully executed as a continuous process for many years
already. Driven by the high cost of the enzyme (glucose isomerase), the development of
very stable immobilized enzymes has ensured that almost all the HFCS is produced in
this way (Bhosale, Rao, & Desphande, 1996). Similarly to the production of HFCS, a
high substrate concentration is used during synthesis of the GOS. This high substrate
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concentration is desired since it is well known that this favours the synthesis of GOS
(Boon, Janssen, & van ‘t Riet, 2000; Gosling, Barber, Kentish, & Gras, 2011; Huerta,
Vera, Guerrero, Wilson, & Illanes, 2011; Torres et al., 2010) and also contributes to the
stability of the enzyme (Back, Oakenfull, & Smith, 1979; Klibanov, 1983). However, the
solubility of glucose (~47 g / 74 g per 100 g of solution at 20 and 60 °C, respectively
(Alves, Almeida e Silva, & Giulietti, 2007)) is much higher than that of lactose (~18 g /
37 g per 100 g of solution at 20 and 60 °C, respectively (McSweeney & Fox, 2009;
Machado, Coutinho, & Macedo, 2001)).
The synthesis of GOS using immobilized β-galactosidase described in the literature is in
general carried out in a packed bed reactor (PBR). The advantages of processing in this
way are the continuous synthesis of GOS and limited handling. The high conversion rate
encountered in PBR systems is caused by the high enzyme density in the packed bed. In
order to prepare the substrate solution, lactose is usually dissolved at elevated
temperatures. However, after preparation of a substrate solution the lactose can re-
crystallize during cooling down to the desired reaction temperature. In a Stirred Tank
Reactor (STR) the latter is not necessarily of interest, since after the addition of enzyme
the solubilized substrate will be converted and simultaneously, the crystalline lactose will
go into solution. In a continuous system, however, crystallization can cause huge
problems such as clogging of pipes and pumps.
This study focused on the use of immobilized B. circulans β-galactosidases during the
repeated, short-time conversion of lactose to GOS in a STR. In this work the possibilities
of using B. circulans β-galactosidase immobilized on Eupergit C250L for the batch-
wise synthesis of GOS were investigated. In order to ensure fast conversion of substrate
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in the batch system a high enzyme dosage was used. Immobilization of the enzyme
allowed for short reaction times and repeated use of the enzyme. Using the immobilized
enzyme, it is desirable to make a product with the same composition as in the process
with the free enzyme. For this reason the obtained products in the immobilized process
were compared and checked for both mutual similarity, as well as similarity to GOS
synthesized with free enzyme and a commercial GOS product (Vivinal® GOS) which is
also produced with free enzyme (FrieslandCampina Domo, 2007). Also the productivities
of the immobilized system in different set-ups were compared to the productivity of the
free enzyme.
3.2.1 Materials
Eupergit C250L (methacrylic carrier with epoxide functionality) was a kind gift from
Evonik (Darmstadt, Germany). Bacillus circulans β-galactosidase preparation (Biolacta
N5) was from Amano Enzyme Inc. (Nagoya, Japan). The protein content was 13%;
determined by Quick StartTM Bradford protein assay Kit (Bio-Rad, Veenendaal, the
Netherlands). Lactose (Lactochem) was kindly supplied by DMV Fonterra Excipients
(Goch, Germany). Vivinal® GOS was obtained from FrieslandCampina (Amersfoort, The
Netherlands). All other chemicals were purchased from Sigma-Aldrich (Steinheim,
Germany).
3.2.2 Enzyme activity measurement
The β-galactosidase activity was determined by measuring the glucose release in a lactose
solution by using a glucose oxidase / peroxidase (GOPOD) kit (Megazyme; Bray,
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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 (LU), where
1 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 the 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 swirling was applied to prevent accumulation of the
immobilized enzyme at the bottom of the tube. The activity of the immobilized enzyme
was expressed as LU.g-1 immobilized material.
3.2.3 Immobilization procedure Eupergit C250L
Eupergit C250L beads were washed with excess water and dried over a glass filter by
vacuum suction. The last washing step was performed with 0.2 M potassium phosphate
buffer pH 7.5. Enzyme solutions (various concentrations) were prepared by dissolving
Biolacta N5 in 1 M potassium phosphate buffer, pH 8.5. The carrier beads were added to
the enzyme solution and incubated at room temperature in a shaker/incubator (VWR,
Amsterdam, The Netherlands) under gentle shaking (70 rpm) for 24 h. Enzyme to carrier
ratios of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.75 were used in order to find the combination that led
to the highest activity / efficiency. During immobilization, samples of the supernatant
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were withdrawn and checked for activity with the GOPOD assay. After 24 h, the beads
were washed with equal amounts of water, 0.5 M NaCl and again water to remove non-
covalently bound protein. After washing the beads were incubated in ethanolamine /
phosphate buffer pH 7.5 to block the remaining epoxide groups and give the carrier beads
a more hydrophilic surface. Finally, the beads were washed with excess water and stored
in 0.1 M potassium phosphate buffer, pH 6.0. The stability of the free enzyme at pH
values of 8.5 and higher and at high salt concentration was checked by incubation in 1 M
sodium phosphate buffer at various pH values.
