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Galacto-oligosaccharide synthesis using chemically modified β-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin
International Dairy Journal 54 (2016) 50e57
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Galacto-oligosaccharide synthesis using chemically modified b-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin Milica Carevi c a, Marija Corovi c a, Mladen Mihailovi c a, Katarina Banjanac a, a b ckovi c , Dejan Bezbradica a, * Ana Milisavljevi c , Dusan Veli a
Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2015 Received in revised form 21 October 2015 Accepted 23 October 2015 Available online 18 November 2015
The goal of this study was to establish an efficient immobilisation protocol for b-galactosidase from Aspergillus oryzae onto the polystyrenic macroporous resin Purolite® A-109 for better utilisation of its transglactosylation activity and application in galacto-oligosaccharide (GOS) synthesis. This was achieved by improving simple ionic adsorption by carboxyl group activation on the enzyme surface with carbodiimide, enabling covalent immobilisation. This yielded significantly increased operational stability, assayed as GOS synthesis, in a batch reactor, and even more prominently, in a fluidised bed reactor (73% activity retained after 10 cycles). The immobilised enzyme showed two very beneficial advantages over the free enzyme for future applications: higher affinity towards catalysing transgalactosylation than towards hydrolysis and shift of pH optimum towards more acidic conditions. GOS synthesis performed under the optimum conditions obtained (400 g L1 lactose, pH 4.5, 50 C) yielded 87 g L1 and 100 g L1 for batch and fluidised bed reactors, respectively. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction
b-Galactosidase (EC 3.2.1.23), also known as a lactase, is considered to be one of the most widely used industrial enzymes. Traditionally, it has been broadly exploited in the dairy industry due to its capability to hydrolyse lactose, thus providing alleviation of lactose maldigestion. Additionally, several other drawbacks regarding lactose in industrial utilisation, such as low solubility and low sweetness, are mitigated by b-galactosidase treatment. However, with increasing appreciation of products with highly beneficial effects on human health, significant attention has been drawn to the production of galacto-oligosaccharides (GOS). GOS represent non-digestible oligosaccharides formed by b-galactosidase catalysed transfer of galactosyl residues onto lactose molecules under specific conditions (Cardelle-Cobas, Villamiel, Olano, & Corzo, 2008). GOS are recognised as highly potent probiotic food ingredients that promote growth and metabolism of the intestinal
* Corresponding author. Tel.: þ38 111 3303 727. E-mail address: [email protected] (D. Bezbradica). http://dx.doi.org/10.1016/j.idairyj.2015.10.002 0958-6946/© 2015 Elsevier Ltd. All rights reserved.
microbiota, namely bifidobacteria and lactobacilli, leading to several positive health effects such as reduction of cholesterol level, vitamin production, calcium absorption enhancement, and colon cancer prevention (Cardelle-Cobas et al., 2008; Fischer & Kleinschmidt, 2015; Guerrero et al., 2014; Sangwan, Tomar, Ali, Singh, & Singh, 2015). Recently, in view of the potential of b-galactosidase transgalactosylation, a new field of development for galactosecontaining compounds, in which different sugars, alcohols, phenolic substances or glucosides serve as galactose moiety acceptors, is being extensively studied due to their promising role in the food industry, cosmetics and medicine (Black et al., 2014; Carevi c et al., 2015; Khatami, Zokaee Ashtiani, Bonakdarpour, & Mehrdad, 2014; Yan, Li, & Zong, 2014). Despite the fact that b-galactosidase is abundant in nature, the most common enzyme sources are microorganisms, especially those considered to be Generally Recognised As Safe (GRAS) and hence allowed to be used in the food and pharmaceutical industry. In this study the acidic b-galactosidase produced by Aspergillus oryzae was used, since it features a high specific activity, broad pH
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range and thermal stability, as well as outstanding transgalactosylation properties (Guerrero, Vera, Plou, & Illanes, 2011). In spite of the fact that enzymatic lactose hydrolysis and transgalactosylation with b-galactosidase from A. oryzae are well established on an industrial level, development of novel and potent immobilised preparations is still highly desired. Owing to the new insights into enzyme structure and characteristics, and progress in the field of material science enabling tailoring adequate immobilisation supports, it is possible to develop advantageous immobilised enzyme preparations that could possess higher thermal and operational stability, enable broader range of options for reactor design and better operational control, and easier product recovery, since protein contamination is principally avoided. Various methods for the immobilisation of b-galactosidase, including entrapment, cross-linking, adsorption, and covalent binding have already been proposed. Nevertheless, immobilisation by physical adsorption onto solid carriers proved to be the most compelling strategy to improve the operational stability and reusability, thus ensuring process cost effectiveness. Although adsorption has a major drawback, i.e., the fact that enzymes may be leached from the support during the reaction, its frequent employment was ensured through a simple protocol, mild immobilisation conditions, minimal damage of catalytic activity, and a wide variety of inexpensive materials that can serve as enzyme carriers with the possibility of simple regeneration. So far, different carriers have been used for the purpose of enzyme immobilisation, such as Eupergit® (Da Silva Campello, Trindade, go, De Medeiros Burkert, & Burkert, 2012; Warmerdam et al., Re 2014), Duolite (Fischer et al., 2013; Gürdas¸, Güleç, & Mutlu, 2012), Celite (Ansari & Husain, 2012) and Sepabeads® (Gaur, Pant, Jain, & Khare, 2006). In this study, an anion exchange resin with weak base functional groups (primary amine), Purolite® A109 was employed. To our knowledge, this highly promising polystyrenic (styrene-divinylbenzene copolymer) resin has not been used as carrier for bgalactosidase immobilisation, even though its non-toxicity and biocompatibility ensures its safe application in the food and pharmaceutical industry. Along with the exceptional thermal and chemical stability, this resin features excellent mechanical properties, high porosity and sphere bead shape with a mean particle diameter of 400 mm (Mihailovi c et al., 2014), which allows simple manipulation, easy enzyme recovery and enables its application in both batch and packed/fluidised bed reactors. The immobilisation of b-galactosidase from A. oryzae had been optimised and transgalactosylation potential of immobilised enzyme was evaluated in the synthesis of galacto-oligosaccharides in batch and fluidised bed reactors.
2. Materials and methods 2.1. Materials
b-Galactosidase from A. oryzae (8 units mg1 solid) used in this study was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Purolite® A109, a macroporous anion exchange resin, employed for enzyme immobilisation was kind donation from Purolite (Bala Cynwyd, PA, USA). Bovine serum albumin (BSA), 2nitrophenyl-b-D-galactopyranoside (O-NPG), Coomassie Brilliant Blue G-250 and lactose were obtained from Sigma Chemical Co. Acetonitrile and water were HPLC grade and purchased from JT Baker (Center Valley, PA, USA). All other reagents used were of analytical grade, and purchased from Centrohem (Stara Pazova, Serbia).
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2.2. b-Galactosidase immobilisation Prior to immobilisation, the Purolite® A109 (copolymer of styrene and divinylbenzene) should be “wetted” as a consequence of its highly hydrophobic nature. This procedure was performed in accordance with the method of Mihailovi c et al. (2014). The support obtained was kept at 4 C until use. Immobilisation of b-galactosidase was performed by suspending 20 mg of previously prepared Purolite® A109 into 1 mL of enzyme solution (1e20 mg mL1) in the corresponding buffer (sodium acetate buffer, pH 4e5, and sodium phosphate buffer, pH 6e7). Immobilisations were performed under mild stirring on roller shaker (IKA® Roller 6 basic, Werke GmbH and Co. KG, Staufen, Germany) at 25 C. During immobilisation, to evaluate immobilisation kinetics, samples of suspensions were taken and vacuum filtered. The filtrate obtained was assayed for both remaining enzyme activity and protein concentration, while the immobilised preparation was washed twice with 0.1 M sodium acetate buffer (pH 4.5) and then assayed for activity. All experiments were carried out in duplicate; the results with corresponding standard deviations are presented throughout the paper. 2.3. Enzyme desorption After the immobilisation, desorption of enzyme was performed by treating the immobilised preparation with 1 M CaCl2 and 1% (v/v) Triton X-100 solution for 1 h. Samples of supernatants and immobilised preparations were taken and enzyme activity and protein concentration were determined. 2.4. Enzyme modification To chemically modify the enzyme, 10 mL of the enzyme solution (5 mg mL1) in 0.1 M sodium acetate buffer (pH 4.75) was mixed with the amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) to reach the final concentration of 0.01 M and left on a roller shaker for 1.5 h. Afterwards, 2.2 mL of 0.5 M hydroxylamine was added and the mixture (total volume 12.2 mL) was dialysed overnight using cellulose membrane dialysis tubing (typical molecular mass cut-off, 14 kDa; Sigma Chemical Co.) against 2 L of 0.1 M sodium acetate buffer at 4 C. Finally, the modified enzyme solution obtained was used for immobilisation. 2.5. Support modification To obtain the cyanuric chloride functionalised carrier, 1 g of purified Purolite A109 was incubated with 5 g of 2,4,6-trichloro1,3,5-triazine (cyanuric chloride) dissolved in 250 mL of acetone with small amount of triethylamine. The mixture was stirred for 2 h at 0 C (Banjanac et al., 2014). After the reaction was finished, the carrier was filtered, washed with acetone twice and dried in a vacuum oven at 40 C for 24 h. To obtain the epoxy functionalised carrier, 1 g of purified Purolite A109 was incubated with 3 mL of 1 M sodium hydroxide and 330 mL of epichlorohydrin. The reaction was carried out stirred at 55 C for 0.5 h, and afterwards the carrier was filtered, washed twice with acetone, and dried in a vacuum oven at 40 C for 24 h (Mihailovi c et al., 2014). 2.6. b-Galactosidase activity assay To determine b-galactosidase hydrolytic activity, 10 mM onitrophenol-b-D-galactopyranoside (O-NPG) in 0.1 M sodium acetate buffer (pH 4.5) was used as a substrate. The reaction between 0.1 mL of free enzyme solution or supernatant and 0.9 mL substrate was allowed to proceed at least 2 min, while o-nitrophenol (O-NP)
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released in reaction was measured spectrophotometrically (Ultrospec 3300 pro, Amersham Biosciences, Freiburg, Germany) every 30 s at 410 nm and activity was determined from the slope of the graph constructed. One unit is defined as the amount of the enzyme that catalyses the liberation of 1 mmol O-NP per min under the assay conditions. The molar extinction coefficient for O-NP was determined to be 1357 dm3 mol1 cm1. Likewise, the activity assay for immobilised enzyme was performed by suspending 20 mg of immobilised preparation in 2 mL of substrate and stirring the mixture with a magnetic stirrer set at 1000 rpm.
