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β-galactosidase stability at high substrate concentrations

β-galactosidase stability at high substrate concentrations Enzymatic synthesis of galacto-oligosaccharides is usually performed at high initial substrate concentrations since higher yields are obtained. We report here on the stability of β-galactosidase from Bacillus circulans at 25, 40, and 60°C in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. In buffer, the half-life time was 220 h and 13 h at 25 and 40°C, respectively, whereas the enzyme was completely inactivated after two hours at 60°C. In systems with 5.0 and 30% (w/w) lactose, a mechanistic model was used to correct the oNPG converting activity for the presence of lactose, glucose, galactose, and oligosaccharides in the activity assay. Without correction, the stability at 5.0% (w/w) lactose was overestimated, while the stability at 30% (w/w) lactose was underestimated. The inactivation constant k was strongly dependent on temperature in buffer, whereas only a slight increase in k was d d found with temperature at high substrate concentrations. The enzyme stability was found to increase strongly with the initial substrate concentrations. The inactivation energy E appeared to be lower at high initial substrate concentrations. Keywords: β-galactosidase; Bacillus circulans; Galacto-oligosaccharides; Concentrated systems; Enzyme activity; Enzyme stability Introduction protein structure of lysozyme in the presence of For enzymatic production processes it is of interest to osmolytes. De Cordt et al. (1994) described the influence use highly concentrated conditions, since energy, of high concentrations of polyalcohols and carbohy- water, and material costs can be saved. However, the drates on the enzymestabilitybysubstratebinding activity and stability of enzymes are often investigated or preferential hydration. They observed various situ- in aqueous systems, which may lead to irrelevant ations in which the presence of inert crowding data. The enzyme activity of β-galactosidases, which agents increases the thermo-stability of proteins is used in the production of galacto-oligosaccharides (Perham et al. 2007; Stagg et al. 2007; Zhou et al. 2008). (GOS), in highly concentrated systems was studied Recently, Yadav (2013) described that the presence of before (Warmerdam et al. 2013a) and was found to sucrose and trehalose strongly increased the half-life be strongly influenced by the concentration of reactants time of α-amylase. and products. The high concentration of reactants GOS are usually produced with β-galactosidase at high and products may not only lead to more reactions temperatures and at high substrate concentrations in taking place, but it will also lead to molecular crow- industry. An advantage of reactions at high temperatures ding, which can have large effects on enzyme activity is the improved solubility of the substrates which makes (Minton 2001; Ellis 2001). higher substrate concentrations possible (Bruins et al. 2001). Besides the enzyme activity, their stability can as well However, the inactivation of the enzyme is faster as well be strongly affected by molecular crowding (Minton (Bruins et al. 2003). 2001; Ellis 2001). In 1985, Arakawa and Timasheff The stability of β-galactosidase from Bacillus circulans (1985) have already described the stabilization of the was investigated before in systems with low lactose con- centration or in absence of lactose (Mozaffar et al. 1984). Vetere and Paoletti (1998), and Song et al. (2011a) studied * Correspondence: anja.janssen@wur.nl Food Process Engineering Group, Wageningen University, PO Box 8129, the stability of several isoforms of β-galactosidase from Wageningen, EV 6700, The Netherlands © 2013 Warmerdam et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Warmerdam et al. SpringerPlus 2013, 2:402 Page 2 of 8 http://www.springerplus.com/content/2/1/402 Bacillus circulans in aqueous systems. They found monohydrate, and disodium hydrogen phosphate were that the enzyme preparation was (partly) stable up purchased from Merck (Darmstadt, Germany). to 50°C. Thestabilityof free β-galactosidase from McIlvaine’s buffer was prepared by adding together Bacillus circulans in systems with high lactose con- 0.1 M citric acid and 0.2 M disodium hydrogen phosphate centrations, which are usually used in production in the right ratio to achieve a pH of 6.0. systems, has to our knowledge never been investi- gated before. Lactose conversion When using high initial substrate concentrations, it is The stability of Biolacta N5 was investigated in a 0, important to investigate the effect of reactants in the 5.0, and 30% (w/w) lactose-in-buffer solution in a activity assay. Baks et al. (2006) found that starch and its temperature controlled batch reactor with an anchor hydrolysis products may have large effects on the stirrer at 150 rpm. A certain mass of lactose monohy- Ceralpha activity assay. This assay is comparable to the drate and a certain mass of buffer were weighted, so activity assay used for β-galactosidases with oNPG as an that a final concentration of lactose was obtained on artificial substrate. Lactose and (some of) its conversion a weight basis of 5% and 30% (w/w). 30% (w/w) lac- products are substrate for β-galactosidase as well as tose is close to the solubility at 50°C. The lactose was oNPG: they act as acceptor molecule for the enzyme- dissolved at approximately 60°C prior to cooling the galactose complex, and they act as inhibitors and solution to the desired temperature. The initial reac- competitors (Warmerdam et al. 2013a; Borralho et al. tion volume was 25 mL. Temperatures were kept at 2002) (Warmerdam A, Zisopoulos FK, Boom RM, -1 25, 40, or 60°C. A volume of 1.0 mL of 2.0 g∙L Janssen AEM: Kinetic characterization of β-galactosidases, Biolacta N5 was added once the temperature was submitted). In addition, galactose and glucose are constant. Samples were taken at 30 s, 5, 10, 15, 30, usually found to be inhibitors for β-galactosidases 60, 120, 240, 360 minutes and 22, and 24 hours for (Warmerdam et al. 2013a; Greenberg and Mahoney determination of the carbohydrate composition (100 μL 1982; Macfarlane et al. 2008; Prenosil et al. 1987) sample) and for determination of the enzyme activity (Warmerdam A, Zisopoulos FK, Boom RM, Janssen (210 μL sample). The final reaction volume was 21 mL. AEM: Kinetic characterization of β-galactosidases, submitted). Because of the interactions of these carbohy- drates, it is important to correct the oNPG activity Sample handling for determination of the carbohydrate measurements for their presence. composition The aim of this study is therefore to investigate the The sample (100 μL) taken from the reactor for deter- stability of β-galactosidase from Bacillus circulans at mination of the carbohydrate composition was directly various temperatures both in buffer, and in systems added into an Eppendorf tube with 50 μL of 5% (w/w) with initially 5.0 and 30% (w/w) lactose. The remaining H SO to inactivate the enzyme. Subsequently, the 2 4 enzyme activity is measured via the oNPG activity samples were stored at −20°C until further preparation. assay. The activity measurements