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refolding
Enzyme and Microbial Technology 49 (2011) 388–394
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Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency César A. Godoy a , Blanca de las Rivas c , Dejan Bezbradica b , Juan M. Bolivar a,b,c , Fernando López-Gallego a,b,c , Gloria Fernandez-Lorente c , Jose M. Guisan a,∗ a
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia c Departamento de Microbiologia, Instituto de Fermentaciones Industriales, CSIC, C/Juan de la Cierva 3, 2006 CSIC, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 17 June 2011 Accepted 23 June 2011 Keywords: Additives Enzyme reactivation Refolding Lipase Cysteine oxidation Immobilization
a b s t r a c t Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25–30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface’s chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Enzymes are useful catalysts for many industrially relevant processes [1–3], but their moderate stability under industrial conditions hampers their widespread implementation as cost-effective biocatalysts [4,5]. Preparation of very robust enzyme derivatives would greatly improve their industrial applicability. However, immobilized enzymes on solid carriers are used under different operational conditions (e.g., at neutral pH values and mild temperatures in the presence of inert organic solvents) that can lead to enzyme inactivation through deleterious conformational changes [6]. In this way, after full enzyme inactivation, the enzyme may be completely unfolded and further refolded into its initial conformation, thus recovering quantitatively the catalytic activity. Thereby,
Abbreviations: BTL2, Lipase 2 from Geobacillus thermocathenolatus; CNBR-BTL2, BTL2 immobilized on agarose activated with cyanogen bromide groups; Gx-BTL2, BTL2 immobilized on agarose activated with glyoxylgroups. ∗ Corresponding author. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: [email protected], [email protected] (J.M. Guisan). 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.06.018
the recovery of enzymatic activity after operational inactivation is a breakthrough for facilitating the implementation of enzymes on industrial scale [7]. In this context, enzyme unfolding/refolding has been applied to reactivate those enzymes that have been inactivated over long periods of operation [6,8]. Nevertheless, most protocols for enzyme reactivation by unfolding/refolding have been carried out with soluble enzymes; there are only a few examples of this process taking place in the solid phase. Recently, different immobilized enzymes were reactivated after their inactivation under different operational conditions (temperature, pH, organic solvents, etc.) [9–12]. The refolding was carried out in the solid phase under aqueous conditions and resulted that those enzymes immobilized by intense covalent attachment to the solid supports were refolded much better than those immobilized through a few covalent bonds. Hence, intense covalent attachments may prevent complete unfolding and preserve “residual” 3D conformations that may act as starting points for guiding the protein toward successful refolding as judged by the recovered activity. Since the lipases have broad substrate specificity accompanied in many instances by a high regio- and/or enantioselectivity [13–15], they have been broadly used in biocatalysis. This family
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of enzymes undergoes interfacial activation mechanisms, and they are active in the presence of hydrophobic surfaces such as drops of fat. When lipases are in aqueous media their active sites are completely shielded by normally one ␣-helix called “lid” [12,16,17]. However, in hydrophobic environments, normally in the surroundings of hydrophobic substrates, this “lid” is opened to allow substrate molecules get into the lipase binding pocket for catalysis to take place. This interfacial activation mechanism implies thereby two lipase conformations, open and close, and the equilibrium between them is shifted to favour either the open or closed conformation depending on media conditions [16,17]. Hence, reactivation of lipases by unfolding/refolding process should restore this complex movement to recover initial activity once the enzyme is refolded. Lipase from Geobacillus thermocatenulatus [18] (BTL2) has been heterologously overexpressed in Escherichia coli [19,20]. This is a thermo-alkalophilic enzyme able to perform some interesting reactions, such as resolution of esters yielding enantiomerically pures alcohols or acids [21–23]. Open structure of BTL2 has been recently crystallized and determined [24,25]. Hence, details of transition between the closed and the open form of BTL2 are now well-known [25]. The close-open transition involves a huge conformational change and such change induced by interfacial activation is so far quite complex in terms of structural reorganization of the protein. Furthermore, in the 3D structure, two metal binding domains (one for Ca2+ and one for Zn2+ ) [25,26] have been found playing important roles for both activity and stability (Fig. 1). Therefore, this protein requires organizing many structural domains to be folded in an active form which would mean a bigger challenge in order to successfully refold its tertiary structure. In previous studies, BTL2 stability was increased by multipoint covalent immobilization on glyoxyl-agarose (Gx-BTL2), while it was not when it was immobilized on CNBr-agarose (CNBr-BTL2) [27]. So, enzyme stabilization relied on the higher 3D rigidification achieved by multipoint covalent attachment.