defined conditions (lactose concentration 400 g L1, pH 4.5) to enable comparison with the aforementioned batch reactor. The reaction mixture was heated to 50 C prior to being pumped into the bioreactor using a peristaltic pump set at a constant flow rate of 2 mL min1. Samples were taken at different times, prepared in accordance with the procedure described in Section 2.2, and analysed using HPLC.
2.7. Protein assay
An operational stability study for the immobilised enzyme preparations obtained was performed in both reactor configurations under the conditions described in Sections 2.8 and 2.10. After taking the samples for analysis, the remaining reaction mixture was separated by filtration, and the immobilised enzyme preparations were thoroughly washed three times with the corresponding buffer, and dried until the next use. Moreover, to assay the enzyme leakage from the immobilisation support, protein concentration was measured (as described in Section 2.7) after every cycle.
The concentration of protein in enzyme solutions and supernatants was determined according to the procedure of Bradford (1976) using bovine serum albumin as the standard. 2.8. Production of galacto-oligosaccharides All reactions were performed at 55 C in conical flasks on an orbital shaker (IKA® KS 4000i control, Werke GmbH and Co.) set at 200 rpm. The reaction mixture comprised 100e600 g L1 lactose in 0.1 M sodium acetate buffer (pH 4.5) and 100 mg of immobilised preparation (4 IU, measured with O-NPG). In reactions carried out with the soluble enzyme, the concentration was adjusted to same b-galactosidase activity (4 IU, measured with O-NPG). Samples were taken at different times, and the reaction was stopped by heating samples at 100 C for 10 min to inactivate the enzyme. Samples were appropriately diluted with deionised water, centrifuged (12,000 g for 10 min), filtered through a 0.2 mm syringe filter and then analysed by high performance liquid chromatography (HPLC). Control samples, without enzyme, were prepared in parallel by exposure to the same temperature treatment; no galacto-oligosaccharide reaction products were detected in these control samples. All experiments were carried out in duplicate and average values of the product concentrations obtained are presented in the figures; all standard deviations were less than 5%. 2.9. High performance liquid chromatography analysis For quantitative analysis of samples, a Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) HPLC system was used. Galacto-oligosaccharide determinations were performed using a Thermo Scientific carbohydrate column (Hyper REZ XP Carbohydrate Ca2þ, 300 mm 7.7 mm, 8 mm) at 80 C. Deionised water was used as the mobile phase with an elution rate of 0.6 mL min1 during the analysis. Detection was performed using an RI detector (RefractoMax 520, ERC, Riemerling, Germany). All data acquisition and processing was done using Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific). For constructing calibration curves for quantification of monosaccharides, disaccharides, trisaccharides and tetrasaccharides, both galactose and glucose, lactose, maltotriose, and maltotetraose, respectively, were used (Warmerdam et al., 2014). Throughout this study, the GOS concentration represents the sum of trisaccharides and tetrasaccharides, since the concentration of higher oligosaccharides was negligible.
2.11. Operational stability study
3. Results and discussion 3.1. Adsorption of b-galactosidase on Purolite® A-109 In this study, optimisation of immobilisation of b-galactosidase from A. oryzae via adsorption onto commercial polystyrenic macroporous resin Purolite® A109 with primary amine groups was performed. In a preliminary experiment, the adsorption kinetics were analysed by monitoring the activity of immobilised enzyme and protein immobilisation yield over 24 h. It was observed (Fig. 1) that both outputs exhibited a sharp increase at an early stage, followed by slower stage that after 7 h led to equilibrium (33.79 IU g1 support; all offered enzyme protein was bound). Such a trend and almost constant specific activity indicate that the enzyme was attached in an active conformation throughout the immobilisation (Fig. 1). All subsequent experiments were performed with the adopted immobilisation time (7 h), since ANOVA analysis (P << 0.001) showed that the immobilisation time exhibited a highly significant effect on the studied factors.
2.10. Galacto-oligosaccharide production in a fluidised bed reactor The performance of the immobilised enzyme preparation obtained was also assessed in continuous mode operation using a fluidised bed reactor with recirculation, comprising a cylindrical glass column (136 mm length 9 mm inner diameter) with an external water jacket. The reaction was conducted under previously
Fig. 1. Immobilisation kinetics of b-galactosidase: (-) activity of immobilised enzyme (IU g1 support), (:) protein immobilisation yield (%), and (C) specific activity (IU mg1 protein). Immobilisation was performed in 0.1 M sodium acetate buffer at pH 4.5 with an initial enzyme concentration of 5 mg mL1. Results are presented as mean values ± standard deviation (n ¼ 2).
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Purolite® A-109 consists of a hydrophobic scaffold and charged amino groups, indicating that immobilisation can occur by several mechanisms, whether by formation of hydrophobic interactions, via ionic and dipoleedipole interactions, or by hydrogen bond formation. Therefore, the effects of pH and ionic strength of immobilisation solutions on the enzyme binding process were studied to give an insight into the nature of the enzymeesupport interactions. The immobilisation conducted in higher ionic strength buffers yielded considerably less active preparations within the whole pH range used (pH 4e7), in comparison with those performed in low molarity buffers (Fig. 2A). Therefore, it can be presumed that immobilisation predominantly occurred via the simple mechanism of ionic binding, where positively charged carrier amine groups interacted with negatively charged carboxylic groups on the enzyme surface. This hypothesis was confirmed by the desorption study, since at high ionic strength around 80% of activity was desorbed (Fig. 2B), as a consequence of hampered electrostatic interactions. Hydrophobic
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adsorption did not occur, despite the highly hydrophobic nature of the carrier surface, since insignificant activity was desorbed with surfactant (less than 3%). When immobilisation was performed at low ionic strength, pH had a significant influence on immobilisation efficiency (P < 0.05), since immobilised activity steeply decreased with an increase in pH above pH 5 (Fig. 2A). The result obtained corroborates previous studies that reported maximum activity retention by ionic adsorption taking place during immobilisation at a pH range of pH 4e5, where the highest activity exhibited was explained in terms of both favourable conformational structure of the immobilised enzyme molecules and the amount of immobilised molecules (Guidini, Fischer, Santana, Cardoso, & Ribeiro, 2010; Gürdas¸ et al., 2012). In the next experiment, the influence of the protein concentration offered was studied. As clearly depicted in Fig. 3, and additionally proved by the ANOVA statistical analysis, the offered protein concentration exhibited a highly significant effect on each of the immobilisation parameters (P < 0.05). With an increase in offered protein concentration, the activity of immobilised enzyme and protein loading increased, while protein immobilisation yield steeply decreased after attaining a maximum (approximately 100%) to about 10 mg protein per g support (Fig. 3). A further increase in offered protein concentration led to lower efficiency of the immobilisation procedure, because protein loading became very close to the saturation level for the support, which appeared to be around 20 mg g1 support (0.19 mmol g1 support; Fig. 3). 3.2. Characterisation of immobilised enzyme Bearing in mind that the enzyme microenvironment can exhibit a large influence on the enzyme activity expressed due to the enzyme and support surface charge (Gürdas¸ et al., 2012; Monier, 2013), the activity of both free and immobilised enzyme was tested as a function of pH and temperature; these profiles are presented in Fig. 4. There was no difference in temperature optimum between free and immobilised enzyme (Fig. 4A), and the values obtained were in accordance with the literature, around 50 C. However, the immobilised preparation exhibited a slightly broader temperature optimum range, indicating that stabilisation
Fig. 2. Panel A: the effect of immobilisation buffer pH and ionic strength on expressed activity of immobilised enzyme; immobilisations were performed for 7 h using initial b-galactosidase concentration of 5 mg mL1 in corresponding buffer solution (0.1 M buffers e black bars, 1 M buffers e white bars). Panel B: desorption of immobilised preparation performed in strong ionic strength solution of CaCl2 (1 M) and 1% Triton X100 solution (B). Results are presented as mean values ± standard deviation (n ¼ 2).