are corrected for Before HPLC analysis, the enzyme was removed from the effect of the carbohydrates present in the reaction the samples by filtering the samples at 14,000 × g at 18°C mixture. for 30 minutes using pretreated Amicon® ultra-0.5 centri- fugal filter devices (Millipore Corporation, Billerica, MA, Materials and methods United States) with a cut-off of 10 kDa in a Beckman Materials Coulter Allegra X-22R centrifuge. The pretreatment Lactose monohydrate (Lactochem), Vivinal-GOS and a of the filters consisted out of two centrifugation β-galactosidase from Bacillus circulans called Biolacta steps: first, 500 μL of Milli-Q water was centrifuged N5 (Daiwa Kasei K. K., Japan) were gifts from at 14,000 × g at 18°C for 15 minutes; and second, FrieslandCampina (Beilen, The Netherlands). Biolacta the filters were placed up-side-down in the tube and N5 was previously found to have a total protein content of centrifuged at 14,000 × g at 18°C for 5 minutes. After 19 ± 3% (Warmerdam et al. 2013b). In all calculations, the filtration, the samples were neutralized with 5% (w/w) total enzyme concentration was assumed to be equal to sodium hydroxide. the total protein concentration, because the actual enzyme concentration is not known. Sulphuric acid, sodium hydroxide, o-nitrophenyl β-D-galactopyranoside Measurement of the carbohydrate composition (oNPG), o-nitrophenol (oNP), D(+)-glucose, D(+)-galactose, The filtered samples were analysed with HPLC using maltotriose, maltotetraose, maltopentaose, maltohexaose, a Rezex RSO oligosaccharide column (Phenomenex, and maltoheptaose were purchased from Sigma-Aldrich Amstelveen, the Netherlands) at 80°C. The column (Steinheim, Germany). Sodium carbonate, citric acid was eluted with Milli-Q water at a flow rate of Warmerdam et al. SpringerPlus 2013, 2:402 Page 3 of 8 http://www.springerplus.com/content/2/1/402 -1 -1 0.3 mL/min. The eluent was monitored with a refractive protein∙L or in mmol protein∙L with the reaction index detector. rate constants k , k , k , k , k , k , k ,and k in 1 a1 a2 3 a3 a4 a5 6 -1 The standards that were used for calibration of the mmol oNP∙L∙(mmol X∙gprotein∙s) or in mmol -1 column were lactose, glucose, galactose, maltotriose, oNP∙L∙(mmol X∙mmol protein∙s) , respectively, with X maltotetraose, maltopentaose, maltohexaose, and being the corresponding reactant. The inhibition constant maltoheptaose. Galacto-oligosaccharides up to a degree of K is in mM. polymerization of 7 were assumed to have the same The respective parameters for Biolacta N5 were deter- response as the glucose-oligomers with an equal mined in previous work (Warmerdam A, Zisopoulos degree of polymerization. This was confirmed with FK, Boom RM, Janssen AEM: Kinetic characterization mass balances. of β-galactosidases, submitted) and are shown in Table 1. Enzyme activity measurements To investigate the effect of the present reactants com- The oNPG activity measurements, adapted from pared to when no reactants are added in the activity Nakanishi et al. (Nakanishi et al. 1983), were performed assay, we want to normalize this initial rate with the ini- immediately after the sample was taken from the reac- tial rate that would have been obtained without addition tion mixture. An Eppendorf tube with 790 μL of 0.25% of carbohydrates, which is given by equation 2: (w/w) oNPG-in-buffer was preheated in an Eppendorf Thermomixer at 40°C and 600 rpm for 10 minutes. k½ oNPG 1 þ k ½ oNPGþk ½ H20 0;oNP a1 a2 Subsequently, 210 μL of sample was added and these ½ c ¼0 k½ oNPGþk ½ lacþk½ oligo ½ gal 1 3 6 v 1 þ þ mixtures were incubated for another 10 minutes at 40°C 0;oNP k ½ oNPGþK ½ H20þk ½ lacþk ½ gluþk ½ gal K a1 a2 a3 a4 a5 i and 600 rpm. A volume of 1.0 mL of 10% (w/w) Na CO 2 3 ð2Þ solution was added to stop the reaction and, afterwards, the absorbance of oNP was measured at 420 nm. The ½ c ¼0 where v is the initial rate of oNP formation without 0;oNP oNP concentration was determined using the law of addition of carbohydrates C. At each time point, the Lambert-Beer of which the extinction coefficient was -1 -1 concentrations of reactants used in this equation is the determined to be 4576 M ∙cm .The oNP formation concentration that has been measured with HPLC. was found to be linear during the first 10 minutes of the reaction. This initial rate of oNP formation was -1 -1 Activity measurements corrected for the presence of expressed in mmol∙min ∙gprotein .Measurements carbohydrates were performed in duplicate and the average enzyme The activity measurements were corrected for the effect activity was used. of lactose, glucose, galactose, and oligosaccharides on the activity assay with equation 3: Modeling the effect of carbohydrates on the activity assay The effect of carbohydrates on the oNPG activity assay A measured A ¼ ð3Þ corrected v 0;oNP can be described with a mechanistic model; we refer ½ c ¼0 0;oNP to previous work for the mechanistic description of the model (Warmerdam A, Zisopoulos FK, Boom RM, where A and A are the enzyme activity measured corrected Janssen AEM: Kinetic characterization of β-galactosidases, calculated directly from the absorbance measurements submitted) (equation 1). This model accounts for the (see “Enzyme activity measurements”), and the enzyme use of oNPG as substrate (k ) as well as acceptor (k ), the use of water as acceptor (k ), the use of a1 a2 lactose (lac) as substrate (k ) as well as acceptor (k ), 3 a3 Table 1 Parameters for Biolacta N5 in the conversion of the use of glucose (glu) as acceptor (k ), the use of a4 oNPG, lactose, glucose, galactose, and oligosaccharides galactose (gal) as acceptor (k ) aswell asinhibitor a5 -1 k (mmol oNP∙L∙(mmol oNPG∙g protein∙s) ) 0.10 (K ), and the use of oligosaccharides (oligo) as substrate -1 k (mmol oNP∙L∙(mmol lactose∙g protein∙s) ) 0.012 (k ) as follows: -1 k (mmol oNP∙L∙(mmol oligos∙g protein∙s) ) 0.077 v k½ oNPG -1 0;oNP 1 k (mmol oNP∙L∙(mmol oNPG∙g protein∙s) ) 0.0063 a1 k½ oNPGþk ½ lacþk½ oligo ½ gal 1 3 6 -1 1 þ þ k (mmol oNP∙L∙(mmol H O∙g protein∙s) ) 0.0042 k ½ oNPGþk ½ H20þk ½ lacþk ½ gluþk ½ gal K a2 2 a1 a2 a3 a4 a5 i -1 k (mmol oNP∙L∙(mmol glucose∙g protein∙s) ) 0.00092 ð1Þ a4 -1 k (mmol oNP∙L∙(mmol galactose∙g protein∙s) ) 0.023 a5 where v is the initial rate of oNP formation in mM 0,oNP K (mM) 255 -1 oNP∙s , E is the initial enzyme concentration in g 0 Warmerdam et al. SpringerPlus 2013, 2:402 Page 4 of 8 http://www.springerplus.com/content/2/1/402 Figure 1 Stability of Biolacta N5 at various substrate concentrations. Residual enzyme activityof Biolacta N5 in (A) buffer, (B) 5.0% (w/w) -1 lactose, (C) 30% (w/w) lactose at ■ 25, 40, xand ▲ 60°C and pH 6.0 with an enzyme concentration of 16 mg∙L . The enzyme activity is measured in the oNPG activity assay. (Lines for guidance). activity corrected for the presence of lactose, glucose, Determination of enzyme stability galactose, and oligosaccharides, respectively. Enzyme inactivation during the running time of the For each sample made with Vivinal-GOS, the concentra- experiment was modelled with a first order inactivation tion of lactose, glucose, galactose, and total oligosaccharide model with: was calculated. The concentrations of lactose, galactose, −k ⋅t glucose and total