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A new heterofunctional amino-vinyl sulfone support to immobilize enzymes: Application to the stabilization of β-galactosidase from Aspergillus oryzae
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
A new heterofunctional amino-vinyl sulfone support to immobilize enzymes: Application to the stabilization of β-galactosidase from Aspergillus oryzae Hadjer Zaaka,b,c, Mohamed Sassic, Roberto Fernandez-Lafuentea, a b c
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Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain Food Biotechnology Division, Biotechnology Research Center (CRBt), Algeria Agrobiotechnology and Nutrition in Semi-Arid Zones Laboratory, Ibn Khalboun University, Algeria
A R T I C L E I N F O
A B S T R A C T
Keywords: Divinylsulfone Heterofunctional supports Enzyme immobilization Enzyme stabilization Ion exchange Enzyme orientation
The paper shows the preparation of a new heterofunctional agarose support: amino-vinylsulfone. This has been employed to immobilize the interesting enzyme β-galactosidase from Aspergillus oryzae. The enzyme cannot be immobilized on just vinylsulfone activated support a pH values ranging from 5.0 to 9.0. Neither the enzyme was immobilized using 200 mM of NaCl on amino-vinylsulfone support. However, the enzyme was readily immobilized at moderate ion strength at pH values from 5.0 to 9.0 via ion exchange on amino-vinylsulfone support, and later some covalent enzyme-support bonds could be formed, more rapidly at alkaline pH value. After optimization of immobilization pH, incubation pH and time, and blocking reagent, several immobilized biocatalysts on amino-vinylsulfone support having 50–80% of the initial activity and a stabilization factor of around 8–15 were prepared, depending on the exact immobilization conditions.
1. Introduction Immobilization of enzymes is a powerful tool to improve enzyme properties [1–4]; a properly designed enzyme immobilization may greatly improve enzyme stability [1–5], but also activity, selectivity [6,7], and resistance to inhibitors or even protein purity [8]. To optimize the stabilization results, a maximum number of bonds between the enzyme and the support must be established [9,10]. This way, all enzyme groups linked to the support reduce their mobility (to the length of the support spacer arm), maintaining their relative positions under any distorting condition. However, to achieve a stabilizing multipoint covalent attachment is precise to select a good support (having large surfaces and a high superficial density of active groups), a good protocol that maximizes the prospects of enzyme-support reaction (that is, favoring enzyme conformation mobility and support/enzyme reactivity) and a proper group in the support [9,10]. An ideal group to get an intense multipoint covalent attachment should be stable under conditions where the enzyme/support reaction is favored (usually alkaline pH value), and also should present low steric hindrances for the reaction with the enzyme groups and react with the maximum variety of nucleophiles of the enzyme surface [11]. The ε-amino of Lys uses to be one of the target groups to this goal, because it is a cationic group frequently placed in the enzyme surface, and it is reactive with many other groups when it is
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in not ionized form [12]. Agarose is an adequate support to evaluate any immobilization protocol, as the support is fully inert if it is not modified, and that is an important feature of a support to immobilize proteins [12]. Glutaraldehyde [13], epoxy [14–17] and glyoxyl [18] supports have been utilized to immobilize-stabilize enzymes via multipoint covalent attachment. The activation of supports with divinylsulfone (DVS) to immobilize proteins and enzymes have been described for a long time [19–27]. However, it has been only recently when DVS activated supports have been described as very suitable supports to produce an extremely intense multipoint covalent attachment using proper immobilization protocols (incubation at alkaline pH value, blocking with suitable nucleophiles), improving the results achieved using glyoxyl supports because vinylsulfone can react with primary amino groups and also with imidazole, thiol or phenol groups. Vinylsulfone groups are also very stable under a wide range of pH values, have not steric problems for reaction with the protein groups, etc. [28]. The only problem is that the spacer arm is longer than that of the glyoxyl group, and the rigidification effect of each additional bond is therefore lower, and in some instances even having more enzyme-support bonds, the stabilization obtained is lower than using glyoxyl supports [29,30]. Moreover, in some instances some proteins that become immobilized on glyoxyl supports are not immobilized on supports activated with DVS, or they are rapidly inactivated, without any clear reason [31].
Corresponding author at: ICP CSIC, C/Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. E-mail address: rfl@icp.csic.es (R. Fernandez-Lafuente).
http://dx.doi.org/10.1016/j.procbio.2017.09.020 Received 1 August 2017; Received in revised form 23 August 2017; Accepted 20 September 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Zaak, H., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.09.020
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suspension were added to 2.5 mL of substrate solution. One unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 μmoL of oNPG per minute under the conditions described previously.
The concept of heterofunctional supports has opened new possibilities to the enzyme immobilization [11]. The idea is to develop supports having two different kinds of groups, one whose function is to produce the first enzyme immobilization (usually a physical adsorption), and other groups that are the responsible ones for an intense enzyme-support multipoint covalent attachment. In some cases, this is inherent to the support activation protocol (e.g., glutaraldehyde that is prepared using an anion exchanger support) [32]. In other cases, they are prepared with this objective, like epoxy-amino [33], glyoxyl-amino supports [34], or in the case of lipases, octyl-glyoxyl, octyl-epoxide or octyl-glutaraldehyde supports [35–42]; even a mix of octyl moieties with cationic or anionic groups has been proposed to get mixed lipases adsorptions [43,44]. Octyl-DVS has been also presented, with very good results, enhancing the stabilization results obtained using octyl-glyoxyl [45]. Interestingly, some lipases that cannot be immobilized on supports activated with DVS were successfully covalently immobilized on these heterofunctional DVS activated supports, after the lipase adsorption via interfacial activation [45]. In this new paper, the preparation of amino-DVS activated agarose beads and its use in the immobilization of the enzyme β-galactosidase from Aspergillus oryzae will be studied. This glycosylated enzyme [46] is difficult to stabilize via multipoint immobilization; the best results have been reported using amino-epoxy supports and the stabilization was a moderate factor of 12 after optimization [47]. The enzyme has interest in hydrolysis of acid milk whey, and also in the production of galactooligosaccharides or modification of galactose with other alcohols [48–53]. It has been shown that this enzyme may be immobilized via ion exchange at different pH values. This alters the enzyme orientation on the support and therefore their final features, e.g. stability [54]. This immobilization pH has been shown to have a great impact in the enzyme stability when immobilized using just aminated supports via ion exchange, now that covalent immobilization is intended, this immobilization pH affect may be quite different as the number of enzymesupport covalent linkages or the enzyme area involved in the covalent immobilization may play a significant role on the final enzyme stability. Thus, the protocol of covalent immobilization of the enzyme will be optimized on supports activated with divinylsulfone (monofunctional and heterofunctional amino-DVS): immobilization pH value, incubation pH value and time and, finally, blocking agent nature will be the studied variables. The results obtained using agarose activated with DVS and agarose-amino activated with DVS will be compared, using the enzyme immobilized on aminated supports as a reference.