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In the current research, the enzyme reactivation parameters of both insoluble derivatives described above have been studied after unfolding/refolding. Here we present a solid-phase unfolding/refolding protocol to reactivate BTL2 under reducing conditions. We have shed light on inactivation mechanism during unfolding that relies mainly on oxidation state of the protein. The refolding/unfolding process recovered both initial specific activity and enantioselectivity for both achiral and chiral substrates. Finally, we studied different variables that affected to refolding efficiencies, such as additives during the refolding, nature of support surface of insoluble derivatives, and refolding pH. 2. Materials and methods 2.1. Materials and conventional protocols All the materials were of analytical grade. Lipase expression, purification and lipase colorimetric assay proceeded as described [27]. Sited directed mutagenesis was performed according to conventional proceedings. For more details see supporting information. 2.2. Incubation of BTL2 catalyst in 8 M guanidine Experiments were performed as previously described by Soler et al. [28]. Two wet grams of each immobilized preparation were washed 10 times with 10 volumes of 8 M guanidine at 25 ◦ C and the desired pH value (0.1 M sodium acetate was used at pH 5, 0.1 M sodium phosphate at pH 7, or 0.1 sodium bicarbonate at pH 9). In some instances, 25 mM DTT was added to the guanidine solution. Then, the immobilized enzyme was suspended in 20 mL of the same solution. Periodically, samples of 50 L were withdrawn and the activity was determined as stated above. We also checked that the presence of diluted guanidine did not affect the enzyme activity. 2.3. Reincubation of the unfolded BTL2 catalyst in aqueous medium Insoluble enzyme preparations previously unfolded by incubation in guanidine solutions were washed 10 times with 10 volumes of aqueous buffer (with or without DTT) to eliminate guanidine and were then suspended in the same buffer at the desired temperature. Periodically, samples were withdrawn and the activity was determined as stated above. The buffer in some cases contained different additives such as DTT and trehalose. 2.4. Determination of the short-term reactivation rate Derivatives were unfolded by incubation in 8 M guanidine and 25 mM DTT. Later on enzyme inactivation, unfolding solution was removed by filtration without washing. Those derivatives were resuspended on the media where the activity assay was carried out. In situ activities of the derivatives were determined at different time intervals (0–25 s, 25–50 s, 50–75 s, 75–100 s, 100–150 s, 150–200 s, 200–300 s and 300–400 s) in the presence or absence of different additives. The maximum of activity to be recovered in the presence of one additive was that observed with CNBr-BTL2 derivative without being unfolded in the presence of the corresponding additive. Activity values at different times are referred to that maximum activity defined above. The activity grew in asymptote way up to a maximum, thereby activities were empirically adjusted to a second order equation to calculate the maximum recovered activity and the refolding rate (time to reach 50% of the maximum recovered activity).
3. Results 3.1. Unfolding of immobilized BTL2 derivatives in guanidine and further refolding in aqueous media
Fig. 1. Structure of the open form of BTL2 (see Ref. [25]). BTL2 is shown in cartoon representation where lid, C94, C294 and cations (Zn2+ and Ca2+ ), are depicted accordingly labelled. Both side chain from C64 and C294 are shown in sticks. Both metals are represented as spheres.