Fig. 3. Effect of the offered protein concentration on: (C) protein loading (mg g1 support), (-) activity of immobilised enzyme (IU g1 support), and (:) protein immobilisation yield (%). Immobilisations were performed for 7 h using corresponding initial b-galactosidase concentration in 0.1 M sodium acetate buffer solution (pH 5). Results are presented as mean values ± standard deviation (n ¼ 2).
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contamination control, which is of tremendous importance in immobilised enzyme reactors operating at high sugar contents. 3.3. Chemical modification of support or enzyme for promotion of covalent immobilisation Besides obtaining a high overall catalytic activity of immobilised preparation, the key role of an immobilisation process is to provide satisfactory stability. Since it is well-known that an enzyme immobilised by formation of covalent bonds usually has a higher stability than that of an enzyme adsorbed on to a support surface through weak forces (Bernal, Sierra, & Mesa, 2014; Bezbradica, pez-Gallego et al., 2013; Rodrigues Mateo, & Guisan, 2014; Lo et al., 2014), further experiments were focused on attempts to obtain covalent immobilisation of A. oryzae b-galactosidase using chemical modifications of the support or the enzyme. Two types of support modification were applied: epichlorhydrine modification for the introduction of reactive epoxy groups on support surface (epoxy-Purolite) and cyanuril chloride modification for introduction of active chlorine (cc-Purolite). It was observed that the activity of b-galactosidase immobilised on modified supports was lower by far than in the previous experiment with adsorption of the enzyme (Fig. 5). In both cases, immobilisation occurred predominantly due to reactions with amino groups on enzyme surface, hence it can be assumed that immobilisation via regions rich with these residues is unfavourable, although such an approach gave very good results in our previous investigations in lipase immobilisation (Mihailovi c et al., 2014). On the other hand, when b-galactosidase was chemically modified with carbodiimide to activate its carboxylic groups, to enable formation of covalent bonds with amino groups of unmodified Purolite, the activity of the immobilised enzyme was similar to that of adsorbed enzyme, indicating that immobilisation via carboxylic groups of the enzyme is by far more favourable. 3.4. The application of immobilised b-galactosidase in galactooligosaccharide synthesis The transgalactosylation activity of the immobilised enzyme was evaluated through monitoring the synthesis of GOS; this
Fig. 4. Effect of (A) temperature and (B) pH on (C) free and (-) immobilised bgalactosidase activity using the O-NPG hydrolysis reaction; for temperature determinations the activity was measured in 0.1 M sodium acetate buffer (pH 4.5), whilst for pH determinations the activity was measured at 50 C. Results are presented as mean values ± standard deviation (n ¼ 2).
of enzyme occurred by means of immobilisation. The pH optimum shift between immobilised and free enzyme was more pronounced. The optimum pH value of the free enzyme was found to be around 4.5, whereas for the immobilised enzyme it shifted towards more acidic pH values in the range pH 3.5e4.0 (Fig. 4B). Data reporting similar behaviour are rather scarce, although Mohy Eldin, El-Aassar, El Zatahry, and Al-Sabah (2014) reported an alteration of optimum pH towards a more acidic value upon covalent immobilisation of bgalactosidase onto amino-functionalised polyvinyl chloride microspheres, which they explained by the non-uniform distribution of hydrogen ions between the bulk solution and the amino groups on the carrier surface. On the other hand, examples of shifting the optimum towards neutral pH are more common. For instance, in a study by Gürdas¸ et al. (2012), the optimum pH of immobilised enzyme was shifted up 0.5 units to a more alkaline value compared with that of the free enzyme. Lowering of the pH optimum observed in our study could be valuable because it allows better
Fig. 5. Activity of different immobilised enzyme preparations: Purolite, unmodified enzyme onto Purolite; mPurolite, modified enzyme onto Purolite; epoxy-Purolite, unmodified enzyme onto epoxy-Purolite; cc-Purolite, unmodified enzyme onto ccPurolite. Results are presented as mean values ± standard deviation (n ¼ 2).
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application of b-galactosidase has gained considerable interest because of the recognition of the physiological (prebiotic) potential of GOS. Free b-galactosidase and two immobilised preparations were assayed in terms of GOS synthesis under the same conditions (4 IU enzyme, 400 g L1 lactose, pH 4.5 and 50 C), and the time course of these reactions is presented in Fig. 6A. The two immobilised enzyme preparations showed the same trend of GOS formation throughout the whole reaction range, yielding approximately 80 g L1 after 14 h, while with the free enzyme a slightly lower GOS concentration (67 g L1) was reached. Hence, it can be concluded that the accessibility of the substrate towards the immobilised enzymes was not hampered and that immobilisation did not introduce presumed mass transfer limitations during GOS formation. Moreover, it seems that immobilisation of enzyme on Purolite shifts selectivity of b-galactosidase towards transgalactosylation, since equal levels of the hydrolytic activity of the enzyme were put into the reaction mixture. It was previously established that the first step in b-galactosidase-catalysed reactions is the formation of a galactosyleenzyme complex and the type of reaction is determined by galactosyl acceptor; if water is acceptor, hydrolysis occurs and if lactose (or another saccharide) is acceptor transgalactosylation occurs (Cardelle-Cobas et al., 2008; Guerrero et al., 2011). It is plausible that transgalactosylation is favoured by
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the immobilised enzyme due to the hydrophobic nature of the support scaffold, which leads to a lower water concentration in the enzyme microenvironment than is the case in the reaction with the free enzyme. The effect of chemical modification on the operational stability of immobilised enzyme was assayed in a study based on performing 10 consecutive GOS synthesis reaction cycles with two different reactor configurations. In the batch reactor, the activity of immobilised unmodified enzyme continuously decreased, reaching around half of the initial activity in third cycle, and finally was completely inactivated in the last cycle. In the fluidised bed reactor a less steep inactivation was observed, reaching around half of initial activity after 5 cycles, and retaining around 10% of initial activity after 10 cycles (Fig. 7). The modified enzyme preparation exhibited a significantly higher stability in comparison with the unmodified enzyme preparation, with activity retained after 10 cycles of around 75% and 50% for the fluidised bed and the batch reactor, respectively. These results may be explained by a reduction in protein structure mobility after covalent immobilisation, since more rigid forms are usually less susceptible to damaging effects of the environment (Osman et al., 2014). Potential enzyme leakage was monitored by measuring protein content after each cycle, and no protein was detected in the reaction mixture. Therefore, activity
Fig. 6. Panel A: kinetics of GOS synthesis catalysed by b-galactosidase from A. oryzae: modified enzyme immobilisation preparation (-), unmodified enzyme immobilisation preparation ( ), free enzyme ( ). Panel B, concentration profiles during GOS synthesis reaction with initial lactose concentration of 400 g L1: disaccharides (C), GOS (-), glucose (:) and galactose (;). Panel C, dependence of GOS concentration upon different initial lactose concentrations: 50 g L1 (-), 100 g L1 (C), 200 g L1 (:), 300 g L1 (;), 400 g L1 (A), 600 g L1 (<). Panel D, kinetics of GOS synthesis in batch (-) and fluidised bed ( ) reactor. Results are presented as mean values ± standard deviation (n ¼ 2).
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Fig. 7. Operational stability of the immobilised preparations: modified (black bars) and unmodified (white bars) b-galactosidase onto Purolite in (A) batch reactor and (B) fluidised bed reactor. Results are presented as mean values ± standard deviation (n ¼ 2).