2.3. Preparation of divinylsulfone activated agarose beads A volume of 7.5 mL of divinylsulfone was added to 200 mL of 333 mM sodium carbonate buffer at pH 11.0 and vigorously stirred until the solution became homogeneous, then 10 g of agarose or MANAE-agarose beads was added and let under gentle stirring for 60 min. Then, the support was washed with an excess of distilled water and stored at 4 °C. 2.4. Immobilization of the enzyme The immobilization was carried out employing 20 oNPG units of free beta-galactosidase activity per g of wet support (1 mg of enzyme per gram of support). This low loading was used to prevent diffusion limitations that could distort the results. The commercial samples of the enzyme were dissolved in the corresponding volume of 50 mM sodium acetate at pH 5.0, sodium phosphate at pH 7.0 or sodium carbonate at pH 9.0. Using DVS activated supports, after the immobilization, the insolubilized enzymes were incubated at different pH values and finally the remaining vinyl sulfone groups were blocked by incubation for 24 h at room temperature in 2 M of different nucleophiles (ethylenediamine, glycine, aspartic acid, histidine, cysteine or mercaptoethanol) at pH 8.0. Finally, the immobilized enzyme preparations were washed with an excess of distilled water and stored at 4 °C. 2.5. Thermal inactivations of the different enzyme preparations To check the stability of the different enzyme immobilized derivatives, 1 g of each immobilized enzyme preparation was suspended in 10 mL of 25 mM sodium acetate at pH 5.0, sodium phosphate at pH 7.0 or sodium carbonate at pH 9.0 at different temperatures. Periodically, samples were withdrawn and the activity was measured using oNPG. Half-lives were calculated from the observed inactivation courses. 3. Results 3.1. Immobilization of the β-galactosidase from Aspergillus oryzae on DVS activated supports Fig. S1 shows that the β-galactosidase from Aspergillus oryzae was not immobilized on agarose activated with DVS at all the studied pH values (5.0, 7.0 and 9.0). Considering the high reactivity of the support with different lateral chains of aminoacids [28] this was an unexpected result, but the failure of immobilization on this support has been previously reported with other enzymes [31]. However, the enzyme was readily immobilized on MANAE-agarose at the 3 studied pH values (Fig. S2) [54]. The enzyme may be released from MANAE-agarose using 250 mM NaCl, and the enzyme was not immobilized on the support under these ion strength. Therefore, we decided to try the heterofunctional DVS-MANAEagarose to immobilize the β-galactosidase from Aspergillus oryzae. This support efficiently immobilizes the β-galactosidase (Fig. 1) at the 3 pH values. If the immobilized enzyme was incubated in 250 mM NaCl immediately after immobilization, more than 90% of the enzyme was desorbed at pH 5.0, 30–40% of the enzyme was desorbed if the immobilization was performed at pH 7.0, while less than 5% of the enzyme was released from the support if the immobilization was performed at pH 9.0. These results suggested that there were a percentage of just ionically adsorbed enzyme molecules, and that after the ionic adsorption some enzyme molecules established a (or several) covalent bond(s), more efficiently at alkaline pH value, less efficiently at acid pH value, in accordance with the reactivity of vinylsulfone groups with the
2. Materials and methods 2.1. Materials β-Galactosidase from A. oryzae (20 Units oNPG/mg of protein), onitrophenyl-β-galactopyranoside (oNPG) and o-nitrophenol (oNP) were purchased from Sigma–Aldrich (St. Louis, USA). Divinylsulfone, ethylenediamine, glycine, aspartic acid, histidine, L-cysteine and 2-mercaptoethanol were purchased from Sigma Chemical Co. (St. Louis, MO). Agarose beads 4% (w/v) matrix was from Agarose Bead Technologies (ABT, Spain) and MANAE-agarose [55] support was prepared as previously described with some modifications (reaction time was prolonged to 24 h), as this permitted increasing the amount of amino groups by a 30%. All other reagents were of analytical grade. 2.2. Standard determination of enzyme activity This assay was performed by measuring the increase in absorbance at 380 nm produced by the release of oNP in the hydrolysis of 10 mM oNPG in 100 mM sodium acetate at pH 4.5 and 25 °C (the calculated extinction coefficient was 10,493 M−1cm−1 under these conditions [54]). To start the reaction, 50–100 μL of the enzyme solution or 2
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Fig. 1. Effect of the immobilization pH on the immobilization courses of β-galactosidase on DVS-MANAE agarose. Experiments have been performed at 25 °C, using 5 mM of the different buffers. Other specifications are described in Methods section. Panel A: (pH 5.0), Panel B: (pH 7.0), Panel C: (pH 9.0). Squares (reference free enzyme solution), Triangles (suspension), Circles (supernatant).
activity, to a value of 45% of the initial activity. This increase on enzyme activity of enzymes during incubation on DVS activated supports only when immobilized in one specific condition has been previously reported. This may be related to the different orientations achieved in immobilizations of the enzyme via ion exchange at different pH values [54], and that the conformational changes induced by the covalent attachment drive to a more active enzyme conformation when involve that specific enzyme area. During the blocking with Gly (Fig. 2b), the activity of the enzyme immobilized at pH 9.0 suffers first a certain decrease in enzyme activity, perhaps because the no fully blocked support may still covalently react with the enzyme, and later, when the support is mostly blocked the enzyme activity increased, leaving a final activity of 85% of the initial one while the enzyme immobilized at pH 5.0 and 7.0 suffered decrease in its activity to 30% and 45 respectively. Regarding stabilities, the enzyme immobilized at pH 9.0 was the most stable when inactivated at pH 5.0 (where the enzyme have more activity), while the enzyme immobilized at pH 5.0 was the most stable at pH 7.0 (Fig. 3). Considering both, activity and stability, we decided that the immobilization at pH 9.0 was the preferred one.
moieties of the protein [28]. The use of the immobilized enzyme after incubation at pH 9 and blocking with Gly (see below) to perform a SDSPAGE experiment did not show any protein band (not shown results), confirming that all enzyme molecules were covalently immobilized (non-shown results). Moreover, β-galactosidase was no immobilized on any of the DVSMANAE-agarose beads at any pH value using 250 mM NaCl in the immobilization media (Fig. S3). This confirmed that the first step of the immobilization was the ion exchange of the enzyme on the support, and later some covalent bonds could be formed. These results together suggest that the enzyme was unable to become immobilized directly on DVS supports (both, amino-DVS or just DVS), and that the previous ionic exchange of the enzyme on the support was required in this specific case. However, after the ion exchange, the nucleophilic groups in the enzyme surface reacted with the vinylsulfone groups in the support, establishing covalent bonds. 3.2. Optimization of immobilized enzyme activity/stability The previous results showed that the enzyme was rapidly immobilized via ion exchange. Moreover, it has been shown that the ion exchange of this enzyme on MANAE agarose beads gives enzyme preparations with different stabilities. In this case, the activity was almost fully preserved at pH 5.0 and 7.0, while at pH 9.0 the activity decreased to 52% (Fig. 1). The optimization of the properties of the immobilized preparations (activity/stability) on supports activated with divinylsulfone involves at least 3 different steps where the pH value plays a very relevant role [28]: the immobilization pH (that may alter the area of the protein involved in the ion exchange), the incubation pH and time (that will determine the intensity of the multipoint covalent attachment) and the blocking agent (that will determine the enzymesupport interactions during operation).