oligosaccharides are 19 dm%, 1 dm%, k ¼ k ⋅ e ð4Þ t 0 21 dm%, and 59 dm% in Vivinal-GOS. Oligosaccharides were assumed to be mainly trisaccharides with a molecular where k and k are the reaction rates at time zero and 0 t -1 weight of 504 g/mol. time t in h, k is the enzyme inactivation constant in h , Figure 2 Carbohydrate profiles at an initial lactose concentration of 5.0% (w/w) at ■ 25, 40, and ▲ 60°C and pH 6.0 with an enzyme -1 concentration of 16 mg∙L .A. Disaccharide conversion; B. GOS (all oligosaccharides larger than DP2) production; C. Glucose production; D. Galactose production. Figure corresponds with Figure 1B. Warmerdam et al. SpringerPlus 2013, 2:402 Page 5 of 8 http://www.springerplus.com/content/2/1/402 Figure 3 Carbohydrate profiles at an initial lactose concentration of 30% (w/w) at ■ 25, 40, and ▲ 60°C and pH 6.0 with an enzyme -1 concentration of 16 mg∙L .A. Disaccharide conversion; B. GOS (all oligosaccharides larger than DP2) production; C. Glucose production; D. Galactose production. Figure corresponds with Figure 1C. and t is the running time at which the sample was taken The half-life time of the enzyme t can be determined in hours. The enzyme inactivation constant k and with equation 6: the reaction rate at time zero k were determined by 1nðÞ 2 linearization of equation 4. t ¼ ð6Þ The inactivation energy E can be determined with the Arrhenius relation, equation 5: Results and discussion Ea − Effect of temperature and initial lactose concentration on R⋅T k ¼ k ⋅ e ð5Þ d ∞ enzyme stability -1 where k and k (the Arrhenius constant) are in s , Figure 1 shows the specific oNPG converting activity of d ∞ -1 -1 R is the gas constant in J∙mol ∙K ,and T is the Biolacta N5 after incubation in buffer (A), in 5.0% (w/w) temperature in K. lactose (B), and in 30% (w/w) lactose (C) at 25, 40, and Figure 4 Corrected stability of Biolacta N5 at various substrate concentrations. Residual enzyme activity of Biolacta N5 in (A) buffer, (B) 5.0% ______ -1 (w/w), and (C) 30% (w/w) lactose at ■, 25; ◇,---40; and ▲, - - 60°C and pH 6.0 with an enzyme concentration of 16 mg∙L ,corrected forthe influence of lactose, galactose, glucose, and oligosaccharides. Symbols represent measured data, (dashed) lines represent modeled data. Warmerdam et al. SpringerPlus 2013, 2:402 Page 6 of 8 http://www.springerplus.com/content/2/1/402 Table 2 The initial oNP formation rate k of Biolacta N5 Table 4 The half-life time t in h of Biolacta N5 in hours 0 ½ -1 -1 in mmol∙min ∙g protein at various initial lactose at various initial lactose concentrations and temperatures concentrations and temperatures, together with its 95% [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) confidence interval 25°C 220 16 29 [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) 40°C 13 17 29 25°C 9.1 ± 1.0 11 ± 1 12 ± 1 60°C 0.048 0.82 16 40°C 9.0 ± 1.4 9.9 ± 1.2 13 ± 1 60°C 12 ± 1 8.9 ± 0.6 12 ± 0 composition in the samples was determined and the effect of these reactants on the activity assay was determined with equation 2. 60°C. The observed data are not corrected yet for the presence of lactose, glucose, galactose, and Vivinal-GOS. Carbohydrate profiles The initial activity in buffer was approximately -1 -1 13 mmol∙min ∙g enzyme , while the initial activities During the stability experiments described in Figure 1B -1 -1 were approximately 10 and 4 mmol∙min ∙g enzyme in and C, also samples were taken to determine the sugar composition. The results are presented in 5.0 and 30% (w/w) lactose, respectively. The reduction in the initial activity with an increasing lactose con- Figures 2 and 3. The concentrations of disaccharide centration is caused by the competition of lactose (A), GOS (B), glucose (C) and galactose (D) are shown. (that is present in the samples) with oNPG in the activity assay, as will be discussed later. The carbohydrate content changed in time, and varied In buffer, the enzyme was stable at 25°C, but lost 84% considerably between the initially different lactose con- centrations. At an initial lactose concentration of 5.0% of its activity after 24 hours of incubation at 40°C, and was completely inactivated after two hours at 60°C. This (w/w), the carbohydrate concentrations hardly changed complete inactivation in buffer at 60°C was expected: anymore after 6 hours of reaction at 60°C. At 25 and 40°C the GOS content showed an optimum around Mozaffar et al. (1984), Vetere and Paoletti (1998), and Song et al. (2011b) described that its isoforms are stable 6 hours of incubation, indicating hydrolysis of the up to at most 50°C for one hour. The stability at elevated desired product at longer incubation times. Also a considerable amount of galactose was present after temperatures improved considerably in the presence of lactose. In a 5.0% (w/w) lactose solution, it took 24 hours of reaction. At an initial lactose concentra- six hours of incubation at 60°C before most of the tion of 30% (w/w), GOS synthesis continued at all temperatures, including 60°C, until at least 22 hours activity was lost, while in a 30% (w/w) lactose solu- tion, 27% of the enzyme activity was left after of reaction (Figure 3B). Only small amounts of galact- 24 hours at 60°C. ose were formed at all temperatures. The galactose production (indicating hydrolysis) was substantial at The measured activity in Figure 1B and C after 24 hours of reaction at 25°C was lower than at 40°C. an initial lactose concentration of 5.0% (w/w) because One would expect a better stability at a lower of a high availability of water molecules, whereas no significant amounts of galactose were observed at an temperature. These unexpected stability values are the result of the presence of reactants during the ac- initial lactose concentration of 30% (w/w). It is clear tivity assay. These reactants interfere with the activ- that both the initial lactose concentration as well as the reaction temperature had a strong effect on the ity measurements similarly as was described by (Baks et al. 2006) (Warmerdam A, Zisopoulos FK, carbohydrate composition. Boom RM, Janssen AEM: Kinetic characterization of β-galactosidases, submitted). Therefore, the carbohydrate Correction for the presence of carbohydrates in stability experiments The specific enzyme activity that was shown in Figure 1B -1 and C was evaluated once more. The influence of Table 3 The inactivation constant k of Biolacta N5 in h lactose, galactose, glucose, and oligosaccharides on at various initial lactose concentrations and temperatures, together with its 95% confidence interval the oNPG activity assay was taken into account using equations 2 and 3. The carbohydrate content [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) differed considerably during lactose conversion at various 25°C 0.0032 ± 0.0112 0.043 ± 0.023 0.024 ± 0.011 conditions (Figures 2 and 3) and the carbohydrates 40°C 0.054 ± 0.040 0.041 ± 0.025 0.024 ± 0.015 have a strong effect on the oNPG activity assay 60°C 15 ± 3 0.85 ± 0.19 0.043 ± 0.006 (Warmerdam et al. 2013a) (Warmerdam A, Zisopoulos Warmerdam et al. SpringerPlus 2013, 2:402 Page 7 of 8 http://www.springerplus.com/content/2/1/402 FK, Boom RM, Janssen AEM: Kinetic characterization enzyme in buffer is strongly