Chaotropic agents such as concentrated guanidine solutions have been extensively used to promote unfolding of aggregated or misfolded free enzymes [29–31]. In the context of operationally inactivated enzymes, their unfolding by such reagents would bring them to a suitable starting point for further enzyme reactivation proceedings. Equivalent amounts of BTL2 were immobilized per unit weight of both supports. As it had been previously described [27], immobilization on both Gx-agarose and CNBr-agarose matrixes resulted in approximately 60–65% of BTL2 expressed activity, meaning that both derivatives presented equivalent activity and protein load.
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3.2. Effect of DTT on reactivation efficiency
Fig. 2. Reactivation courses of different immobilized BTL2 derivatives by incubation in aqueous solution. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, afterwards the derivatives were incubated with 0.1 M sodium phosphate at pH 7 under the same conditions. The activity was monitored in both inactivation and reactivation stage. Black circles: Gx-BTL2; white rhombus: CNBrBTL2.
Gx-BTL2 and CNBr-BTL2 derivatives were inactivated by incubation in saturated guanidine solutions. When enzymes are incubated with chaotropic reagents as guanidine, they are unfolded because water is sequestered by such reagent [30]. Thus, enzyme inactivation is due to protein unfolding in the presence of these molecules. In this way, both immobilized derivatives lost rapidly their activities, expressing less than 15% of activity after 1 h. After unfolding, insoluble derivatives were extensively washed in order to completely eliminate guanidine and then incubated in aqueous buffer at pH 7 and 25 ◦ C (Fig. 2). Both lipase derivatives slowly recovered about 30% of their initial activity in 2.5 h and longer incubation times did not promote higher reactivation percentages. These values are much lower than those observed in unfolding/refolding of others enzymes [9,12]. Moreover, different loads of derivatives had negligible effect on reactivation efficiencies; thereby unfolding/refolding showed no dependence on protein load for both Gx-agarose and CNBr-agarose matrixes. Because of the cation dependence of this lipase, EDTA or TPEN were added to remove the structural cations during the unfolding process. Afterwards, the refolding process was carried out either in the presence or absence of both cations (Zn2+ and Ca2+ ) to study their effects on the enzyme folding. None of these two metals had measurable effect on the final refolding efficiency because improvement of neither recovered activity nor the refolding rate was detected (data not shown).
In addition to conformational changes, chemical modifications may also inactivate enzymes during their operational performance. Furthermore, those modifications can directly affect protein folding avoiding optimal refolding and thereby resulting in low reactivation efficiencies. In order to determine the cysteines’ role in enzyme refolding, we carried out the complete unfolding/refolding process in the presence of mild reducing agents such as dithiothreitol (DTT). This reducing compound was chosen to address this issue because it has been reported as very good reducing agent to prevent cysteine oxidation or disulfide bond formation [32]. When both unfolding and refolding steps were carried out at neutral pH under reducing conditions (DTT added to the media), Gx-BTL2 recovered over 70% of its initial catalytic activity whereas CNBr-BTL2 recovered around 60% (Fig. 3A). The effect of DTT also preserved enzyme activity because when BTL2 was unfolded by guanidine, the catalyst inactivation proceeded more slowly. Moreover, the addition of DTT seemed to be important during the unfolding step to prevent cysteine oxidation because when unfolding was carried out with DTT, this reducing agent was not needed during refolding to achieve higher reactivation efficiencies (Fig. 3B). On the contrary, the addition of DTT to the refolding step also led to high reactivation efficiencies albeit unfolding was carried in the absence of DTT (Fig. 3B). Oxidation events involving Cys residues were thus demonstrated to play a crucial role in enzyme reactivation. They are prevented to be oxidized in reducing environments during the unfolding step or enzyme oxidation state is reverted by reducing agent (DTT) addition to the refolding step. Expectedly, the addition of metals to the unfolding/refolding process in reducing conditions did have no effect on