3.2.2. Effect of incubation pH and time on immobilized enzyme performance After immobilization at pH 9.0, the immobilized enzymes were incubated at pH 8.0, 9.0 and 10.0. Fig. S4 shows that the expressed activity was more or less the same at all the studied pH values (around 60%). The stability of the biocatalyst was higher when the incubations were performed at pH 10.0 (Fig. S4). Considering the activity and stability, we decided that pH 10.0 gave a reasonable activity and an optimal stabilization. Next, the effect of the incubation time before the blocking step was analyzed at pH 10.0. Fig. S5 shows how the activity and stability of the immobilized enzyme increased until 3 h of incubation, and then both decreased. That way, an incubation time of 3 h seemed to be the optimal considering both activity and stability.
3.2.1. Effect of immobilization pH during enzyme incubation at pH 9.0 Initially, the enzymes immobilized at the different pH values were incubated at pH 9.0 for 3 h, and later blocked with Gly. Fig. 2a shows that the enzymes suffer a rapid initial decrement on its activity when incubated under these conditions (around 30% of the activity is maintained), the enzyme immobilized at pH 9.0 increased later its
3.2.3. Effect of the blocking reagent As an end point to the reaction between the enzyme and the support, we assayed different nucleophiles that will alter the final properties of the support. Gly was the blocking reagent used as standard 3
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Fig. 2. Panel A. Effect of the immobilization pH on the activity of the immobilized β-galactosidase on DVS-MANAE agarose. Experiments have been carried out at 25 °C and, after enzyme immobilization, the immobilized enzymes were incubated at pH 9.0, using 50 mM sodium carbonate. Panel B. Effect of the blocking with 2 M Gly at pH 8.0 on the activity of β-galactosidase immobilized at different pH values and incubated for 3 h at pH 9.0. Experiments have been carried out at 25 °C as described in Methods section. Circles: pH 5.0; Triangles: pH 7.0; Squares: pH 9.0.
gave the best values when the enzyme was immobilized at pH 5.0 or 9.0, while aspartic acid was the nucleophile that permitted a higher stability of the enzyme when immobilized at pH 7.0. However, differences were not very significant, except when the blocking was with mercaptoethanol, where the enzyme stability also dropped. The optimal preparation could be different depending on the weight that activity or stability may have for a specific process. After immobilization at pH 9.0, it is clear that the blocking with Gly is the one that permit simultaneously a higher activity and stability; in fact it is the most stable preparation. If the immobilization is performed at pH 7.0, the blocking with Cys permitted the highest activity (around 80%), but the half-live is 2/3 of that of the biocatalysts blocked with Asp. The blocking of the enzyme immobilized at pH 5.0 with Cys and Asp gave the best activity recovery (around 65%), while the blocking with Gly have a 22%. However, in stabilization the blocking with Gly gave the best values, almost doubling to that of the blocking with Cys and a 20% higher than when using Asp.
Fig. 3. Effect of the immobilization pH on the inactivation courses at pH 5.0 and 56 °C of different β-galactosidase preparations. The immobilized enzymes were incubated at pH 9.0 for 3 h and blocked with 2 M Gly at pH 8.0. Other details are in Methods section. Circles: pH 5.0; Triangles: pH 7.0; Squares: pH 9.0.
3.3. Stability of the optimal preparations until this point and it introduced a cationic and an anionic group on the support. We have also assayed ethylenediamine that will introduce two cationic groups, His that will has two cationic (one weak) and one anionic group and aspartic acid that will introduce two anionic and one cationic moieties. Also, we used two thiolated compounds, mercaptoethanol that will not introduce additional ionic groups, and Cys, that will introduced one cationic and one anionic group. Both thiolated compounds may have some reduction power, which may affect the disulfide bridges of the enzyme. The blocking reagent will affect the final interactions between enzyme and support, that way the effect of the blocking agent may be different depending on the immobilization pH and the inactivation conditions. Thus, the enzyme was immobilized at pH 5.0, 7.0 and 9.0, incubated 3 h at pH 10.0 and blocked with this collection of nucleophiles and inactivated at pH 5.0 and 7.0. Regarding the activity (Table 1 S), the most active immobilized preparation among the enzyme immobilized at pH 9.0 was that blocked with Gly. The enzyme immobilized at pH 7.0 was more active when blocked with Cys, in fact a 50% more active than the most active enzyme immobilized at pH 9.0. The most active biocatalyst was the enzyme immobilized at pH 5.0 and blocked with Asp shortly followed by the blocking with Cys. On the other hand, the blocking with mercaptoethanol gave the lowest values of activity at all the studied pH values, reaching a minimum value if the enzyme was immobilized at pH 7.0. The incubation of previously Gly blocked enzyme preparations with mercaptoethanol gave significant enzyme inactivations, suggesting that the main cause of the decrease in enzyme activity is the reduction of some disulfide bonds caused by this reagent. These results clearly show that the area of the enzyme involved in the immobilization is different depending on the immobilization pH value, and that the blocking agent may alter the enzyme-support interactions greatly affecting the final enzyme activity in a different way depending on the involved area. Regarding the enzyme stabilities, Gly
Fig. 4 shows the inactivation courses of the optimal preparation, considering just stability (using Gly when the enzyme has been immobilized at pH 5.0 or 9.0, and Asp when the enzyme was immobilized at pH 7.0), for each different immobilization pH compared to the free enzyme and the enzyme just adsorbed on MANAE support at pH 5.0. Stabilization factors ranged 12–15 depending on the immobilization pH. It is remarkable that the large differences on stabilities observed with the just ionically exchange enzymes are now almost annulled, and even inverted. Now, a slightly higher stability may be observed after immobilization at pH 9 and a lower one when the enzyme was
Fig. 4. Inactivation courses at pH 5.0 and 56 °C of optimized β-galactosidase preparations. Other specifications are described in Methods section. Empty Squares: Free enzyme, Asterisk: Enzyme immobilized on MANAE at pH 5.0; Solid Circles: enzyme was immobilized on DVS-MANAE at pH 5.0, incubated 3 h at pH 10.0 and blocked with 2 M Gly; Solid Triangles: enzyme was immobilized on DVS-MANAE at pH 7.0, incubated 3 h at pH 10.0 and blocked with 2 M Asp; Solid Squares: enzyme was immobilized on DVS-MANAE at pH 9.0, incubated 3 h at pH 10.0 and blocked with 2 M Gly.