dependent on the of β-galactosidases, submitted). The corrected enzyme temperature, whereas the inactivation in a system activities are shown in Figure 4. with 30% (w/w) lactose initially is hardly dependent After correction for the presence of carbohydrates on the temperature. The inactivation energy E , in the oNPG activity assay, the values of the specific shown in Table 5, decreased with increasing substrate enzyme activity on the Y-axis of Figure 4 are differ- concentration. This is similar to what was found by ent. The enzyme activities at time zero are more or De Cordt et al. (1994). The higher stability of the less similar (Table 2). The amount of added enzyme enzyme is might be caused by molecular crowding or by was the same in all experiments, thus a similar complexation with the substrate or with a remaining enzyme activity was indeed expected. Another aspect galactose moiety. is theincreasein the activityat40°Cina5.0% (w/w) A higher thermostability at high substrate concen- lactose solution at longer incubation times. After trations is very favourable in the production of GOS correction this increase in activity is not present any- by β-galactosidases from B.circulans.Athighsub- more and the activity decreased in time. After correc- strate concentrations, the reaction temperature can tion theactivitydecrease versustimeat25and 40°C be higher than the enzyme’s stable ranges that were is more or less similar. reported before in aqueous solutions, and it can be equal/ The corrected data were used to fit the first order closer to their optimal temperatures (Mozaffar et al. 1984; inactivation model (equation 4). The best fit is shown Vetere and Paoletti 1998; Song et al. 2011a), which in Figure 4. The inactivation constant and the half- will result in a higher enzyme stability. life time are shown in Tables 3 and 4. In buffer the half-life time at 25°C is about 4600 times higher as Conclusions compared to the half-life time at 60°C. In 5.0% (w/w) β-Galactosidase from Bacillus circulans was found to be lactose this value is only twenty times higher, while at quite stable against temperature at high substrate con- 30% (w/w) lactose there is only a factor of two differ- centrations. For a proper conclusion on the remaining ence in the half-life time. At 25°C the enzyme enzyme activity versus time it was important to correct appeared to be most stable in buffer, however, at 40 the enzyme activity measurements for the presence of and 60°C the enzyme is most stable at elevated various reactants. lactose concentrations. The half-life time of the Without correcting the enzyme activity at 5.0% (w/w) enzyme (Table 4) (strongly) increased with increasing lactose, the actual stability was overestimated, whereas substrate concentration at 40 and 60°C. not correcting the enzyme activity at 30% (w/w) lactose Figure 5 shows the linearized Arrhenius plot of ln resulted in an underestimation of the actual stability of (k ) as a function of 1/T. The inactivation of the β-galactosidase from Bacillus circulans. A high initial lactose concentration had a large positive effect on the enzyme stability. The improved stability in more concentrated sys- tems is very interesting for production conditions. The utilization of more concentrated systems for en- zymatic conversions is economically more interesting in order to avoid the unnecessary use of water, to save energy as a smaller volume needs to be heated, and to save on capital expenditures as less equipment is necessary. Table 5 Inactivation energy E for various lactose concentrations [lactose] E -1 [% (w/w)] [kJ∙mol ] 0 200 Figure 5 Linearized Arrhenius plot of ln(k ) as a function of 1/T. ______ 5.0 72 Symbols: ◇, 0% (w/w) lactose; ■, - - - 5.0% (w/w) lactose, and ▲, - - 30% (w/w) lactose. 30 14 Warmerdam et al. SpringerPlus 2013, 2:402 Page 8 of 8 http://www.springerplus.com/content/2/1/402 Competing interests Warmerdam A, Wang J, Boom RM, Janssen AEM (2013a) Effects of carbohydrates The authors declare that they have no competing interests. on the oNPG converting activity of β-galactosidases. J Agric Food Chem 61:6458–6464 Warmerdam A, Paudel E, Jia W, Boom RM, Janssen AEM (2013b) Characterization Authors’ contributions of β-galactosidase isoforms from Bacillus circulans and their contribution to AW: designed the experiments, performed the experiments, analyzed the GOS production. Appl Biochem Biotechnol 170:340–358 data, and wrote the paper. RMB: designed the experiments, and wrote the Yadav JK (2013) Macromolecular crowding enhances catalytic efficiency and paper. AEMJ: designed the experiments, analyzed the data, and wrote the stability of α-amylase. ISRN Biotechnology 2013:Article ID 737805 paper. All authors read and approved the final manuscript. Zhou HX, Rivas GN, Minton AP (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological Acknowledgements consequenses. Annu Rev Biophys 37:375–397 The authors would like to thank Eric Benjamins, Linqiu Cao, Ellen van Leusen, Albert van der Padt, and Jan Swarts of FrieslandCampina for the valuable doi:10.1186/2193-1801-2-402 scientific discussions. Cite this article as: Warmerdam et al.: β-galactosidase stability at high This project is jointly financed by the European Union, European Regional substrate concentrations. SpringerPlus 2013 2:402. Development Fund and The Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the Delta, the Municipality of Groningen, the Provinces of Groningen, Fryslân and Drenthe as well as the Dutch Carbohydrate Competence Center (CCC WP9). Received: 3 April 2013 Accepted: 20 August 2013 Published: 27 August 2013 References Arakawa T, Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47:411–414 Baks T, Janssen AEM, Boom RM (2006) The effect of carbohydrates on α-amylase activity measurements. 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Biotechnol Bioeng 30:1019–1025 journal and benefi t from: Song J, Abe K, Imanaka H, Imamura K, Minoda M, Yamaguchi S, Nakanishi K (2011a) Causes of the production of multiple forms of β-galactosidase by 7 Convenient online submission Bacillus circulans. Biosci Biotechnol Biochem 75:268–278 7 Rigorous peer review Song J, Imanaka H, Imamura K, Minoda M, Katase T, Hoshi Y, Yamaguchi S, Nakanishi K (2011b) Cloning and expression of a beta-galactosidase gene of 7 Immediate publication on acceptance Bacillus circulans. Biosci Biotechnol Biochem 75:1164–1167 7 Open access: articles freely available online Stagg L, Zhang S-Q, Cheung MS, Wittung-Stafshede P (2007) Molecular crowding 7 High visibility within the fi eld enhances native structure and stability of α/β protein flavodoxin. Proc Natl 7 Retaining the copyright to your article Acad Sci 104:18976–18981 Vetere A, Paoletti S (1998) Separation and characterization of three β- galactosidases from Bacillus circulans. Biochimica et Biophysica Acta (BBA) - Submit your next manuscript at 7 springeropen.com General Subjects 1380:223–231 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png SpringerPlus Springer Journals

β-galactosidase stability at high substrate concentrations

SpringerPlus , Volume 2 (1) – Aug 27, 2013

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Copyright © 2013 by Warmerdam et al.; licensee Springer.