loss throughout this experiment could not be attributed to enzyme detachment, and discrepancies between inactivation in the two reactors can probably be ascribed to different flow regimes and level of turbulence. Taking into account its higher stability (Fig. 7) and rather high activity (Fig. 6A), all further experiments were performed using the modified enzyme immobilised preparation. First of all, to get a better insight in complex nature of GOS synthesis, a representative depiction of the profiles of all reaction components throughout time course of the reaction was obtained (Fig. 6B). The hydrolysis products (glucose and galactose) did not have equal concentration profiles, since molecules of galactose were incorporated into the newly formed disaccharide and oligosaccharide molecules. It can be seen that this difference is more pronounced in the earlier stages of the reaction, when the lactose concentration is rapidly decreasing and GOS synthesis prevails. GOS concentration subsequently decreased towards the end of period observed, and concentrations of glucose and galactose became similar, due to the fact that GOS are also susceptible to
hydrolysis in later stages due to their significant concentration in the reaction mixture and cannot be further replenished by synthesis of equal or higher quantities of GOS (Mahoney, 1998). This can be seen even better in Fig. 6C, where GOS concentration peaks were clearly achieved for samples with lower initial lactose concentrations. In the next set of experiments, the effect of initial lactose concentration on GOS synthesis was analysed (Fig. 6B). It was found that the GOS concentration obtained increased with an increased initial lactose concentration over the whole range examined, and consequently the highest GOS concentration (120 g L1) corresponded to the highest initial lactose concentration (600 g L1). This undoubtedly shows that the transgalactosylation reaction predominates at higher lactose concentrations, lowering the water content, and also making lactose and other sugars more favourable acceptors of the galactosyl moiety (Mahoney, 1998; Prenosil, Stuker, & Bourne, 1987). On the other hand, at low lactose concentrations, water is most likely to be the acceptor so hydrolysis occurs and thus lower GOS yields are obtained (Park & Oh, 2010). In view of the efficiency of the immobilised preparation obtained, our results are comparable with those previously obtained in other studies. For example, Urrutia et al. (2013), using 15 IU mL1 of enzyme preparation at initial lactose concentration of 400 g L1 for 7 h, achieved maximum GOS concentration of 107 g L1 (26.8%, w/w, concentration of GOS to total carbohydrate), while we obtained 78.5 g L1 (23%, w/w) with 75 times less enzyme activity. Likewise, Huerta, Vera, Guerrero, Wilson, and Illanes (2011) reported a GOS yield of approximately 30% using an initial lactose concentration of 546 g L1 and 33 IU g1 lactose. Under similar conditions, 600 g L1 lactose using 6.67 IU g1 lactose we reached maximal GOS yield of 24.1% (w/w). This was, however, slightly less than Neri et al. (2011) reported for b-galactosidase immobilised onto magnetised Dacron, namely 26.2% or 131.0 g L1 GOS. These results clearly indicate that the method developed for b-galactosidase yields a cheap and stable preparation, which could be used in further development of transgalactosylation processes. Finally, since the aim of every immobilisation is to ensure efficient implementation in continuous mode of operation, we tested the immobilised enzyme in a fluidised bed reactor with recirculation. GOS production showed a reaction course similar to that in a batch reactor, ensuring a moderately higher GOS concentration was achieved; around 100 g L1 after 14 h. However, since immobilised enzyme in the fluidised bed exhibited higher operational stability (Fig. 7B), substantial enhancement (around 40%) in the total mass of GOS produced (after 10 successive cycles) was obtained within the fluidised bed reactor application (Fig. 6D). Papers investigating employment of fluidised bed reactors are scarce, but our results are comparable with previously obtained results in similar continual systems, namely packed bed reactors. For example, under similar reaction conditions (40% lactose feed solution, pH 4.5 and 40 C), Jovanovic-Malinovska Fernandes, Winkelhausen, and Fonseca (2012) obtained a GOS yield of around 108 g L1. These findings, therefore, confirm that the immobilised preparation obtained could present a valuable contribution to the development of efficient and stable production of GOS. 4. Conclusions The aim of this study was to optimise the immobilisation of bgalactosidase from A. oryzae on Purolite® A-109, to improve the stability of immobilised enzyme by means of chemical modification and evaluate the immobilised enzyme obtained in transgalactosylation. Although immobilisation occurred predominantly by ionic adsorption, a simple and non-invasive activation of carboxyl groups on the enzyme surface with carbodiimide enabled
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formation of covalent bond between enzyme and support, resulting in a significantly increased operational stability of the immobilised b-galactosidase. The immobilised enzyme showed higher affinity towards catalysing transgalactosylation than hydrolysis reaction. Moreover, immobilised enzyme exhibited very good operational stability in a fluidised bed reactor, which indicates good prospects for its application in continuous bioprocesses. Since the use of bgalactosidases in synthesis of GOS is gaining increasing importance, the findings of this study could be a valuable contribution to the development of an efficient and stable production method for physiologically active galactoside in immobilised enzyme reactors. Acknowledgement This research was financed by the Ministry of Education, Science and Technological Development of Republic of Serbia through funding Project III 46010. References Ansari, S. A., & Husain, Q. (2012). Lactose hydrolysis from milk/whey in batch and continuous processes by concanavalin A-Celite 545 immobilized Aspergillus oryzae b galactosidase. Food and Bioproducts Processing, 90, 351e359. Banjanac, K., Mihailovi c, M., Prlainovi c, N., Stojanovi c, M., Carevi c, M., Marinkovi c, A., et al. (2014). Cyanuric chloride functionalized silica nanoparticles for covalent immobilization of lipase. Journal of Chemical Technology and Biotechnology. http://dx.doi.org/10.1002/jctb.4595. Bernal, C., Sierra, L., & Mesa, M. (2014). Design of b-galactosidase/silica biocatalysts: impact of the enzyme properties and immobilization pathways on their catalytic performance. Engineering in Life Sciences, 14, 85e94. Bezbradica, D. I., Mateo, C., & Guisan, J. M. (2014). Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde. Journal of Molecular Catalysis B: Enzymatic, 102, 218e224. Black, B. A., Yan, Y., Galle, S., Hu, Y., Curtis, J. M., & G€ anzle, M. G. (2014). Characterisation of novel galactosylated chitin-oligosaccharides and chitosan-oligosaccharides. International Dairy Journal, 39, 330e335. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Cardelle-Cobas, A., Villamiel, M., Olano, A., & Corzo, N. (2008). Study of galactooligosaccharide formation from lactose using Pectinex Ultra SP-L. Journal of the Science of Food and Agriculture, 88, 954e961. Carevi c, M., Veli ckovi c, D., Stojanovi c, M., Milosavi c, N., Rogniaux, H., Ropartz, D., et al. (2015). Insight in the regioselective enzymatic transgalactosylation of salicin catalyzed by b-galactosidase from Aspergillus oryzae. Process Biochemistry, 50, 782e788. go, T. V., De Medeiros Burkert, J. F., & Da Silva Campello, G., Trindade, R. A., Re Burkert, C. A. V. (2012). Immobilization of b-galactosidase from Kluyveromyces ® lactis on Eupergit c and properties of the biocatalyst. International Journal of Food Engineering, 8. http://dx.doi.org/10.1515/1556-3758.2760. Fischer, J., Guidini, C. Z., Santana, L. N. S., de Resende, M. M., Cardoso, V. L., & Ribeiro, E. J. (2013). Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized b-galactosidase from Aspergillus oryzae. Journal of Molecular Catalysis B: Enzymatic, 85e86, 178e186. Fischer, C., & Kleinschmidt, T. (2015). Synthesis of galactooligosaccharides using sweet and acid whey as a substrate. International Dairy Journal, 48, 15e22. Gaur, R., Pant, H., Jain, R., & Khare, S. K. (2006). Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae b-galactosidase. Food Chemistry, 97, 426e430. Guerrero, C., Vera, C., Novoa, C., Dumont, J., Acevedo, F., & Illanes, A. (2014). Purification of highly concentrated galacto-oligosaccharide preparations by selective fermentation with yeasts. International Dairy Journal, 39, 78e88.
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Guerrero, C., Vera, C., Plou, F., & Illanes, A. (2011). Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial b-galactosidases. Journal of Molecular Catalysis B: Enzymatic, 72, 206e212. Guidini, C. Z., Fischer, J., Santana, L. N. S., Cardoso, V. L., & Ribeiro, E. J. (2010). Immobilization of Aspergillus oryzae b-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking. Biochemical Engineering Journal, 52, 137e143. Gürdas¸, S., Güleç, H. A., & Mutlu, M. (2012). Immobilization of Aspergillus oryzae bgalactosidase onto Duolite A568 resin via simple adsorption mechanism. Food and Bioprocess Technology, 5, 904e911. Huerta, L. M., Vera, C., Guerrero, C., Wilson, L., & Illanes, A. (2011). Synthesis of galacto-oligosaccharides at very high lactose concentrations with immobilized b-galactosidases from Aspergillus oryzae. Process Biochemistry, 46, 245e252. Jovanovic-Malinovska, R., Fernandes, P., Winkelhausen, E., & Fonseca, L. (2012). Galacto-oligosaccharides synthesis from lactose and whey by b-galactosidase immobilized in PVA. Applied Biochemistry and Biotechnology, 168, 1197e1211. Khatami, S., Zokaee Ashtiani, F., Bonakdarpour, B., & Mehrdad, M. (2014). The enzymatic production of lactulose via transglycosylation in conventional and non-conventional media. International Dairy Journal, 34, 74e79. pez-Gallego, F., Fernandez-Lorente, G., Rocha-Martin, J., Bolivar, J. M., Mateo, C., & Lo Guisan, J. M. (2013). Stabilization of enzymes by multipoint covalent immobilization on supports activated with glyoxyl groups. Methods in Molecular Biology, 1051, 59e71. Mahoney, R. R. (1998). Galactosyl-oligosaccharide formation during lactose hydrolysis: a review. Food Chemistry, 63, 147e154. Mihailovi c, M., Stojanovi c, M., Banjanac, K., Carevi c, M., Prlainovi c, N., Milosavi c, N., et al. (2014). Immobilization of lipase on epoxy-activated Purolite® A109 and its post-immobilization stabilization. Process Biochemistry, 49, 637e646. Mohy Eldin, M. S., El-Aassar, M. R., El Zatahry, A. A., & Al-Sabah, M. M. B. (2014). Covalent immobilization of b-galactosidase onto amino-functionalized polyvinyl chloride microspheres: enzyme immobilization and characterization. Advances in Polymer Technology, 33. Monier, M. (2013). Immobilization of b-galactosidase from Escherichia coli onto modified natural silk fibers. Journal of Applied Polymer Science, 130, 2923e2931. ~o, V. M., Cardoso, S. M., Silva, A. M. S., Domingues, M. D. R. M., Neri, D. F. M., Balca Torres, D. P. M., et al. (2011). Characterisation of galactooligosaccharides produced by b-galactosidase immobilised onto magnetised Dacron. International Dairy Journal, 21, 172e178. Osman, A., Symeou, S., Trisse, V., Watson, K. A., Tzortzis, G., & Charalampopoulos, D. (2014). Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial b-galactosidase (BbgIV) immobilised on DEAE-Cellulose, QSepharose and amino-ethyl agarose. Biochemical Engineering Journal, 82, 188e199. Park, A. R., & Oh, D. K. (2010). Galacto-oligosaccharide production using microbial bgalactosidase: current state and perspectives. Applied Microbiology and Biotechnology, 85, 1279e1286. Prenosil, J. E., Stuker, E., & Bourne, J. R. (1987). Formation of oligosaccharides during enzymatic lactose hydrolysis. Part 1. State of art. Biotechnology and Bioengineering, 30, 1019e1025. Torres, R., & FernandezRodrigues, R. C., Barbosa, O., Ortiz, C., Berenguer-Murcia, A., Lafuente, R. (2014). Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools. RSC Advances, 4, 38350e38374. Sangwan, V., Tomar, S. K., Ali, B., Singh, R. R. B., & Singh, A. K. (2015). Galactooligosaccharides reduce infection caused by Listeria monocytogenes and modulate IgG and IgA levels in mice. International Dairy Journal, 41, 58e63. Urrutia, P., Rodriguez-Colinas, B., Fernandez-Arrojo, L., Ballesteros, A. O., Wilson, L., Illanes, A., et al. (2013). Detailed analysis of galactooligosaccharides synthesis with -galactosidase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 61, 1081e1087. Warmerdam, A., Benjamins, E., de Leeuw, T. F., Broekhuis, T. A., Boom, R. M., & Janssen, A. E. M. (2014). Galacto-oligosaccharide production with immobilized b-galactosidase in a packed-bed reactor vs. free b-galactosidase in a batch reactor. Food and Bioproducts Processing, 92, 383e392. Yan, L. Q., Li, N., & Zong, M. H. (2014). First enzymatic galactosylation of acyclic nucleoside drugs by b-galactosidase: synthesis of water-soluble b-D-galactosidic prodrugs. Biotechnology and Bioprocess Engineering, 19, 586e591.