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immobilized at pH 5. Stabilization is not impressive, but improves the results using other strategies like glutaraldehyde [56] or amino-epoxydes [47]. The large size of the protein, the glycosylation that difficulty the enzyme/support interaction and the large spacer arm of the MANAE-DVS group may explain this relatively short stabilization. 4. Conclusions This is the first example of the use of an amino support activated with DVS to prepare a heterofunctional support useful to immobilize an enzyme that failed to become immobilized in standard supports activated with DVS. β-galactosidase from Aspergillus oryzae cannot be immobilized on DVS activated agarose, neither can be immobilized on MANAE-DVS at high ionic strength, but it is readily immobilized on MANAE-DVS if a previous enzyme immobilization via ion exchange is permitted. The incubation at alkaline pH value permitted a rapid formation of covalent bonds between the enzyme and the support. Even with the high reactivity of vinylsulfone group, the first step of the immobilization of the enzyme is clearly the ion exchange. Only after the enzyme first immobilization, the enzyme is able to covalently react with the support. And them the reaction is quite rapid. After optimization, a stabilization factor of 15 has been achieved, compared to the enzyme ionically adsorbed at pH 5.0 (optimal value for enzyme immobilization [54]). Acknowledgements We gratefully recognize the support from the MINECO from Spanish Government, (projects numbers CTQ2013-41507-R and CTQ201786170-R). Hadjer Zaak thanks the Algerian Ministry of higher education and scientific research for her fellowship. The suggestions from Drs Sanchez and Villalonga (Department of Analytical Chemistry, Faculty of Chemistry, UCM) are gratefully recognized. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2017.09.020. References [1] R.A. Sheldon, S. Van, Pelt, enzyme immobilisation in biocatalysis: why, what and how, Chem. Soc. Rev. 42 (2013) 6223–6235. [2] R.K. Singh, M.K. Tiwari, R. Singh, J.-K. Lee, From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes, Int. J. Mol. Sci. 14 (2013) 1232–1277. [3] A.A. Homaei, R. Sariri, F. Vianello, R. Stevanato, Enzyme immobilization: an update, J. Chem. Biol. 6 (2013) 185–205. [4] U. Guzik, K. Hupert-Kocurek, D. Wojcieszynska, Immobilization as a strategy for improving enzyme properties- application to oxidoreductases, Molecules 19 (2014) 8995–9018. [5] R. Fernandez-Lafuente, Stabilization of multimeric enzymes: strategies to prevent subunit dissociation, Enzyme Microb. Technol. 45 (2009) 405–418. [6] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev. 42 (2013) 6290–6307. [7] F. Secundo, Conformational changes of enzymes upon immobilization, Chem. Soc. Rev. 42 (2013) 6250–6261. [8] O. Barbosa, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R.C. Rodrigues, R. FernandezLafuente, Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts, Biotechnol. Adv. 33 (2015) 435–456. [9] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007) 1451–1463. [10] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Potential of different enzyme immobilization strategies to improve enzyme performance, Adv. Synth. Catal. 353 (2011) 2885–2904. [11] O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R.C. Rodrigues, R. FernandezLafuente, Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties, Biomacromolecules 14 (2013) 2433–2462. [12] P. Zucca, R. Fernandez-Lafuente, E. Sanjust, Agarose and its derivatives as supports for enzyme immobilization, Molecules 21 (2016) 1577.
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galactosidase from Aspergillus oryzae, Biotechnol. Prog. 19 (2003) 1056–1060. [48] D.F.M. Neri, V.M. Balcão, R.S. Costa, I.C.A.P. Rocha, E.M.F.C. Ferreira, D.P.M. Torres, L.R.M. Rodrigues, L.B. Carvalho Jr, J.A. Teixeira, Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol, Food Chem. 115 (2009) 92–99. [49] N. Albayrak, S.-T. Yang, Immobilization of β-galactosidase on fibrous matrix by polyethyleneimine for production of galacto-oligosaccharides from lactose, Biotechnol. Prog. 18 (2002) 240–251. [50] B. Rodriguez-Colinas, L. Fernandez-Arrojo, A.O. Ballesteros, F.J. Plou, Galactooligosaccharides formation during enzymatic hydrolysis of lactose: towards a prebiotic-enriched milk, Food Chem. 145 (2014) 388–394. [51] C. Vera, C. Guerrero, A. Illanes, R. Conejeros, Fed-batch synthesis of galacto-oligosaccharides with Aspergillus oryzae β-galactosidase using optimal control strategy, Biotechnol. Prog. 30 (2014) 59–67. [52] C. Guerrero, C. Vera, F. Acevedo, A. Illanes, Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation, J. Biotechnol. 209 (2015) 31–40. [53] S.A. Ansari, R. Satar, F. Alam, M.H. Alqahtani, A.G. Chaudhary, M.I. Naseer, S. Karim, I.A. Sheikh, Cost effective surface functionalization of silver nanoparticles for high yield immobilization of Aspergillus oryzae β-galactosidase and its application in lactose hydrolysis, Process Biochem. 47 (2012) 2427–2433. [54] T.L. De Albuquerque, S. Peirce, N. Rueda, A. Marzocchella, L.R.B. Gonçalves, M.V.P. Rocha, R. Fernandez-Lafuente, Ion exchange of β-galactosidase: the effect of the immobilization pH on enzyme stability, Process Biochem. 51 (2016) 875–880. [55] R. Fernandez-Lafuente, C.M. Rosell, V. Rodriguez, C. Santana, G. Soler, A. Bastida, J.M. Guisan, Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method, Enzyme Microb. Technol. 15 (1993) 546–550. [56] S. Zaak, M. de-Albuquerque, R. Fernandez-Lafuente, Exploiting the versatility of aminated supports activated with glutaraldehyde to immobilize β-galactosidase from Aspergillus oryzae, Catalysts 7 (2017) 250.