3.2.4 Immobilization activity
The loading activity, YA, was defined as the activity of the enzyme coupled to the carrier
(LU.g-1 of carrier). YA was calculated as the difference between the activity (LU) in the
initial enzyme solution and the activity in the filtrate and washing solution (the liquid
collected after washing the constructs) after the immobilization. The loading activity was
calculated according to Eq. (1)
. % = ./0/1.2023.404
./0/ (1)
where A0 is the activity of the initial immobilization solution (LU.mL-1); V0 the initial
solution volume (mL); Af the activity in the filtrate (LU.mL-1); Vf the filtrate volume
(mL); Aw the activity in the washing solution (LU.mL-1) and Vw the washing solution
volume (mL)
5% = 67
87 (2)
where CA is the activity of the immobilized enzyme (LU.g-1).
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3.2.5 Experimental set-up of a stirred tank reactor (STR) for GOS Synthesis
For the conversion of lactose to GOS, generally an enzyme dosage of 15 LU per g of
lactose was chosen. The immobilized enzyme was added to the lactose slurry. For the
slurry, 70 g of lactose was added to 50 mL of 0.1 M potassium phosphate buffer, pH 6.3.
This slurry was stirred using an overhead stirrer and heated to 58 °C in a jacketed glass
reactor vessel and maintained for at least 1 h before starting the synthesis reaction.
During the reaction, samples were withdrawn from the reaction mixture and immediately
filtered through a syringe filter (0.45 µm syringe filter (Whatman (GE Healthcare),
Diegem, Belgium) in order to separate the enzyme from the product. In preliminary trials,
the filtrate was checked for enzyme activity. Since the samples contain a large amount of
glucose, the GOPOD method was not applicable. Therefore ortho-nitrophenyl-β-D-
galactoside (o-NPG) was used a substrate. No activity was detected in the filtrate.
Samples were appropriately diluted and analyzed the same day or stored at -18 °C for
further analysis. After each run, the GOS mixture was separated from the immobilized
enzyme by filtration over a sintered glass filter. The beads were extensively washed with
demineralized water. The residual activity was measured and the beads were stored in 0.1
M potassium phosphate buffer until use the next day. Consecutive batch-wise synthesis
reactions were carried out under the same conditions, but differed in incubation time and
whether or not the enzyme was replenished. Initially, incubation times of 7 to 8 h were
applied (exp. I). In the subsequent set-up (exp. II) fresh immobilized enzyme was added
after each batch reaction to compensate for the loss of immobilized enzyme material due
to sampling as observed previously (exp. I). Reduction of the incubation time was
performed in the following experimental set-up (exp. III) where the reaction time was
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kept constant in order to monitor the inactivation of the immobilized enzyme. In the final
set-up (exp. IV) the inactivation of the enzyme was taken into account in order to obtain a
constant GOS yield for each batch reaction. The incubation times were gradually
increased to incubation times between 3 – 4 h.
3.2.6 Productivity
The enzymatic productivity, QEnz, is defined as the total amount of GOS produced per g
of protein. QEnz of the enzyme was calculated using Eq. (3):
9:;< = =>?@
=ABC (3)
where MGOS is the total mass of GOS in g; and MBN5 is the mass of Biolacta N5 protein in
g. The enzymatic productivity rate QEnz,t is defined as the total amount of GOS produced
per g of protein per h. QEnz,t of the enzyme was calculated using Eq. 4:
9:;<,E = =>?@
where t is the running time in h.