Contents lists available at ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Galacto-oligosaccharide synthesis using chemically modified b-galactosidase from Aspergillus oryzae immobilised onto macroporous amino resin Milica Carevi c a, Marija Corovi c a, Mladen Mihailovi c a, Katarina Banjanac a, a b ckovi c , Dejan Bezbradica a, * Ana Milisavljevi c , Dusan Veli a
Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000 Belgrade, Serbia
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 May 2015 Received in revised form 21 October 2015 Accepted 23 October 2015 Available online 18 November 2015
The goal of this study was to establish an efficient immobilisation protocol for b-galactosidase from Aspergillus oryzae onto the polystyrenic macroporous resin Purolite® A-109 for better utilisation of its transglactosylation activity and application in galacto-oligosaccharide (GOS) synthesis. This was achieved by improving simple ionic adsorption by carboxyl group activation on the enzyme surface with carbodiimide, enabling covalent immobilisation. This yielded significantly increased operational stability, assayed as GOS synthesis, in a batch reactor, and even more prominently, in a fluidised bed reactor (73% activity retained after 10 cycles). The immobilised enzyme showed two very beneficial advantages over the free enzyme for future applications: higher affinity towards catalysing transgalactosylation than towards hydrolysis and shift of pH optimum towards more acidic conditions. GOS synthesis performed under the optimum conditions obtained (400 g L1 lactose, pH 4.5, 50 C) yielded 87 g L1 and 100 g L1 for batch and fluidised bed reactors, respectively. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction
b-Galactosidase (EC 3.2.1.23), also known as a lactase, is considered to be one of the most widely used industrial enzymes. Traditionally, it has been broadly exploited in the dairy industry due to its capability to hydrolyse lactose, thus providing alleviation of lactose maldigestion. Additionally, several other drawbacks regarding lactose in industrial utilisation, such as low solubility and low sweetness, are mitigated by b-galactosidase treatment. However, with increasing appreciation of products with highly beneficial effects on human health, significant attention has been drawn to the production of galacto-oligosaccharides (GOS). GOS represent non-digestible oligosaccharides formed by b-galactosidase catalysed transfer of galactosyl residues onto lactose molecules under specific conditions (Cardelle-Cobas, Villamiel, Olano, & Corzo, 2008). GOS are recognised as highly potent probiotic food ingredients that promote growth and metabolism of the intestinal
* Corresponding author. Tel.: þ38 111 3303 727. E-mail address: [email protected] (D. Bezbradica). http://dx.doi.org/10.1016/j.idairyj.2015.10.002 0958-6946/© 2015 Elsevier Ltd. All rights reserved.
microbiota, namely bifidobacteria and lactobacilli, leading to several positive health effects such as reduction of cholesterol level, vitamin production, calcium absorption enhancement, and colon cancer prevention (Cardelle-Cobas et al., 2008; Fischer & Kleinschmidt, 2015; Guerrero et al., 2014; Sangwan, Tomar, Ali, Singh, & Singh, 2015). Recently, in view of the potential of b-galactosidase transgalactosylation, a new field of development for galactosecontaining compounds, in which different sugars, alcohols, phenolic substances or glucosides serve as galactose moiety acceptors, is being extensively studied due to their promising role in the food industry, cosmetics and medicine (Black et al., 2014; Carevi c et al., 2015; Khatami, Zokaee Ashtiani, Bonakdarpour, & Mehrdad, 2014; Yan, Li, & Zong, 2014). Despite the fact that b-galactosidase is abundant in nature, the most common enzyme sources are microorganisms, especially those considered to be Generally Recognised As Safe (GRAS) and hence allowed to be used in the food and pharmaceutical industry. In this study the acidic b-galactosidase produced by Aspergillus oryzae was used, since it features a high specific activity, broad pH
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range and thermal stability, as well as outstanding transgalactosylation properties (Guerrero, Vera, Plou, & Illanes, 2011). In spite of the fact that enzymatic lactose hydrolysis and transgalactosylation with b-galactosidase from A. oryzae are well established on an industrial level, development of novel and potent immobilised preparations is still highly desired. Owing to the new insights into enzyme structure and characteristics, and progress in the field of material science enabling tailoring adequate immobilisation supports, it is possible to develop advantageous immobilised enzyme preparations that could possess higher thermal and operational stability, enable broader range of options for reactor design and better operational control, and easier product recovery, since protein contamination is principally avoided. Various methods for the immobilisation of b-galactosidase, including entrapment, cross-linking, adsorption, and covalent binding have already been proposed. Nevertheless, immobilisation by physical adsorption onto solid carriers proved to be the most compelling strategy to improve the operational stability and reusability, thus ensuring process cost effectiveness. Although adsorption has a major drawback, i.e., the fact that enzymes may be leached from the support during the reaction, its frequent employment was ensured through a simple protocol, mild immobilisation conditions, minimal damage of catalytic activity, and a wide variety of inexpensive materials that can serve as enzyme carriers with the possibility of simple regeneration. So far, different carriers have been used for the purpose of enzyme immobilisation, such as Eupergit® (Da Silva Campello, Trindade, go, De Medeiros Burkert, & Burkert, 2012; Warmerdam et al., Re 2014), Duolite (Fischer et al., 2013; Gürdas¸, Güleç, & Mutlu, 2012), Celite (Ansari & Husain, 2012) and Sepabeads® (Gaur, Pant, Jain, & Khare, 2006). In this study, an anion exchange resin with weak base functional groups (primary amine), Purolite® A109 was employed. To our knowledge, this highly promising polystyrenic (styrene-divinylbenzene copolymer) resin has not been used as carrier for bgalactosidase immobilisation, even though its non-toxicity and biocompatibility ensures its safe application in the food and pharmaceutical industry. Along with the exceptional thermal and chemical stability, this resin features excellent mechanical properties, high porosity and sphere bead shape with a mean particle diameter of 400 mm (Mihailovi c et al., 2014), which allows simple manipulation, easy enzyme recovery and enables its application in both batch and packed/fluidised bed reactors. The immobilisation of b-galactosidase from A. oryzae had been optimised and transgalactosylation potential of immobilised enzyme was evaluated in the synthesis of galacto-oligosaccharides in batch and fluidised bed reactors.
2. Materials and methods 2.1. Materials
b-Galactosidase from A. oryzae (8 units mg1 solid) used in this study was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Purolite® A109, a macroporous anion exchange resin, employed for enzyme immobilisation was kind donation from Purolite (Bala Cynwyd, PA, USA). Bovine serum albumin (BSA), 2nitrophenyl-b-D-galactopyranoside (O-NPG), Coomassie Brilliant Blue G-250 and lactose were obtained from Sigma Chemical Co. Acetonitrile and water were HPLC grade and purchased from JT Baker (Center Valley, PA, USA). All other reagents used were of analytical grade, and purchased from Centrohem (Stara Pazova, Serbia).