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10.1186/2193-1801-2-402
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Abstract

Enzymatic synthesis of galacto-oligosaccharides is usually performed at high initial substrate concentrations since higher yields are obtained. We report here on the stability of β-galactosidase from Bacillus circulans at 25, 40, and 60°C in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. In buffer, the half-life time was 220 h and 13 h at 25 and 40°C, respectively, whereas the enzyme was completely inactivated after two hours at 60°C. In systems with 5.0 and 30% (w/w) lactose, a mechanistic model was used to correct the oNPG converting activity for the presence of lactose, glucose, galactose, and oligosaccharides in the activity assay. Without correction, the stability at 5.0% (w/w) lactose was overestimated, while the stability at 30% (w/w) lactose was underestimated. The inactivation constant k was strongly dependent on temperature in buffer, whereas only a slight increase in k was d d found with temperature at high substrate concentrations. The enzyme stability was found to increase strongly with the initial substrate concentrations. The inactivation energy E appeared to be lower at high initial substrate concentrations. Keywords: β-galactosidase; Bacillus circulans; Galacto-oligosaccharides; Concentrated systems; Enzyme activity; Enzyme stability Introduction protein structure of lysozyme in the presence of For enzymatic production processes it is of interest to osmolytes. De Cordt et al. (1994) described the influence use highly concentrated conditions, since energy, of high concentrations of polyalcohols and carbohy- water, and material costs can be saved. However, the drates on the enzymestabilitybysubstratebinding activity and stability of enzymes are often investigated or preferential hydration. They observed various situ- in aqueous systems, which may lead to irrelevant ations in which the presence of inert crowding data. The enzyme activity of β-galactosidases, which agents increases the thermo-stability of proteins is used in the production of galacto-oligosaccharides (Perham et al. 2007; Stagg et al. 2007; Zhou et al. 2008). (GOS), in highly concentrated systems was studied Recently, Yadav (2013) described that the presence of before (Warmerdam et al. 2013a) and was found to sucrose and trehalose strongly increased the half-life be strongly influenced by the concentration of reactants time of α-amylase. and products. The high concentration of reactants GOS are usually produced with β-galactosidase at high and products may not only lead to more reactions temperatures and at high substrate concentrations in taking place, but it will also lead to molecular crow- industry. An advantage of reactions at high temperatures ding, which can have large effects on enzyme activity is the improved solubility of the substrates which makes (Minton 2001; Ellis 2001). higher substrate concentrations possible (Bruins et al. 2001). Besides the enzyme activity, their stability can as well However, the inactivation of the enzyme is faster as well be strongly affected by molecular crowding (Minton (Bruins et al. 2003). 2001; Ellis 2001). In 1985, Arakawa and Timasheff The stability of β-galactosidase from Bacillus circulans (1985) have already described the stabilization of the was investigated before in systems with low lactose con- centration or in absence of lactose (Mozaffar et al. 1984). Vetere and Paoletti (1998), and Song et al. (2011a) studied * Correspondence: anja.janssen@wur.nl Food Process Engineering Group, Wageningen University, PO Box 8129, the stability of several isoforms of β-galactosidase from Wageningen, EV 6700, The Netherlands © 2013 Warmerdam et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Warmerdam et al. SpringerPlus 2013, 2:402 Page 2 of 8 http://www.springerplus.com/content/2/1/402 Bacillus circulans in aqueous systems. They found monohydrate, and disodium hydrogen phosphate were that the enzyme preparation was (partly) stable up purchased from Merck (Darmstadt, Germany). to 50°C. Thestabilityof free β-galactosidase from McIlvaine’s buffer was prepared by adding together Bacillus circulans in systems with high lactose con- 0.1 M citric acid and 0.2 M disodium hydrogen phosphate centrations, which are usually used in production in the right ratio to achieve a pH of 6.0. systems, has to our knowledge never been investi- gated before. Lactose conversion When using high initial substrate concentrations, it is The stability of Biolacta N5 was investigated in a 0, important to investigate the effect of reactants in the 5.0, and 30% (w/w) lactose-in-buffer solution in a activity assay. Baks et al. (2006) found that starch and its temperature controlled batch reactor with an anchor hydrolysis products may have large effects on the stirrer at 150 rpm. A certain mass of lactose monohy- Ceralpha activity assay. This assay is comparable to the drate and a certain mass of buffer were weighted, so activity assay used for β-galactosidases with oNPG as an that a final concentration of lactose was obtained on artificial substrate. Lactose and (some of) its conversion a weight basis of 5% and 30% (w/w). 30% (w/w) lac- products are substrate for β-galactosidase as well as tose is close to the solubility at 50°C. The lactose was oNPG: they act as acceptor molecule for the enzyme- dissolved at approximately 60°C prior to cooling the galactose complex, and they act as inhibitors and solution to the desired temperature. The initial reac- competitors (Warmerdam et al. 2013a; Borralho et al. tion volume was 25 mL. Temperatures were kept at 2002) (Warmerdam A, Zisopoulos FK, Boom RM, -1 25, 40, or 60°C. A volume of 1.0 mL of 2.0 g∙L Janssen AEM: Kinetic characterization of β-galactosidases, Biolacta N5 was added once the temperature was submitted). In addition, galactose and glucose are constant. Samples were taken at 30 s, 5, 10, 15, 30, usually found to be inhibitors for β-galactosidases 60, 120, 240, 360 minutes and 22, and 24 hours for (Warmerdam et al. 2013a; Greenberg and Mahoney determination of the carbohydrate composition (100 μL 1982; Macfarlane et al. 2008; Prenosil et al. 1987) sample) and for determination of the enzyme activity (Warmerdam A, Zisopoulos FK, Boom RM, Janssen (210 μL sample). The final reaction volume was 21 mL. AEM: Kinetic characterization of β-galactosidases, submitted). Because of the interactions of these carbohy- drates, it is important to correct the oNPG activity Sample handling for determination of the carbohydrate measurements for their presence. composition The aim of this study is therefore to investigate the The sample (100 μL) taken from the reactor for deter- stability of β-galactosidase from Bacillus circulans at mination of the carbohydrate composition was directly various temperatures both in