Geobacillus thermocatenulatus lipase, Catal. Today 255 (2015) 21–26. [39] N. Guajardo, C. Bernal, L. Wilson, Z. Cabrera, Selectivity of R-α-monobenzoate glycerol synthesis catalyzed by Candida antarctica lipase B immobilized on heterofunctional supports, Process Biochem. 50 (2015) 1870–1877. [40] V. Vescovi, R.L.C. Giordano, A.A. Mendes, P.W. Tardioli, Immobilized lipases on functionalized silica particles as potential biocatalysts for the synthesis of fructose oleate in an organic solvent/water system, Molecules 22 (2017) 212. [41] Z. Boros, D. Weiser, M. Márkus, E. Abaháziová, Á. Magyar, A. Tomin, B. Koczka, P. Kovács, L. Poppe, Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations, Process Biochem. 48 (2013) 1039–1047. [42] A. Suescun, N. Rueda, J.C.S. Dos Santos, J.J. Castillo, C. Ortiz, R. Torres, O. Barbosa, R. Fernandez-Lafuente, Immobilization of lipases on glyoxyl-octyl supports: improved stability and reactivation strategies, Process Biochem. 50 (2015) 1211–1217. [43] N. Rueda, C.S. Dos Santos, M.D. Rodriguez, T.L. Albuquerque, O. Barbosa, R. Torres, C. Ortiz, R. Fernandez-Lafuente, Reversible immobilization of lipases on octylglutamic agarose beads: a mixed adsorption that reinforces enzyme immobilization, J. Mol. Catal. B: Enzym. 128 (2016) 10–18. [44] N. Rueda, T.L. Albuquerque, R. Bartolome-Cabrero, L. Fernandez-Lopez, R. Torres, C. Ortiz, J.C.S. Dos Santos, O. Barbosa, R. Fernandez-Lafuente, Reversible immobilization of lipases on heterofunctional octyl-Amino agarose beads prevents enzyme desorption, Molecules 21 (2016) 646. [45] T.L.D. Albuquerque, N. Rueda, J.C.S. Dos Santos, O. Barbosa, C. Ortiz, B. Binay, E. Özdemir, L.R.B. Gonçalves, R. Fernandez-Lafuente, Easy stabilization of interfacially activated lipases using heterofunctional divinyl sulfone activated-octyl agarose beabeads. Modulation of the immobilized enzymes by altering their nanoenvironment, Process Biochem. 51 (2016) 865–874. [46] Y. Tanaka, A. Kagamiishi, A. Kiuchi, T. Horiuchi, Purification and properties of βgalactosidase from Aspergillus oryzae, J. Biochem. 77 (1975) 241–247. [47] R. Torres, C. Mateo, G. Fernández-Lorente, C. Ortiz, M. Fuentes, J.M. Palomo, J.M. Guisan, R. Fernández-Lafuente, A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of β-
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
A new heterofunctional amino-vinyl sulfone support to immobilize enzymes: Application to the stabilization of β-galactosidase from Aspergillus oryzae Hadjer Zaaka,b,c, Mohamed Sassic, Roberto Fernandez-Lafuentea, a b c
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Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain Food Biotechnology Division, Biotechnology Research Center (CRBt), Algeria Agrobiotechnology and Nutrition in Semi-Arid Zones Laboratory, Ibn Khalboun University, Algeria
A R T I C L E I N F O
A B S T R A C T
Keywords: Divinylsulfone Heterofunctional supports Enzyme immobilization Enzyme stabilization Ion exchange Enzyme orientation
The paper shows the preparation of a new heterofunctional agarose support: amino-vinylsulfone. This has been employed to immobilize the interesting enzyme β-galactosidase from Aspergillus oryzae. The enzyme cannot be immobilized on just vinylsulfone activated support a pH values ranging from 5.0 to 9.0. Neither the enzyme was immobilized using 200 mM of NaCl on amino-vinylsulfone support. However, the enzyme was readily immobilized at moderate ion strength at pH values from 5.0 to 9.0 via ion exchange on amino-vinylsulfone support, and later some covalent enzyme-support bonds could be formed, more rapidly at alkaline pH value. After optimization of immobilization pH, incubation pH and time, and blocking reagent, several immobilized biocatalysts on amino-vinylsulfone support having 50–80% of the initial activity and a stabilization factor of around 8–15 were prepared, depending on the exact immobilization conditions.
1. Introduction Immobilization of enzymes is a powerful tool to improve enzyme properties [1–4]; a properly designed enzyme immobilization may greatly improve enzyme stability [1–5], but also activity, selectivity [6,7], and resistance to inhibitors or even protein purity [8]. To optimize the stabilization results, a maximum number of bonds between the enzyme and the support must be established [9,10]. This way, all enzyme groups linked to the support reduce their mobility (to the length of the support spacer arm), maintaining their relative positions under any distorting condition. However, to achieve a stabilizing multipoint covalent attachment is precise to select a good support (having large surfaces and a high superficial density of active groups), a good protocol that maximizes the prospects of enzyme-support reaction (that is, favoring enzyme conformation mobility and support/enzyme reactivity) and a proper group in the support [9,10]. An ideal group to get an intense multipoint covalent attachment should be stable under conditions where the enzyme/support reaction is favored (usually alkaline pH value), and also should present low steric hindrances for the reaction with the enzyme groups and react with the maximum variety of nucleophiles of the enzyme surface [11]. The ε-amino of Lys uses to be one of the target groups to this goal, because it is a cationic group frequently placed in the enzyme surface, and it is reactive with many other groups when it is
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in not ionized form [12]. Agarose is an adequate support to evaluate any immobilization protocol, as the support is fully inert if it is not modified, and that is an important feature of a support to immobilize proteins [12]. Glutaraldehyde [13], epoxy [14–17] and glyoxyl [18] supports have been utilized to immobilize-stabilize enzymes via multipoint covalent attachment. The activation of supports with divinylsulfone (DVS) to immobilize proteins and enzymes have been described for a long time [19–27]. However, it has been only recently when DVS activated supports have been described as very suitable supports to produce an extremely intense multipoint covalent attachment using proper immobilization protocols (incubation at alkaline pH value, blocking with suitable nucleophiles), improving the results achieved using glyoxyl supports because vinylsulfone can react with primary amino groups and also with imidazole, thiol or phenol groups. Vinylsulfone groups are also very stable under a wide range of pH values, have not steric problems for reaction with the protein groups, etc. [28]. The only problem is that the spacer arm is longer than that of the glyoxyl group, and the rigidification effect of each additional bond is therefore lower, and in some instances even having more enzyme-support bonds, the stabilization obtained is lower than using glyoxyl supports [29,30]. Moreover, in some instances some proteins that become immobilized on glyoxyl supports are not immobilized on supports activated with DVS, or they are rapidly inactivated, without any clear reason [31].