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2.2. b-Galactosidase immobilisation Prior to immobilisation, the Purolite® A109 (copolymer of styrene and divinylbenzene) should be “wetted” as a consequence of its highly hydrophobic nature. This procedure was performed in accordance with the method of Mihailovi c et al. (2014). The support obtained was kept at 4 C until use. Immobilisation of b-galactosidase was performed by suspending 20 mg of previously prepared Purolite® A109 into 1 mL of enzyme solution (1e20 mg mL1) in the corresponding buffer (sodium acetate buffer, pH 4e5, and sodium phosphate buffer, pH 6e7). Immobilisations were performed under mild stirring on roller shaker (IKA® Roller 6 basic, Werke GmbH and Co. KG, Staufen, Germany) at 25 C. During immobilisation, to evaluate immobilisation kinetics, samples of suspensions were taken and vacuum filtered. The filtrate obtained was assayed for both remaining enzyme activity and protein concentration, while the immobilised preparation was washed twice with 0.1 M sodium acetate buffer (pH 4.5) and then assayed for activity. All experiments were carried out in duplicate; the results with corresponding standard deviations are presented throughout the paper. 2.3. Enzyme desorption After the immobilisation, desorption of enzyme was performed by treating the immobilised preparation with 1 M CaCl2 and 1% (v/v) Triton X-100 solution for 1 h. Samples of supernatants and immobilised preparations were taken and enzyme activity and protein concentration were determined. 2.4. Enzyme modification To chemically modify the enzyme, 10 mL of the enzyme solution (5 mg mL1) in 0.1 M sodium acetate buffer (pH 4.75) was mixed with the amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) to reach the final concentration of 0.01 M and left on a roller shaker for 1.5 h. Afterwards, 2.2 mL of 0.5 M hydroxylamine was added and the mixture (total volume 12.2 mL) was dialysed overnight using cellulose membrane dialysis tubing (typical molecular mass cut-off, 14 kDa; Sigma Chemical Co.) against 2 L of 0.1 M sodium acetate buffer at 4 C. Finally, the modified enzyme solution obtained was used for immobilisation. 2.5. Support modification To obtain the cyanuric chloride functionalised carrier, 1 g of purified Purolite A109 was incubated with 5 g of 2,4,6-trichloro1,3,5-triazine (cyanuric chloride) dissolved in 250 mL of acetone with small amount of triethylamine. The mixture was stirred for 2 h at 0 C (Banjanac et al., 2014). After the reaction was finished, the carrier was filtered, washed with acetone twice and dried in a vacuum oven at 40 C for 24 h. To obtain the epoxy functionalised carrier, 1 g of purified Purolite A109 was incubated with 3 mL of 1 M sodium hydroxide and 330 mL of epichlorohydrin. The reaction was carried out stirred at 55 C for 0.5 h, and afterwards the carrier was filtered, washed twice with acetone, and dried in a vacuum oven at 40 C for 24 h (Mihailovi c et al., 2014). 2.6. b-Galactosidase activity assay To determine b-galactosidase hydrolytic activity, 10 mM onitrophenol-b-D-galactopyranoside (O-NPG) in 0.1 M sodium acetate buffer (pH 4.5) was used as a substrate. The reaction between 0.1 mL of free enzyme solution or supernatant and 0.9 mL substrate was allowed to proceed at least 2 min, while o-nitrophenol (O-NP)
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released in reaction was measured spectrophotometrically (Ultrospec 3300 pro, Amersham Biosciences, Freiburg, Germany) every 30 s at 410 nm and activity was determined from the slope of the graph constructed. One unit is defined as the amount of the enzyme that catalyses the liberation of 1 mmol O-NP per min under the assay conditions. The molar extinction coefficient for O-NP was determined to be 1357 dm3 mol1 cm1. Likewise, the activity assay for immobilised enzyme was performed by suspending 20 mg of immobilised preparation in 2 mL of substrate and stirring the mixture with a magnetic stirrer set at 1000 rpm.
defined conditions (lactose concentration 400 g L1, pH 4.5) to enable comparison with the aforementioned batch reactor. The reaction mixture was heated to 50 C prior to being pumped into the bioreactor using a peristaltic pump set at a constant flow rate of 2 mL min1. Samples were taken at different times, prepared in accordance with the procedure described in Section 2.2, and analysed using HPLC.
2.7. Protein assay
An operational stability study for the immobilised enzyme preparations obtained was performed in both reactor configurations under the conditions described in Sections 2.8 and 2.10. After taking the samples for analysis, the remaining reaction mixture was separated by filtration, and the immobilised enzyme preparations were thoroughly washed three times with the corresponding buffer, and dried until the next use. Moreover, to assay the enzyme leakage from the immobilisation support, protein concentration was measured (as described in Section 2.7) after every cycle.
The concentration of protein in enzyme solutions and supernatants was determined according to the procedure of Bradford (1976) using bovine serum albumin as the standard. 2.8. Production of galacto-oligosaccharides All reactions were performed at 55 C in conical flasks on an orbital shaker (IKA® KS 4000i control, Werke GmbH and Co.) set at 200 rpm. The reaction mixture comprised 100e600 g L1 lactose in 0.1 M sodium acetate buffer (pH 4.5) and 100 mg of immobilised preparation (4 IU, measured with O-NPG). In reactions carried out with the soluble enzyme, the concentration was adjusted to same b-galactosidase activity (4 IU, measured with O-NPG). Samples were taken at different times, and the reaction was stopped by heating samples at 100 C for 10 min to inactivate the enzyme. Samples were appropriately diluted with deionised water, centrifuged (12,000 g for 10 min), filtered through a 0.2 mm syringe filter and then analysed by high performance liquid chromatography (HPLC). Control samples, without enzyme, were prepared in parallel by exposure to the same temperature treatment; no galacto-oligosaccharide reaction products were detected in these control samples. All experiments were carried out in duplicate and average values of the product concentrations obtained are presented in the figures; all standard deviations were less than 5%. 2.9. High performance liquid chromatography analysis For quantitative analysis of samples, a Dionex Ultimate 3000 (Thermo Scientific, Waltham, MA, USA) HPLC system was used. Galacto-oligosaccharide determinations were performed using a Thermo Scientific carbohydrate column (Hyper REZ XP Carbohydrate Ca2þ, 300 mm 7.7 mm, 8 mm) at 80 C. Deionised water was used as the mobile phase with an elution rate of 0.6 mL min1 during the analysis. Detection was performed using an RI detector (RefractoMax 520, ERC, Riemerling, Germany). All data acquisition and processing was done using Chromeleon™ 7.2 Chromatography Data System (Thermo Scientific). For constructing calibration curves for quantification of monosaccharides, disaccharides, trisaccharides and tetrasaccharides, both galactose and glucose, lactose, maltotriose, and maltotetraose, respectively, were used (Warmerdam et al., 2014). Throughout this study, the GOS concentration represents the sum of trisaccharides and tetrasaccharides, since the concentration of higher oligosaccharides was negligible.
2.11. Operational stability study
3. Results and discussion 3.1. Adsorption of b-galactosidase on Purolite® A-109 In this study, optimisation of immobilisation of b-galactosidase from A. oryzae via adsorption onto commercial polystyrenic macroporous resin Purolite® A109 with primary amine groups was performed. In a preliminary experiment, the adsorption kinetics were analysed by monitoring the activity of immobilised enzyme and protein immobilisation yield over 24 h. It was observed (Fig. 1) that both outputs exhibited a sharp increase at an early stage, followed by slower stage that after 7 h led to equilibrium (33.79 IU g1 support; all offered enzyme protein was bound). Such a trend and almost constant specific activity indicate that the enzyme was attached in an active conformation throughout the immobilisation (Fig. 1). All subsequent experiments were performed with the adopted immobilisation time (7 h), since ANOVA analysis (P << 0.001) showed that the immobilisation time exhibited a highly significant effect on the studied factors.
2.10. Galacto-oligosaccharide production in a fluidised bed reactor The performance of the immobilised enzyme preparation obtained was also assessed in continuous mode operation using a fluidised bed reactor with recirculation, comprising a cylindrical glass column (136 mm length 9 mm inner diameter) with an external water jacket. The reaction was conducted under previously
Fig. 1. Immobilisation kinetics of b-galactosidase: (-) activity of immobilised enzyme (IU g1 support), (:) protein immobilisation yield (%), and (C) specific activity (IU mg1 protein). Immobilisation was performed in 0.1 M sodium acetate buffer at pH 4.5 with an initial enzyme concentration of 5 mg mL1. Results are presented as mean values ± standard deviation (n ¼ 2).