buffer, and in systems added into an Eppendorf tube with 50 μL of 5% (w/w) with initially 5.0 and 30% (w/w) lactose. The remaining H SO to inactivate the enzyme. Subsequently, the 2 4 enzyme activity is measured via the oNPG activity samples were stored at −20°C until further preparation. assay. The activity measurements are corrected for Before HPLC analysis, the enzyme was removed from the effect of the carbohydrates present in the reaction the samples by filtering the samples at 14,000 × g at 18°C mixture. for 30 minutes using pretreated Amicon® ultra-0.5 centri- fugal filter devices (Millipore Corporation, Billerica, MA, Materials and methods United States) with a cut-off of 10 kDa in a Beckman Materials Coulter Allegra X-22R centrifuge. The pretreatment Lactose monohydrate (Lactochem), Vivinal-GOS and a of the filters consisted out of two centrifugation β-galactosidase from Bacillus circulans called Biolacta steps: first, 500 μL of Milli-Q water was centrifuged N5 (Daiwa Kasei K. K., Japan) were gifts from at 14,000 × g at 18°C for 15 minutes; and second, FrieslandCampina (Beilen, The Netherlands). Biolacta the filters were placed up-side-down in the tube and N5 was previously found to have a total protein content of centrifuged at 14,000 × g at 18°C for 5 minutes. After 19 ± 3% (Warmerdam et al. 2013b). In all calculations, the filtration, the samples were neutralized with 5% (w/w) total enzyme concentration was assumed to be equal to sodium hydroxide. the total protein concentration, because the actual enzyme concentration is not known. Sulphuric acid, sodium hydroxide, o-nitrophenyl β-D-galactopyranoside Measurement of the carbohydrate composition (oNPG), o-nitrophenol (oNP), D(+)-glucose, D(+)-galactose, The filtered samples were analysed with HPLC using maltotriose, maltotetraose, maltopentaose, maltohexaose, a Rezex RSO oligosaccharide column (Phenomenex, and maltoheptaose were purchased from Sigma-Aldrich Amstelveen, the Netherlands) at 80°C. The column (Steinheim, Germany). Sodium carbonate, citric acid was eluted with Milli-Q water at a flow rate of Warmerdam et al. SpringerPlus 2013, 2:402 Page 3 of 8 http://www.springerplus.com/content/2/1/402 -1 -1 0.3 mL/min. The eluent was monitored with a refractive protein∙L or in mmol protein∙L with the reaction index detector. rate constants k , k , k , k , k , k , k ,and k in 1 a1 a2 3 a3 a4 a5 6 -1 The standards that were used for calibration of the mmol oNP∙L∙(mmol X∙gprotein∙s) or in mmol -1 column were lactose, glucose, galactose, maltotriose, oNP∙L∙(mmol X∙mmol protein∙s) , respectively, with X maltotetraose, maltopentaose, maltohexaose, and being the corresponding reactant. The inhibition constant maltoheptaose. Galacto-oligosaccharides up to a degree of K is in mM. polymerization of 7 were assumed to have the same The respective parameters for Biolacta N5 were deter- response as the glucose-oligomers with an equal mined in previous work (Warmerdam A, Zisopoulos degree of polymerization. This was confirmed with FK, Boom RM, Janssen AEM: Kinetic characterization mass balances. of β-galactosidases, submitted) and are shown in Table 1. Enzyme activity measurements To investigate the effect of the present reactants com- The oNPG activity measurements, adapted from pared to when no reactants are added in the activity Nakanishi et al. (Nakanishi et al. 1983), were performed assay, we want to normalize this initial rate with the ini- immediately after the sample was taken from the reac- tial rate that would have been obtained without addition tion mixture. An Eppendorf tube with 790 μL of 0.25% of carbohydrates, which is given by equation 2: (w/w) oNPG-in-buffer was preheated in an Eppendorf Thermomixer at 40°C and 600 rpm for 10 minutes. k½ oNPG 1 þ k ½ oNPGþk ½ H20 0;oNP a1 a2 Subsequently, 210 μL of sample was added and these ½ c ¼0 k½ oNPGþk ½ lacþk½ oligo ½ gal 1 3 6 v 1 þ þ mixtures were incubated for another 10 minutes at 40°C 0;oNP k ½ oNPGþK ½ H20þk ½ lacþk ½ gluþk ½ gal K a1 a2 a3 a4 a5 i and 600 rpm. A volume of 1.0 mL of 10% (w/w) Na CO 2 3 ð2Þ solution was added to stop the reaction and, afterwards, the absorbance of oNP was measured at 420 nm. The ½ c ¼0 where v is the initial rate of oNP formation without 0;oNP oNP concentration was determined using the law of addition of carbohydrates C. At each time point, the Lambert-Beer of which the extinction coefficient was -1 -1 concentrations of reactants used in this equation is the determined to be 4576 M ∙cm .The oNP formation concentration that has been measured with HPLC. was found to be linear during the first 10 minutes of the reaction. This initial rate of oNP formation was -1 -1 Activity measurements corrected for the presence of expressed in mmol∙min ∙gprotein .Measurements carbohydrates were performed in duplicate and the average enzyme The activity measurements were corrected for the effect activity was used. of lactose, glucose, galactose, and oligosaccharides on the activity assay with equation 3: Modeling the effect of carbohydrates on the activity assay The effect of carbohydrates on the oNPG activity assay A measured A ¼ ð3Þ corrected v 0;oNP can be described with a mechanistic model; we refer ½ c ¼0 0;oNP to previous work for the mechanistic description of the model (Warmerdam A, Zisopoulos FK, Boom RM, where A and A are the enzyme activity measured corrected Janssen AEM: Kinetic characterization of β-galactosidases, calculated directly from the absorbance measurements submitted) (equation 1). This model accounts for the (see “Enzyme activity measurements”), and the enzyme use of oNPG as substrate (k ) as well as acceptor (k ), the use of water as acceptor (k ), the use of a1 a2 lactose (lac) as substrate (k ) as well as acceptor (k ), 3 a3 Table 1 Parameters for Biolacta N5 in the conversion of the use of glucose (glu) as acceptor (k ), the use of a4 oNPG, lactose, glucose, galactose, and oligosaccharides galactose (gal) as acceptor (k ) aswell asinhibitor a5 -1 k (mmol oNP∙L∙(mmol oNPG∙g protein∙s) ) 0.10 (K ), and the use of oligosaccharides (oligo) as substrate -1 k (mmol oNP∙L∙(mmol lactose∙g protein∙s) ) 0.012 (k ) as follows: -1 k (mmol oNP∙L∙(mmol oligos∙g protein∙s) ) 0.077 v k½ oNPG -1 0;oNP 1 k (mmol oNP∙L∙(mmol oNPG∙g protein∙s) ) 0.0063 a1 k½ oNPGþk ½ lacþk½ oligo ½ gal 1 3 6 -1 1 þ þ k (mmol oNP∙L∙(mmol H O∙g protein∙s) ) 0.0042 k ½ oNPGþk ½ H20þk ½ lacþk ½ gluþk ½ gal K a2 2 a1 a2 a3 a4 a5 i -1 k (mmol oNP∙L∙(mmol glucose∙g protein∙s) ) 0.00092 ð1Þ a4 -1 k (mmol oNP∙L∙(mmol galactose∙g protein∙s) ) 0.023 a5 where v is the initial rate of oNP formation in mM 0,oNP K (mM) 255 -1 oNP∙s , E is the initial enzyme concentration in g 0 Warmerdam et al. SpringerPlus 2013, 2:402 Page 4 of 8 http://www.springerplus.com/content/2/1/402 Figure 1 Stability of Biolacta N5 at various substrate concentrations. Residual enzyme activityof Biolacta N5 in (A) buffer, (B) 5.0% (w/w) -1 lactose, (C) 30% (w/w) lactose at ■ 25, 40, xand ▲ 60°C and pH 6.0 with an enzyme concentration of 16 mg∙L . The enzyme activity is measured in the oNPG activity assay. (Lines for guidance). activity corrected for the presence of lactose, glucose, Determination of enzyme stability galactose, and oligosaccharides, respectively. Enzyme inactivation during the running time of the For each sample made with Vivinal-GOS, the concentra- experiment was modelled with a first order inactivation tion of lactose, glucose, galactose, and total oligosaccharide model with: was calculated. The concentrations of lactose, galactose, −k ⋅t glucose and total oligosaccharides are 19 dm%, 1 dm%, k ¼ k ⋅ e ð4Þ t 0 21 dm%, and 59 dm% in Vivinal-GOS. Oligosaccharides were assumed to be mainly trisaccharides with a molecular where k and k are the reaction rates at time zero and 0 t -1 weight of 504 g/mol. time t in h, k is the enzyme inactivation constant in h , Figure 2 Carbohydrate profiles at an initial lactose concentration of 5.0% (w/w) at ■ 25, 40, and ▲ 60°C and pH 6.0 with an enzyme -1 concentration of 16 mg∙L .A. Disaccharide conversion; B. GOS (all oligosaccharides larger than DP2) production; C. Glucose production; D. Galactose production. Figure corresponds with Figure 1B. Warmerdam et al. SpringerPlus 2013, 2:402 Page 5 of 8 http://www.springerplus.com/content/2/1/402 Figure 3 Carbohydrate profiles at an initial lactose concentration of 30% (w/w) at ■ 25, 40, and ▲ 60°C and pH 6.0 with an enzyme -1 concentration of 16 mg∙L .A. Disaccharide conversion; B. GOS (all oligosaccharides larger than DP2) production; C. Glucose production; D. Galactose production. Figure corresponds with Figure 1C. and t is the running time at which the sample was taken The half-life time of the enzyme t can be determined in hours. The enzyme inactivation constant k and with equation 6: the reaction rate at time zero k were determined by 1nðÞ 2 linearization of equation 4. t ¼ ð6Þ The inactivation energy E can be determined with the Arrhenius relation, equation 5: Results and discussion Ea − Effect of temperature and initial lactose concentration on R⋅T k ¼ k ⋅ e ð5Þ d ∞ enzyme stability -1 where k and k (the Arrhenius constant) are in s , Figure 1 shows the specific oNPG converting activity of d ∞ -1 -1 R is the gas constant in J∙mol ∙K ,and T is the Biolacta N5 after incubation in buffer (A), in 5.0% (w/w) temperature in K. lactose (B), and in 30% (w/w) lactose (C) at 25, 40, and Figure 4 Corrected stability of Biolacta N5 at various substrate concentrations. Residual enzyme activity of Biolacta N5 in (A) buffer, (B) 5.0% ______ -1 (w/w), and (C) 30% (w/w) lactose at ■, 25; ◇,---40; and ▲, - - 60°C and pH 6.0 with an enzyme concentration of 16 mg∙L ,corrected forthe influence of lactose, galactose, glucose, and oligosaccharides. Symbols represent measured data, (dashed) lines represent modeled data. Warmerdam et al. SpringerPlus 2013, 2:402 Page 6 of 8 http://www.springerplus.com/content/2/1/402 Table 2 The initial oNP formation rate k of Biolacta N5 Table 4 The half-life time t in h of Biolacta N5 in hours 0 ½ -1 -1 in mmol∙min ∙g protein at various initial lactose at various initial lactose concentrations and temperatures concentrations and temperatures, together with its 95% [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) confidence interval 25°C 220 16 29 [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) 40°C 13 17 29 25°C 9.1 ± 1.0 11 ± 1 12 ± 1 60°C 0.048 0.82 16 40°C 9.0 ± 1.4 9.9 ± 1.2 13 ± 1 60°C 12 ± 1 8.9 ± 0.6 12 ± 0 composition in the samples was determined and the effect of these reactants on the activity assay was determined with equation 2. 60°C. The observed data are not corrected yet for the presence of lactose, glucose, galactose, and Vivinal-GOS. Carbohydrate profiles The initial activity in buffer was approximately -1 -1 13 mmol∙min ∙g enzyme , while the initial activities During the stability experiments described in Figure 1B -1 -1 were approximately 10 and 4 mmol∙min ∙g enzyme in and C, also samples were taken to determine the sugar composition. The results are presented in 5.0 and 30% (w/w) lactose, respectively. The reduction in the initial activity with an increasing lactose con- Figures 2 and 3. The concentrations of disaccharide centration is caused by the competition of lactose (A), GOS (B), glucose (C) and galactose (D) are shown. (that is present in the samples) with oNPG in the activity assay, as will be discussed later. The carbohydrate content changed in time, and varied In buffer, the enzyme was stable at 25°C, but lost 84% considerably between the initially different lactose con- centrations. At an initial lactose concentration of 5.0% of its activity after 24 hours of incubation at 40°C, and was completely inactivated after two hours at 60°C. This (w/w), the carbohydrate concentrations hardly changed complete inactivation in buffer at 60°C was expected: anymore after 6 hours of reaction at 60°C. At 25 and 40°C the GOS content showed an optimum around Mozaffar et al. (1984), Vetere and Paoletti (1998), and Song et al. (2011b) described that its isoforms are stable 6 hours of incubation, indicating hydrolysis of the up to at most 50°C for one hour. The stability at elevated desired product at longer incubation times. Also a considerable amount of galactose was present after temperatures improved considerably in the presence of lactose. In a 5.0% (w/w) lactose solution, it took 24 hours of reaction. At an initial lactose concentra- six hours of incubation at 60°C before most of the tion of 30% (w/w), GOS synthesis continued at all temperatures, including 60°C, until at least 22 hours activity was lost, while in a 30% (w/w) lactose solu- tion, 27% of the enzyme activity was left after of reaction (Figure 3B). Only small amounts of galact- 24 hours at 60°C. ose were formed at all temperatures. The galactose production (indicating hydrolysis) was substantial at The measured activity in Figure 1B and C after 24 hours of reaction at 25°C was lower than at 40°C. an initial lactose concentration of 5.0% (w/w) because One would expect a better stability at a lower of a high availability of water molecules, whereas no significant amounts of galactose were observed at an temperature. These unexpected stability values are the result of the presence of reactants during the ac- initial lactose concentration of 30% (w/w). It is clear tivity assay. These reactants interfere with the activ- that both the initial lactose concentration as well as the reaction temperature had a strong effect on the ity measurements similarly as was described by (Baks et al. 2006) (Warmerdam A, Zisopoulos FK, carbohydrate composition. Boom RM, Janssen AEM: Kinetic characterization of β-galactosidases, submitted). Therefore, the carbohydrate Correction for the presence of carbohydrates