Corresponding author at: ICP CSIC, C/Marie Curie 2, Campus UAM-CSIC, Cantoblanco, 28049 Madrid, Spain. E-mail address: rfl@icp.csic.es (R. Fernandez-Lafuente).
http://dx.doi.org/10.1016/j.procbio.2017.09.020 Received 1 August 2017; Received in revised form 23 August 2017; Accepted 20 September 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Zaak, H., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.09.020
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suspension were added to 2.5 mL of substrate solution. One unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 μmoL of oNPG per minute under the conditions described previously.
The concept of heterofunctional supports has opened new possibilities to the enzyme immobilization [11]. The idea is to develop supports having two different kinds of groups, one whose function is to produce the first enzyme immobilization (usually a physical adsorption), and other groups that are the responsible ones for an intense enzyme-support multipoint covalent attachment. In some cases, this is inherent to the support activation protocol (e.g., glutaraldehyde that is prepared using an anion exchanger support) [32]. In other cases, they are prepared with this objective, like epoxy-amino [33], glyoxyl-amino supports [34], or in the case of lipases, octyl-glyoxyl, octyl-epoxide or octyl-glutaraldehyde supports [35–42]; even a mix of octyl moieties with cationic or anionic groups has been proposed to get mixed lipases adsorptions [43,44]. Octyl-DVS has been also presented, with very good results, enhancing the stabilization results obtained using octyl-glyoxyl [45]. Interestingly, some lipases that cannot be immobilized on supports activated with DVS were successfully covalently immobilized on these heterofunctional DVS activated supports, after the lipase adsorption via interfacial activation [45]. In this new paper, the preparation of amino-DVS activated agarose beads and its use in the immobilization of the enzyme β-galactosidase from Aspergillus oryzae will be studied. This glycosylated enzyme [46] is difficult to stabilize via multipoint immobilization; the best results have been reported using amino-epoxy supports and the stabilization was a moderate factor of 12 after optimization [47]. The enzyme has interest in hydrolysis of acid milk whey, and also in the production of galactooligosaccharides or modification of galactose with other alcohols [48–53]. It has been shown that this enzyme may be immobilized via ion exchange at different pH values. This alters the enzyme orientation on the support and therefore their final features, e.g. stability [54]. This immobilization pH has been shown to have a great impact in the enzyme stability when immobilized using just aminated supports via ion exchange, now that covalent immobilization is intended, this immobilization pH affect may be quite different as the number of enzymesupport covalent linkages or the enzyme area involved in the covalent immobilization may play a significant role on the final enzyme stability. Thus, the protocol of covalent immobilization of the enzyme will be optimized on supports activated with divinylsulfone (monofunctional and heterofunctional amino-DVS): immobilization pH value, incubation pH value and time and, finally, blocking agent nature will be the studied variables. The results obtained using agarose activated with DVS and agarose-amino activated with DVS will be compared, using the enzyme immobilized on aminated supports as a reference.
2.3. Preparation of divinylsulfone activated agarose beads A volume of 7.5 mL of divinylsulfone was added to 200 mL of 333 mM sodium carbonate buffer at pH 11.0 and vigorously stirred until the solution became homogeneous, then 10 g of agarose or MANAE-agarose beads was added and let under gentle stirring for 60 min. Then, the support was washed with an excess of distilled water and stored at 4 °C. 2.4. Immobilization of the enzyme The immobilization was carried out employing 20 oNPG units of free beta-galactosidase activity per g of wet support (1 mg of enzyme per gram of support). This low loading was used to prevent diffusion limitations that could distort the results. The commercial samples of the enzyme were dissolved in the corresponding volume of 50 mM sodium acetate at pH 5.0, sodium phosphate at pH 7.0 or sodium carbonate at pH 9.0. Using DVS activated supports, after the immobilization, the insolubilized enzymes were incubated at different pH values and finally the remaining vinyl sulfone groups were blocked by incubation for 24 h at room temperature in 2 M of different nucleophiles (ethylenediamine, glycine, aspartic acid, histidine, cysteine or mercaptoethanol) at pH 8.0. Finally, the immobilized enzyme preparations were washed with an excess of distilled water and stored at 4 °C. 2.5. Thermal inactivations of the different enzyme preparations To check the stability of the different enzyme immobilized derivatives, 1 g of each immobilized enzyme preparation was suspended in 10 mL of 25 mM sodium acetate at pH 5.0, sodium phosphate at pH 7.0 or sodium carbonate at pH 9.0 at different temperatures. Periodically, samples were withdrawn and the activity was measured using oNPG. Half-lives were calculated from the observed inactivation courses. 3. Results 3.1. Immobilization of the β-galactosidase from Aspergillus oryzae on DVS activated supports Fig. S1 shows that the β-galactosidase from Aspergillus oryzae was not immobilized on agarose activated with DVS at all the studied pH values (5.0, 7.0 and 9.0). Considering the high reactivity of the support with different lateral chains of aminoacids [28] this was an unexpected result, but the failure of immobilization on this support has been previously reported with other enzymes [31]. However, the enzyme was readily immobilized on MANAE-agarose at the 3 studied pH values (Fig. S2) [54]. The enzyme may be released from MANAE-agarose using 250 mM NaCl, and the enzyme was not immobilized on the support under these ion strength. Therefore, we decided to try the heterofunctional DVS-MANAEagarose to immobilize the β-galactosidase from Aspergillus oryzae. This support efficiently immobilizes the β-galactosidase (Fig. 1) at the 3 pH values. If the immobilized enzyme was incubated in 250 mM NaCl immediately after immobilization, more than 90% of the enzyme was desorbed at pH 5.0, 30–40% of the enzyme was desorbed if the immobilization was performed at pH 7.0, while less than 5% of the enzyme was released from the support if the immobilization was performed at pH 9.0. These results suggested that there were a percentage of just ionically adsorbed enzyme molecules, and that after the ionic adsorption some enzyme molecules established a (or several) covalent bond(s), more efficiently at alkaline pH value, less efficiently at acid pH value, in accordance with the reactivity of vinylsulfone groups with the
2. Materials and methods 2.1. Materials β-Galactosidase from A. oryzae (20 Units oNPG/mg of protein), onitrophenyl-β-galactopyranoside (oNPG) and o-nitrophenol (oNP) were purchased from Sigma–Aldrich (St. Louis, USA). Divinylsulfone, ethylenediamine, glycine, aspartic acid, histidine, L-cysteine and 2-mercaptoethanol were purchased from Sigma Chemical Co. (St. Louis, MO). Agarose beads 4% (w/v) matrix was from Agarose Bead Technologies (ABT, Spain) and MANAE-agarose [55] support was prepared as previously described with some modifications (reaction time was prolonged to 24 h), as this permitted increasing the amount of amino groups by a 30%. All other reagents were of analytical grade. 2.2. Standard determination of enzyme activity This assay was performed by measuring the increase in absorbance at 380 nm produced by the release of oNP in the hydrolysis of 10 mM oNPG in 100 mM sodium acetate at pH 4.5 and 25 °C (the calculated extinction coefficient was 10,493 M−1cm−1 under these conditions [54]). To start the reaction, 50–100 μL of the enzyme solution or 2
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Fig. 1. Effect of the immobilization pH on the immobilization courses of β-galactosidase on DVS-MANAE agarose. Experiments have been performed at 25 °C, using 5 mM of the different buffers. Other specifications are described in Methods section. Panel A: (pH 5.0), Panel B: (pH 7.0), Panel C: (pH 9.0). Squares (reference free enzyme solution), Triangles (suspension), Circles (supernatant).