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Purolite® A-109 consists of a hydrophobic scaffold and charged amino groups, indicating that immobilisation can occur by several mechanisms, whether by formation of hydrophobic interactions, via ionic and dipoleedipole interactions, or by hydrogen bond formation. Therefore, the effects of pH and ionic strength of immobilisation solutions on the enzyme binding process were studied to give an insight into the nature of the enzymeesupport interactions. The immobilisation conducted in higher ionic strength buffers yielded considerably less active preparations within the whole pH range used (pH 4e7), in comparison with those performed in low molarity buffers (Fig. 2A). Therefore, it can be presumed that immobilisation predominantly occurred via the simple mechanism of ionic binding, where positively charged carrier amine groups interacted with negatively charged carboxylic groups on the enzyme surface. This hypothesis was confirmed by the desorption study, since at high ionic strength around 80% of activity was desorbed (Fig. 2B), as a consequence of hampered electrostatic interactions. Hydrophobic
53
adsorption did not occur, despite the highly hydrophobic nature of the carrier surface, since insignificant activity was desorbed with surfactant (less than 3%). When immobilisation was performed at low ionic strength, pH had a significant influence on immobilisation efficiency (P < 0.05), since immobilised activity steeply decreased with an increase in pH above pH 5 (Fig. 2A). The result obtained corroborates previous studies that reported maximum activity retention by ionic adsorption taking place during immobilisation at a pH range of pH 4e5, where the highest activity exhibited was explained in terms of both favourable conformational structure of the immobilised enzyme molecules and the amount of immobilised molecules (Guidini, Fischer, Santana, Cardoso, & Ribeiro, 2010; Gürdas¸ et al., 2012). In the next experiment, the influence of the protein concentration offered was studied. As clearly depicted in Fig. 3, and additionally proved by the ANOVA statistical analysis, the offered protein concentration exhibited a highly significant effect on each of the immobilisation parameters (P < 0.05). With an increase in offered protein concentration, the activity of immobilised enzyme and protein loading increased, while protein immobilisation yield steeply decreased after attaining a maximum (approximately 100%) to about 10 mg protein per g support (Fig. 3). A further increase in offered protein concentration led to lower efficiency of the immobilisation procedure, because protein loading became very close to the saturation level for the support, which appeared to be around 20 mg g1 support (0.19 mmol g1 support; Fig. 3). 3.2. Characterisation of immobilised enzyme Bearing in mind that the enzyme microenvironment can exhibit a large influence on the enzyme activity expressed due to the enzyme and support surface charge (Gürdas¸ et al., 2012; Monier, 2013), the activity of both free and immobilised enzyme was tested as a function of pH and temperature; these profiles are presented in Fig. 4. There was no difference in temperature optimum between free and immobilised enzyme (Fig. 4A), and the values obtained were in accordance with the literature, around 50 C. However, the immobilised preparation exhibited a slightly broader temperature optimum range, indicating that stabilisation
Fig. 2. Panel A: the effect of immobilisation buffer pH and ionic strength on expressed activity of immobilised enzyme; immobilisations were performed for 7 h using initial b-galactosidase concentration of 5 mg mL1 in corresponding buffer solution (0.1 M buffers e black bars, 1 M buffers e white bars). Panel B: desorption of immobilised preparation performed in strong ionic strength solution of CaCl2 (1 M) and 1% Triton X100 solution (B). Results are presented as mean values ± standard deviation (n ¼ 2).
Fig. 3. Effect of the offered protein concentration on: (C) protein loading (mg g1 support), (-) activity of immobilised enzyme (IU g1 support), and (:) protein immobilisation yield (%). Immobilisations were performed for 7 h using corresponding initial b-galactosidase concentration in 0.1 M sodium acetate buffer solution (pH 5). Results are presented as mean values ± standard deviation (n ¼ 2).
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contamination control, which is of tremendous importance in immobilised enzyme reactors operating at high sugar contents. 3.3. Chemical modification of support or enzyme for promotion of covalent immobilisation Besides obtaining a high overall catalytic activity of immobilised preparation, the key role of an immobilisation process is to provide satisfactory stability. Since it is well-known that an enzyme immobilised by formation of covalent bonds usually has a higher stability than that of an enzyme adsorbed on to a support surface through weak forces (Bernal, Sierra, & Mesa, 2014; Bezbradica, pez-Gallego et al., 2013; Rodrigues Mateo, & Guisan, 2014; Lo et al., 2014), further experiments were focused on attempts to obtain covalent immobilisation of A. oryzae b-galactosidase using chemical modifications of the support or the enzyme. Two types of support modification were applied: epichlorhydrine modification for the introduction of reactive epoxy groups on support surface (epoxy-Purolite) and cyanuril chloride modification for introduction of active chlorine (cc-Purolite). It was observed that the activity of b-galactosidase immobilised on modified supports was lower by far than in the previous experiment with adsorption of the enzyme (Fig. 5). In both cases, immobilisation occurred predominantly due to reactions with amino groups on enzyme surface, hence it can be assumed that immobilisation via regions rich with these residues is unfavourable, although such an approach gave very good results in our previous investigations in lipase immobilisation (Mihailovi c et al., 2014). On the other hand, when b-galactosidase was chemically modified with carbodiimide to activate its carboxylic groups, to enable formation of covalent bonds with amino groups of unmodified Purolite, the activity of the immobilised enzyme was similar to that of adsorbed enzyme, indicating that immobilisation via carboxylic groups of the enzyme is by far more favourable. 3.4. The application of immobilised b-galactosidase in galactooligosaccharide synthesis The transgalactosylation activity of the immobilised enzyme was evaluated through monitoring the synthesis of GOS; this
Fig. 4. Effect of (A) temperature and (B) pH on (C) free and (-) immobilised bgalactosidase activity using the O-NPG hydrolysis reaction; for temperature determinations the activity was measured in 0.1 M sodium acetate buffer (pH 4.5), whilst for pH determinations the activity was measured at 50 C. Results are presented as mean values ± standard deviation (n ¼ 2).
of enzyme occurred by means of immobilisation. The pH optimum shift between immobilised and free enzyme was more pronounced. The optimum pH value of the free enzyme was found to be around 4.5, whereas for the immobilised enzyme it shifted towards more acidic pH values in the range pH 3.5e4.0 (Fig. 4B). Data reporting similar behaviour are rather scarce, although Mohy Eldin, El-Aassar, El Zatahry, and Al-Sabah (2014) reported an alteration of optimum pH towards a more acidic value upon covalent immobilisation of bgalactosidase onto amino-functionalised polyvinyl chloride microspheres, which they explained by the non-uniform distribution of hydrogen ions between the bulk solution and the amino groups on the carrier surface. On the other hand, examples of shifting the optimum towards neutral pH are more common. For instance, in a study by Gürdas¸ et al. (2012), the optimum pH of immobilised enzyme was shifted up 0.5 units to a more alkaline value compared with that of the free enzyme. Lowering of the pH optimum observed in our study could be valuable because it allows better
Fig. 5. Activity of different immobilised enzyme preparations: Purolite, unmodified enzyme onto Purolite; mPurolite, modified enzyme onto Purolite; epoxy-Purolite, unmodified enzyme onto epoxy-Purolite; cc-Purolite, unmodified enzyme onto ccPurolite. Results are presented as mean values ± standard deviation (n ¼ 2).
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application of b-galactosidase has gained considerable interest because of the recognition of the physiological (prebiotic) potential of GOS. Free b-galactosidase and two immobilised preparations were assayed in terms of GOS synthesis under the same conditions (4 IU enzyme, 400 g L1 lactose, pH 4.5 and 50 C), and the time course of these reactions is presented in Fig. 6A. The two immobilised enzyme preparations showed the same trend of GOS formation throughout the whole reaction range, yielding approximately 80 g L1 after 14 h, while with the free enzyme a slightly lower GOS concentration (67 g L1) was reached. Hence, it can be concluded that the accessibility of the substrate towards the immobilised enzymes was not hampered and that immobilisation did not introduce presumed mass transfer limitations during GOS formation. Moreover, it seems that immobilisation of enzyme on Purolite shifts selectivity of b-galactosidase towards transgalactosylation, since equal levels of the hydrolytic activity of the enzyme were put into the reaction mixture. It was previously established that the first step in b-galactosidase-catalysed reactions is the formation of a galactosyleenzyme complex and the type of reaction is determined by galactosyl acceptor; if water is acceptor, hydrolysis occurs and if lactose (or another saccharide) is acceptor transgalactosylation occurs (Cardelle-Cobas et al., 2008; Guerrero et al., 2011). It is plausible that transgalactosylation is favoured by
55
the immobilised enzyme due to the hydrophobic nature of the support scaffold, which leads to a lower water concentration in the enzyme microenvironment than is the case in the reaction with the free enzyme. The effect of chemical modification on the operational stability of immobilised enzyme was assayed in a study based on performing 10 consecutive GOS synthesis reaction cycles with two different reactor configurations. In the batch reactor, the activity of immobilised unmodified enzyme continuously decreased, reaching around half of the initial activity in third cycle, and finally was completely inactivated in the last cycle. In the fluidised bed reactor a less steep inactivation was observed, reaching around half of initial activity after 5 cycles, and retaining around 10% of initial activity after 10 cycles (Fig. 7). The modified enzyme preparation exhibited a significantly higher stability in comparison with the unmodified enzyme preparation, with activity retained after 10 cycles of around 75% and 50% for the fluidised bed and the batch reactor, respectively. These results may be explained by a reduction in protein structure mobility after covalent immobilisation, since more rigid forms are usually less susceptible to damaging effects of the environment (Osman et al., 2014). Potential enzyme leakage was monitored by measuring protein content after each cycle, and no protein was detected in the reaction mixture. Therefore, activity
Fig. 6. Panel A: kinetics of GOS synthesis catalysed by b-galactosidase from A. oryzae: modified enzyme immobilisation preparation (-), unmodified enzyme immobilisation preparation ( ), free enzyme ( ). Panel B, concentration profiles during GOS synthesis reaction with initial lactose concentration of 400 g L1: disaccharides (C), GOS (-), glucose (:) and galactose (;). Panel C, dependence of GOS concentration upon different initial lactose concentrations: 50 g L1 (-), 100 g L1 (C), 200 g L1 (:), 300 g L1 (;), 400 g L1 (A), 600 g L1 (<). Panel D, kinetics of GOS synthesis in batch (-) and fluidised bed ( ) reactor. Results are presented as mean values ± standard deviation (n ¼ 2).