in stability experiments The specific enzyme activity that was shown in Figure 1B -1 and C was evaluated once more. The influence of Table 3 The inactivation constant k of Biolacta N5 in h lactose, galactose, glucose, and oligosaccharides on at various initial lactose concentrations and temperatures, together with its 95% confidence interval the oNPG activity assay was taken into account using equations 2 and 3. The carbohydrate content [lactose]▶ ▼T 0% (w/w) 5.0% (w/w) 30% (w/w) differed considerably during lactose conversion at various 25°C 0.0032 ± 0.0112 0.043 ± 0.023 0.024 ± 0.011 conditions (Figures 2 and 3) and the carbohydrates 40°C 0.054 ± 0.040 0.041 ± 0.025 0.024 ± 0.015 have a strong effect on the oNPG activity assay 60°C 15 ± 3 0.85 ± 0.19 0.043 ± 0.006 (Warmerdam et al. 2013a) (Warmerdam A, Zisopoulos Warmerdam et al. SpringerPlus 2013, 2:402 Page 7 of 8 http://www.springerplus.com/content/2/1/402 FK, Boom RM, Janssen AEM: Kinetic characterization enzyme in buffer is strongly dependent on the of β-galactosidases, submitted). The corrected enzyme temperature, whereas the inactivation in a system activities are shown in Figure 4. with 30% (w/w) lactose initially is hardly dependent After correction for the presence of carbohydrates on the temperature. The inactivation energy E , in the oNPG activity assay, the values of the specific shown in Table 5, decreased with increasing substrate enzyme activity on the Y-axis of Figure 4 are differ- concentration. This is similar to what was found by ent. The enzyme activities at time zero are more or De Cordt et al. (1994). The higher stability of the less similar (Table 2). The amount of added enzyme enzyme is might be caused by molecular crowding or by was the same in all experiments, thus a similar complexation with the substrate or with a remaining enzyme activity was indeed expected. Another aspect galactose moiety. is theincreasein the activityat40°Cina5.0% (w/w) A higher thermostability at high substrate concen- lactose solution at longer incubation times. After trations is very favourable in the production of GOS correction this increase in activity is not present any- by β-galactosidases from B.circulans.Athighsub- more and the activity decreased in time. After correc- strate concentrations, the reaction temperature can tion theactivitydecrease versustimeat25and 40°C be higher than the enzyme’s stable ranges that were is more or less similar. reported before in aqueous solutions, and it can be equal/ The corrected data were used to fit the first order closer to their optimal temperatures (Mozaffar et al. 1984; inactivation model (equation 4). The best fit is shown Vetere and Paoletti 1998; Song et al. 2011a), which in Figure 4. The inactivation constant and the half- will result in a higher enzyme stability. life time are shown in Tables 3 and 4. In buffer the half-life time at 25°C is about 4600 times higher as Conclusions compared to the half-life time at 60°C. In 5.0% (w/w) β-Galactosidase from Bacillus circulans was found to be lactose this value is only twenty times higher, while at quite stable against temperature at high substrate con- 30% (w/w) lactose there is only a factor of two differ- centrations. For a proper conclusion on the remaining ence in the half-life time. At 25°C the enzyme enzyme activity versus time it was important to correct appeared to be most stable in buffer, however, at 40 the enzyme activity measurements for the presence of and 60°C the enzyme is most stable at elevated various reactants. lactose concentrations. The half-life time of the Without correcting the enzyme activity at 5.0% (w/w) enzyme (Table 4) (strongly) increased with increasing lactose, the actual stability was overestimated, whereas substrate concentration at 40 and 60°C. not correcting the enzyme activity at 30% (w/w) lactose Figure 5 shows the linearized Arrhenius plot of ln resulted in an underestimation of the actual stability of (k ) as a function of 1/T. The inactivation of the β-galactosidase from Bacillus circulans. A high initial lactose concentration had a large positive effect on the enzyme stability. The improved stability in more concentrated sys- tems is very interesting for production conditions. The utilization of more concentrated systems for en- zymatic conversions is economically more interesting in order to avoid the unnecessary use of water, to save energy as a smaller volume needs to be heated, and to save on capital expenditures as less equipment is necessary. Table 5 Inactivation energy E for various lactose concentrations [lactose] E -1 [% (w/w)] [kJ∙mol ] 0 200 Figure 5 Linearized Arrhenius plot of ln(k ) as a function of 1/T. ______ 5.0 72 Symbols: ◇, 0% (w/w) lactose; ■, - - - 5.0% (w/w) lactose, and ▲, - - 30% (w/w) lactose. 30 14 Warmerdam et al. SpringerPlus 2013, 2:402 Page 8 of 8 http://www.springerplus.com/content/2/1/402 Competing interests Warmerdam A, Wang J, Boom RM, Janssen AEM (2013a) Effects of carbohydrates The authors declare that they have no competing interests. on the oNPG converting activity of β-galactosidases. J Agric Food Chem 61:6458–6464 Warmerdam A, Paudel E, Jia W, Boom RM, Janssen AEM (2013b) Characterization Authors’ contributions of β-galactosidase isoforms from Bacillus circulans and their contribution to AW: designed the experiments, performed the experiments, analyzed the GOS production. Appl Biochem Biotechnol 170:340–358 data, and wrote the paper. RMB: designed the experiments, and wrote the Yadav JK (2013) Macromolecular crowding enhances catalytic efficiency and paper. AEMJ: designed the experiments, analyzed the data, and wrote the stability of α-amylase. ISRN Biotechnology 2013:Article ID 737805 paper. All authors read and approved the final manuscript. Zhou HX, Rivas GN, Minton AP (2008) Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological Acknowledgements consequenses. Annu Rev Biophys 37:375–397 The authors would like to thank Eric Benjamins, Linqiu Cao, Ellen van Leusen, Albert van der Padt, and Jan Swarts of FrieslandCampina for the valuable doi:10.1186/2193-1801-2-402 scientific discussions. Cite this article as: Warmerdam et al.: β-galactosidase stability at high This project is jointly financed by the European Union, European Regional substrate concentrations. SpringerPlus 2013 2:402. Development Fund and The Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the Delta, the Municipality of Groningen, the Provinces of Groningen, Fryslân and Drenthe as well as the Dutch Carbohydrate Competence Center (CCC WP9). Received: 3 April 2013 Accepted: 20 August 2013 Published: 27 August 2013 References Arakawa T, Timasheff SN (1985) The stabilization of proteins by osmolytes. Biophys J 47:411–414 Baks T, Janssen AEM, Boom RM (2006) The effect of carbohydrates on α-amylase activity measurements. 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