The volumetric productivity Qv of the batch reactor, averaged over the running time of
the reaction, was determined using Eq. 5:
90 = =>?@
3.2.7 Analysis of GOS
For the analysis of the degree of polymerization (DP), the samples were analyzed with
Size Exclusion Liquid Chromatography (HPLC-SEC) using a Rezex RSO
oligosaccharide column (Phenomenex, Amstelveen, the Netherlands) at 80 °C. The
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column was eluted with Milli-Q water at a flow rate of 0.3 mL.min-1. The eluent was
monitored with a refractive index detector. The standards that were used for calibration of
the column were galactose, glucose and GOS. The samples for analysis of the GOS
content and GOS composition were filtered through a 0.2 µm syringe filter (Whatman)
and diluted to approximately 10 ppm and analyzed with High Performance Anion
Exchange Chromatography (HPAEC) using a CarboPac PA1: 250 x 4 mm anion-
exchange column (Dionex, Sunnyvale, CA, USA) at 30ºC. The column was eluted with a
gradient of Eluent A (0.2 M sodium hydroxide) and Eluent B (0.1 M sodium hydroxide +
0.5 M sodium acetate) at a flow rate of 1.0 mL.min-1. The eluent was monitored with a
pulsed amperometric detector (PAD). The standards that were used for calibration of the
column were lactose and lactulose. GOS yield was calculated as follows:
% GOS = 100% - % Lactose - % Glucose - % Galactose
3.2.8 Kinetic measurements
The Km values of native and immobilized enzyme were determined by measurements of
enzyme activity with various concentrations of substrates (1 mM to 9 mM) in a 0.1 M
sodium acetate buffer pH 6.0 at 30 °C. Briefly, 100 µL of enzyme solution was added to
4.0 mL of a lactose solution and periodically 100 µL samples were withdrawn from the
reaction mixture and 100 µL of 0.1 M NaOH was added to inactivate the enzyme. After 5
min 100 µL 0.1 M HCl was added to neutralize and the glucose concentration was
determined using the GOPOD assay. For immobilized enzyme, a small amount
immobilized enzyme (5 - 10 mg) was accurately weighed into a reaction tube and 100 µL
of 0.1 M sodium acetate buffer pH 6.0 was added. Additionally 4.0 mL of lactose
solution was added. Occasional repeated swirling was applied to prevent accumulation of
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the immobilized enzyme at the bottom of the tube. Periodically 100 µL samples were
withdrawn from the upper supernatant and 100 µL of 0.1 M NaOH was added to
inactivate the enzyme. After 5 min 100 µL 0.1 M HCl was added to neutralize and the
glucose concentration was determined using the GOPOD assay. The activity of the
immobilized enzyme was expressed as LU.g-1 immobilized material. The initial reaction
rates at various substrate concentrations were determined from the linear part of the time
courses. The same activity of free and immobilized enzyme was used in these assays and
the Km values were calculated by using Lineweaver–Burk double reciprocal plots
(Lineweaver and Burk, 1934).
3.3 Results and discussion
β-Galactosidase from B. circulans was successfully immobilized on Eupergit C250L.
The activity of the construct that was selected as the most active was determined to have
an activity of 89 ± 0.2 LU.g-1 immobilisate (0.4 g enzyme per g carrier). Torres and
Batista-Viera (2012) immobilized β-galactosidase from B. circulans under comparable
conditions and found inactivation at higher ionic strength (1.4 M). Further optimization
would contribute to cost savings in terms of enzyme use. In this study, no loss of activity
was observed for the free B. circulans β-galactosidase after 22 h incubation at pH values
up to 9.0 in a 1.0 M potassium phosphate buffer (Table 1). Above those values rapid
inactivation was observed. During immobilization no pH values over 8.5 were applied,
therefore eq. (1) and (2) could be applied without correction for loss of activity. The
results for the activity of the constructs (80.7 ± 6.8 LU.g-1) and the immobilization
efficiencies (19.3 ± 1.2 %) showed good reproducibility of the immobilization procedure.
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For this reason, freshly immobilized enzyme was used in all experiments. The
immobilized enzyme was more stable than the soluble enzyme during incubation in
buffer at various temperatures, which is displayed in Figure 1. It was determined
previously that the enzymes present in Biolacta N5 are rather stable below 50 °C (Song et
al., 2011).
Table 1. Stability of B. circulans β-galactosidase at different pH values in 0.1M potassium phosphate buffer at 25 °C.
Activity [LU.ml-1]
pH 0 min 30 min 60 min 180 min 1320 min
8.5 1.0 1.2 1.1 1.1 1.0
8.8 1.0 1.1 1.1 1.3 1.0
9.0 0.9 1.0 1.0 1.1 1.0
9.2 1.0 1.0 1.0 1.2 0.8
9.4 1.0 1.0 1.0 1.0 0.4
9.6 0.9 0.9 0.7 0.6 -
9.8 0.9 0.7 0.5 0.15 -
10.4 0.9 0.02 - - -
10.6 0.9 0.02 - - -
Above this temperature rapid inactivation of the free enzyme was observed. At 60 °C and
65 °C in buffer the stability of the immobilized enzyme is 250% and 131%, respectively,
compared to the free enzyme (Stability construct % = Half-life immobilized enzyme /
Half-life free enzyme x 100%).
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Figure 1. Thermal stability of B. circulans β-galactosidase immobilized on Eupergit C250L (closed
symbols) as compared to the free enzyme (open symbols), incubated in 0.1M K2HPO4 / KH2PO4 buffer at
50, 55, 60 and 65 °C, respectively.
The immobilized enzyme showed a reduction in Vmax compared to the free enzyme (2.6
µmol.min-1 vs. 3.01 µmol.min-1). On the other hand, the Km increased from 8.2 mM for
free enzyme, to 11.3 mM for the immobilized biocatalyst. The kinetic values from this
study are in line with those described by Hernaiz and Crout (2000), who used the same
enzyme and carrier under the same conditions. The value for the Km found in this study
was slightly lower than the value of Hernaiz and Crout (2000). In contrast, the Vmax
determined in this study was higher, however, all values are in the same order of
0
10
20
30
40
50
60
70
80
90
100
110
0 10 20 30 40 50 60 70 80 90 100
A c ti v it y [ % ]
Time [min]
50 °C
55 °C
60 °C
65 °C
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magnitude. Also, for…