activity, to a value of 45% of the initial activity. This increase on enzyme activity of enzymes during incubation on DVS activated supports only when immobilized in one specific condition has been previously reported. This may be related to the different orientations achieved in immobilizations of the enzyme via ion exchange at different pH values [54], and that the conformational changes induced by the covalent attachment drive to a more active enzyme conformation when involve that specific enzyme area. During the blocking with Gly (Fig. 2b), the activity of the enzyme immobilized at pH 9.0 suffers first a certain decrease in enzyme activity, perhaps because the no fully blocked support may still covalently react with the enzyme, and later, when the support is mostly blocked the enzyme activity increased, leaving a final activity of 85% of the initial one while the enzyme immobilized at pH 5.0 and 7.0 suffered decrease in its activity to 30% and 45 respectively. Regarding stabilities, the enzyme immobilized at pH 9.0 was the most stable when inactivated at pH 5.0 (where the enzyme have more activity), while the enzyme immobilized at pH 5.0 was the most stable at pH 7.0 (Fig. 3). Considering both, activity and stability, we decided that the immobilization at pH 9.0 was the preferred one.
moieties of the protein [28]. The use of the immobilized enzyme after incubation at pH 9 and blocking with Gly (see below) to perform a SDSPAGE experiment did not show any protein band (not shown results), confirming that all enzyme molecules were covalently immobilized (non-shown results). Moreover, β-galactosidase was no immobilized on any of the DVSMANAE-agarose beads at any pH value using 250 mM NaCl in the immobilization media (Fig. S3). This confirmed that the first step of the immobilization was the ion exchange of the enzyme on the support, and later some covalent bonds could be formed. These results together suggest that the enzyme was unable to become immobilized directly on DVS supports (both, amino-DVS or just DVS), and that the previous ionic exchange of the enzyme on the support was required in this specific case. However, after the ion exchange, the nucleophilic groups in the enzyme surface reacted with the vinylsulfone groups in the support, establishing covalent bonds. 3.2. Optimization of immobilized enzyme activity/stability The previous results showed that the enzyme was rapidly immobilized via ion exchange. Moreover, it has been shown that the ion exchange of this enzyme on MANAE agarose beads gives enzyme preparations with different stabilities. In this case, the activity was almost fully preserved at pH 5.0 and 7.0, while at pH 9.0 the activity decreased to 52% (Fig. 1). The optimization of the properties of the immobilized preparations (activity/stability) on supports activated with divinylsulfone involves at least 3 different steps where the pH value plays a very relevant role [28]: the immobilization pH (that may alter the area of the protein involved in the ion exchange), the incubation pH and time (that will determine the intensity of the multipoint covalent attachment) and the blocking agent (that will determine the enzymesupport interactions during operation).
3.2.2. Effect of incubation pH and time on immobilized enzyme performance After immobilization at pH 9.0, the immobilized enzymes were incubated at pH 8.0, 9.0 and 10.0. Fig. S4 shows that the expressed activity was more or less the same at all the studied pH values (around 60%). The stability of the biocatalyst was higher when the incubations were performed at pH 10.0 (Fig. S4). Considering the activity and stability, we decided that pH 10.0 gave a reasonable activity and an optimal stabilization. Next, the effect of the incubation time before the blocking step was analyzed at pH 10.0. Fig. S5 shows how the activity and stability of the immobilized enzyme increased until 3 h of incubation, and then both decreased. That way, an incubation time of 3 h seemed to be the optimal considering both activity and stability.
3.2.1. Effect of immobilization pH during enzyme incubation at pH 9.0 Initially, the enzymes immobilized at the different pH values were incubated at pH 9.0 for 3 h, and later blocked with Gly. Fig. 2a shows that the enzymes suffer a rapid initial decrement on its activity when incubated under these conditions (around 30% of the activity is maintained), the enzyme immobilized at pH 9.0 increased later its
3.2.3. Effect of the blocking reagent As an end point to the reaction between the enzyme and the support, we assayed different nucleophiles that will alter the final properties of the support. Gly was the blocking reagent used as standard 3
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Fig. 2. Panel A. Effect of the immobilization pH on the activity of the immobilized β-galactosidase on DVS-MANAE agarose. Experiments have been carried out at 25 °C and, after enzyme immobilization, the immobilized enzymes were incubated at pH 9.0, using 50 mM sodium carbonate. Panel B. Effect of the blocking with 2 M Gly at pH 8.0 on the activity of β-galactosidase immobilized at different pH values and incubated for 3 h at pH 9.0. Experiments have been carried out at 25 °C as described in Methods section. Circles: pH 5.0; Triangles: pH 7.0; Squares: pH 9.0.
gave the best values when the enzyme was immobilized at pH 5.0 or 9.0, while aspartic acid was the nucleophile that permitted a higher stability of the enzyme when immobilized at pH 7.0. However, differences were not very significant, except when the blocking was with mercaptoethanol, where the enzyme stability also dropped. The optimal preparation could be different depending on the weight that activity or stability may have for a specific process. After immobilization at pH 9.0, it is clear that the blocking with Gly is the one that permit simultaneously a higher activity and stability; in fact it is the most stable preparation. If the immobilization is performed at pH 7.0, the blocking with Cys permitted the highest activity (around 80%), but the half-live is 2/3 of that of the biocatalysts blocked with Asp. The blocking of the enzyme immobilized at pH 5.0 with Cys and Asp gave the best activity recovery (around 65%), while the blocking with Gly have a 22%. However, in stabilization the blocking with Gly gave the best values, almost doubling to that of the blocking with Cys and a 20% higher than when using Asp.
Fig. 3. Effect of the immobilization pH on the inactivation courses at pH 5.0 and 56 °C of different β-galactosidase preparations. The immobilized enzymes were incubated at pH 9.0 for 3 h and blocked with 2 M Gly at pH 8.0. Other details are in Methods section. Circles: pH 5.0; Triangles: pH 7.0; Squares: pH 9.0.