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Fig. 7. Operational stability of the immobilised preparations: modified (black bars) and unmodified (white bars) b-galactosidase onto Purolite in (A) batch reactor and (B) fluidised bed reactor. Results are presented as mean values ± standard deviation (n ¼ 2).
loss throughout this experiment could not be attributed to enzyme detachment, and discrepancies between inactivation in the two reactors can probably be ascribed to different flow regimes and level of turbulence. Taking into account its higher stability (Fig. 7) and rather high activity (Fig. 6A), all further experiments were performed using the modified enzyme immobilised preparation. First of all, to get a better insight in complex nature of GOS synthesis, a representative depiction of the profiles of all reaction components throughout time course of the reaction was obtained (Fig. 6B). The hydrolysis products (glucose and galactose) did not have equal concentration profiles, since molecules of galactose were incorporated into the newly formed disaccharide and oligosaccharide molecules. It can be seen that this difference is more pronounced in the earlier stages of the reaction, when the lactose concentration is rapidly decreasing and GOS synthesis prevails. GOS concentration subsequently decreased towards the end of period observed, and concentrations of glucose and galactose became similar, due to the fact that GOS are also susceptible to
hydrolysis in later stages due to their significant concentration in the reaction mixture and cannot be further replenished by synthesis of equal or higher quantities of GOS (Mahoney, 1998). This can be seen even better in Fig. 6C, where GOS concentration peaks were clearly achieved for samples with lower initial lactose concentrations. In the next set of experiments, the effect of initial lactose concentration on GOS synthesis was analysed (Fig. 6B). It was found that the GOS concentration obtained increased with an increased initial lactose concentration over the whole range examined, and consequently the highest GOS concentration (120 g L1) corresponded to the highest initial lactose concentration (600 g L1). This undoubtedly shows that the transgalactosylation reaction predominates at higher lactose concentrations, lowering the water content, and also making lactose and other sugars more favourable acceptors of the galactosyl moiety (Mahoney, 1998; Prenosil, Stuker, & Bourne, 1987). On the other hand, at low lactose concentrations, water is most likely to be the acceptor so hydrolysis occurs and thus lower GOS yields are obtained (Park & Oh, 2010). In view of the efficiency of the immobilised preparation obtained, our results are comparable with those previously obtained in other studies. For example, Urrutia et al. (2013), using 15 IU mL1 of enzyme preparation at initial lactose concentration of 400 g L1 for 7 h, achieved maximum GOS concentration of 107 g L1 (26.8%, w/w, concentration of GOS to total carbohydrate), while we obtained 78.5 g L1 (23%, w/w) with 75 times less enzyme activity. Likewise, Huerta, Vera, Guerrero, Wilson, and Illanes (2011) reported a GOS yield of approximately 30% using an initial lactose concentration of 546 g L1 and 33 IU g1 lactose. Under similar conditions, 600 g L1 lactose using 6.67 IU g1 lactose we reached maximal GOS yield of 24.1% (w/w). This was, however, slightly less than Neri et al. (2011) reported for b-galactosidase immobilised onto magnetised Dacron, namely 26.2% or 131.0 g L1 GOS. These results clearly indicate that the method developed for b-galactosidase yields a cheap and stable preparation, which could be used in further development of transgalactosylation processes. Finally, since the aim of every immobilisation is to ensure efficient implementation in continuous mode of operation, we tested the immobilised enzyme in a fluidised bed reactor with recirculation. GOS production showed a reaction course similar to that in a batch reactor, ensuring a moderately higher GOS concentration was achieved; around 100 g L1 after 14 h. However, since immobilised enzyme in the fluidised bed exhibited higher operational stability (Fig. 7B), substantial enhancement (around 40%) in the total mass of GOS produced (after 10 successive cycles) was obtained within the fluidised bed reactor application (Fig. 6D). Papers investigating employment of fluidised bed reactors are scarce, but our results are comparable with previously obtained results in similar continual systems, namely packed bed reactors. For example, under similar reaction conditions (40% lactose feed solution, pH 4.5 and 40 C), Jovanovic-Malinovska Fernandes, Winkelhausen, and Fonseca (2012) obtained a GOS yield of around 108 g L1. These findings, therefore, confirm that the immobilised preparation obtained could present a valuable contribution to the development of efficient and stable production of GOS. 4. Conclusions The aim of this study was to optimise the immobilisation of bgalactosidase from A. oryzae on Purolite® A-109, to improve the stability of immobilised enzyme by means of chemical modification and evaluate the immobilised enzyme obtained in transgalactosylation. Although immobilisation occurred predominantly by ionic adsorption, a simple and non-invasive activation of carboxyl groups on the enzyme surface with carbodiimide enabled
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formation of covalent bond between enzyme and support, resulting in a significantly increased operational stability of the immobilised b-galactosidase. The immobilised enzyme showed higher affinity towards catalysing transgalactosylation than hydrolysis reaction. Moreover, immobilised enzyme exhibited very good operational stability in a fluidised bed reactor, which indicates good prospects for its application in continuous bioprocesses. Since the use of bgalactosidases in synthesis of GOS is gaining increasing importance, the findings of this study could be a valuable contribution to the development of an efficient and stable production method for physiologically active galactoside in immobilised enzyme reactors. Acknowledgement This research was financed by the Ministry of Education, Science and Technological Development of Republic of Serbia through funding Project III 46010. References Ansari, S. A., & Husain, Q. (2012). Lactose hydrolysis from milk/whey in batch and continuous processes by concanavalin A-Celite 545 immobilized Aspergillus oryzae b galactosidase. Food and Bioproducts Processing, 90, 351e359. Banjanac, K., Mihailovi c, M., Prlainovi c, N., Stojanovi c, M., Carevi c, M., Marinkovi c, A., et al. (2014). Cyanuric chloride functionalized silica nanoparticles for covalent immobilization of lipase. Journal of Chemical Technology and Biotechnology. http://dx.doi.org/10.1002/jctb.4595. Bernal, C., Sierra, L., & Mesa, M. (2014). Design of b-galactosidase/silica biocatalysts: impact of the enzyme properties and immobilization pathways on their catalytic performance. Engineering in Life Sciences, 14, 85e94. Bezbradica, D. I., Mateo, C., & Guisan, J. M. (2014). Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde. Journal of Molecular Catalysis B: Enzymatic, 102, 218e224. Black, B. A., Yan, Y., Galle, S., Hu, Y., Curtis, J. M., & G€ anzle, M. G. (2014). Characterisation of novel galactosylated chitin-oligosaccharides and chitosan-oligosaccharides. International Dairy Journal, 39, 330e335. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Cardelle-Cobas, A., Villamiel, M., Olano, A., & Corzo, N. (2008). Study of galactooligosaccharide formation from lactose using Pectinex Ultra SP-L. Journal of the Science of Food and Agriculture, 88, 954e961. Carevi c, M., Veli ckovi c, D., Stojanovi c, M., Milosavi c, N., Rogniaux, H., Ropartz, D., et al. (2015). Insight in the regioselective enzymatic transgalactosylation of salicin catalyzed by b-galactosidase from Aspergillus oryzae. Process Biochemistry, 50, 782e788. go, T. V., De Medeiros Burkert, J. F., & Da Silva Campello, G., Trindade, R. A., Re Burkert, C. A. V. (2012). Immobilization of b-galactosidase from Kluyveromyces ® lactis on Eupergit c and properties of the biocatalyst. International Journal of Food Engineering, 8. http://dx.doi.org/10.1515/1556-3758.2760. Fischer, J., Guidini, C. Z., Santana, L. N. S., de Resende, M. M., Cardoso, V. L., & Ribeiro, E. J. (2013). Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized b-galactosidase from Aspergillus oryzae. Journal of Molecular Catalysis B: Enzymatic, 85e86, 178e186. Fischer, C., & Kleinschmidt, T. (2015). Synthesis of galactooligosaccharides using sweet and acid whey as a substrate. International Dairy Journal, 48, 15e22. Gaur, R., Pant, H., Jain, R., & Khare, S. K. (2006). Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae b-galactosidase. Food Chemistry, 97, 426e430. Guerrero, C., Vera, C., Novoa, C., Dumont, J., Acevedo, F., & Illanes, A. (2014). Purification of highly concentrated galacto-oligosaccharide preparations by selective fermentation with yeasts. International Dairy Journal, 39, 78e88.
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