3.3. Stability of the optimal preparations until this point and it introduced a cationic and an anionic group on the support. We have also assayed ethylenediamine that will introduce two cationic groups, His that will has two cationic (one weak) and one anionic group and aspartic acid that will introduce two anionic and one cationic moieties. Also, we used two thiolated compounds, mercaptoethanol that will not introduce additional ionic groups, and Cys, that will introduced one cationic and one anionic group. Both thiolated compounds may have some reduction power, which may affect the disulfide bridges of the enzyme. The blocking reagent will affect the final interactions between enzyme and support, that way the effect of the blocking agent may be different depending on the immobilization pH and the inactivation conditions. Thus, the enzyme was immobilized at pH 5.0, 7.0 and 9.0, incubated 3 h at pH 10.0 and blocked with this collection of nucleophiles and inactivated at pH 5.0 and 7.0. Regarding the activity (Table 1 S), the most active immobilized preparation among the enzyme immobilized at pH 9.0 was that blocked with Gly. The enzyme immobilized at pH 7.0 was more active when blocked with Cys, in fact a 50% more active than the most active enzyme immobilized at pH 9.0. The most active biocatalyst was the enzyme immobilized at pH 5.0 and blocked with Asp shortly followed by the blocking with Cys. On the other hand, the blocking with mercaptoethanol gave the lowest values of activity at all the studied pH values, reaching a minimum value if the enzyme was immobilized at pH 7.0. The incubation of previously Gly blocked enzyme preparations with mercaptoethanol gave significant enzyme inactivations, suggesting that the main cause of the decrease in enzyme activity is the reduction of some disulfide bonds caused by this reagent. These results clearly show that the area of the enzyme involved in the immobilization is different depending on the immobilization pH value, and that the blocking agent may alter the enzyme-support interactions greatly affecting the final enzyme activity in a different way depending on the involved area. Regarding the enzyme stabilities, Gly
Fig. 4 shows the inactivation courses of the optimal preparation, considering just stability (using Gly when the enzyme has been immobilized at pH 5.0 or 9.0, and Asp when the enzyme was immobilized at pH 7.0), for each different immobilization pH compared to the free enzyme and the enzyme just adsorbed on MANAE support at pH 5.0. Stabilization factors ranged 12–15 depending on the immobilization pH. It is remarkable that the large differences on stabilities observed with the just ionically exchange enzymes are now almost annulled, and even inverted. Now, a slightly higher stability may be observed after immobilization at pH 9 and a lower one when the enzyme was
Fig. 4. Inactivation courses at pH 5.0 and 56 °C of optimized β-galactosidase preparations. Other specifications are described in Methods section. Empty Squares: Free enzyme, Asterisk: Enzyme immobilized on MANAE at pH 5.0; Solid Circles: enzyme was immobilized on DVS-MANAE at pH 5.0, incubated 3 h at pH 10.0 and blocked with 2 M Gly; Solid Triangles: enzyme was immobilized on DVS-MANAE at pH 7.0, incubated 3 h at pH 10.0 and blocked with 2 M Asp; Solid Squares: enzyme was immobilized on DVS-MANAE at pH 9.0, incubated 3 h at pH 10.0 and blocked with 2 M Gly.
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immobilized at pH 5. Stabilization is not impressive, but improves the results using other strategies like glutaraldehyde [56] or amino-epoxydes [47]. The large size of the protein, the glycosylation that difficulty the enzyme/support interaction and the large spacer arm of the MANAE-DVS group may explain this relatively short stabilization. 4. Conclusions This is the first example of the use of an amino support activated with DVS to prepare a heterofunctional support useful to immobilize an enzyme that failed to become immobilized in standard supports activated with DVS. β-galactosidase from Aspergillus oryzae cannot be immobilized on DVS activated agarose, neither can be immobilized on MANAE-DVS at high ionic strength, but it is readily immobilized on MANAE-DVS if a previous enzyme immobilization via ion exchange is permitted. The incubation at alkaline pH value permitted a rapid formation of covalent bonds between the enzyme and the support. Even with the high reactivity of vinylsulfone group, the first step of the immobilization of the enzyme is clearly the ion exchange. Only after the enzyme first immobilization, the enzyme is able to covalently react with the support. And them the reaction is quite rapid. After optimization, a stabilization factor of 15 has been achieved, compared to the enzyme ionically adsorbed at pH 5.0 (optimal value for enzyme immobilization [54]). Acknowledgements We gratefully recognize the support from the MINECO from Spanish Government, (projects numbers CTQ2013-41507-R and CTQ201786170-R). Hadjer Zaak thanks the Algerian Ministry of higher education and scientific research for her fellowship. The suggestions from Drs Sanchez and Villalonga (Department of Analytical Chemistry, Faculty of Chemistry, UCM) are gratefully recognized. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2017.09.020. References [1] R.A. Sheldon, S. Van, Pelt, enzyme immobilisation in biocatalysis: why, what and how, Chem. Soc. Rev. 42 (2013) 6223–6235. [2] R.K. Singh, M.K. Tiwari, R. Singh, J.-K. Lee, From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes, Int. J. Mol. Sci. 14 (2013) 1232–1277. [3] A.A. Homaei, R. Sariri, F. Vianello, R. Stevanato, Enzyme immobilization: an update, J. Chem. Biol. 6 (2013) 185–205. [4] U. Guzik, K. Hupert-Kocurek, D. Wojcieszynska, Immobilization as a strategy for improving enzyme properties- application to oxidoreductases, Molecules 19 (2014) 8995–9018. [5] R. Fernandez-Lafuente, Stabilization of multimeric enzymes: strategies to prevent subunit dissociation, Enzyme Microb. Technol. 45 (2009) 405–418. [6] R.C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev. 42 (2013) 6290–6307. [7] F. Secundo, Conformational changes of enzymes upon immobilization, Chem. Soc. Rev. 42 (2013) 6250–6261. [8] O. Barbosa, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R.C. Rodrigues, R. FernandezLafuente, Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts, Biotechnol. Adv. 33 (2015) 435–456. [9] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. Fernandez-Lafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007) 1451–1463. [10] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Potential of different enzyme immobilization strategies to improve enzyme performance, Adv. Synth. Catal. 353 (2011) 2885–2904. [11] O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R.C. Rodrigues, R. FernandezLafuente, Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties, Biomacromolecules 14 (2013) 2433–2462. [12] P. Zucca, R. Fernandez-Lafuente, E. Sanjust, Agarose and its derivatives as supports for enzyme immobilization, Molecules 21 (2016) 1577.
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