refolding efficiency, just as it was observed in non reducing conditions. In this way, the enzyme, after unfolding/refolding process in reducing conditions, kept unaffected in terms of specific activity (26.6 U/mg vs 27.8 U/mg from the wild type), stability against organic solvents and enantioselectivity (E > 100 toward S isomer as well as wild type) for the hydrolysis of rac-␣-methylbenzylacetate [27]. The optimal reactivation was kept after at least five unfolding/refolding cycles. This fact further supports the use of reactivation as another alternative to make longer the biocatalysts life-time. 3.3. The role of Cys during unfolding/refolding In the light of the observed insights, we studied which role was played by each cysteine in the deleterious oxidation process during
Fig. 3. Effect of the presence of 25 mM DTT on the reactivation of different immobilized BTL2 derivatives. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium phosphate at pH 7 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stage. (A) Reactivation course of insoluble BTL2 preparation in the presence of DTT after unfolding with guanidine. Refolding was performed in 25 mM sodium phosphate at pH 7 and 25 ◦ C as described in Section 2. Black circles: Gx-BTL2 derivative; white rhombus: CNBr-BTL2 derivative. Solid lines: the absence of DTT. Dashed lines: the presence of DTT. (B) Dynamic unfolding/refolding of Gx-BTL2 derivative in the presence and absence of DTT at pH 5. The Gx-BTL2 derivative was incubated with guanidine 8 M either in the presence (grey dash line) or absence (black dash line) of DTT. Afterwards, the derivatives were incubated in aqueous media with DTT (grey solid line) or without DTT (dark solid line).
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Fig. 4. Reactivation of different mutants of BTL2 immobilized on CNBr-agarose beads after inactivation by incubation in guanidine. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium phosphate at pH 7 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stages. (A) Full circles: wild type BTL2; empty rhombus: BTL2(C64S/C295S) and (B) full circles: wild type BTL2, empty triangles: BTL2(C64S) and empty squares: BTL2(C295S).
BTL2 unfolding, causing low refolding efficiencies. A double BTL2 mutant with both native cysteines residues replaced by serine was prepared. Such double mutant showed similar biochemical properties in terms of activity and stability than the wild type variant. This double mutant was immobilized on CNBr-agarose (see Section 2), and then unfolded in guanidine solution. Finally, the unfolded derivative was incubated in aqueous solution in order to allow protein refolding. At no reducing conditions (no DTT), 94% of initial activity was recovered for refolded BTL2-(C64S/C295S) unlike wild type that only recovered 30% of its activity under the same conditions (Fig. 4A). Therefore, in the cysteine free BTL2 variant, DTT hardly affected to its refolding efficiency, supporting once more the negative effect of cysteine oxidation on BTL2 reactivation during unfolding/refolding processes. The optimal reactivation of the double mutant lacking cysteines was observed under at least five cycles of unfolding/refolding. Moreover, the enzyme properties of the mutant lacking the two cysteines residues were exactly the same before and after the unfolding/refolding process (data not shown). In addition to the double mutant BTL2-(C64S/C295S), two single mutants of each cysteine were prepared. In BTL2-C295S and BTL2C64S, cysteines were again substituted by serines. As well as in the case of the double mutant the substitution of one single cysteine at position either 295 or 64 negligible affected to the biochemical behaviour of BTL2. They were finally immobilized on CNBr-agarose support, and later on the insoluble derivates of each BTL2 single mutant underwent the unfolding/refolding process. BTL2-C295S variant recovered more than 90% of its initial activity in the absence of DTT (non reducing conditions). Contrarily BTL-C64S could recovered 65% of its initial activity, but reactivation was 2-fold more efficient than the wild type variant (Fig. 4B). Nevertheless, a cysteine in that position still promoted a suboptimal BTL2 refolding. It means that C295 individually had a more negative effect on BTL2 refolding than C64 but the combination of both (wild type enzyme) led to a much worse reactivation yield (35%). In order to discover whether this effect was specific for these two cysteines, the cysteine pair was restored in the single mutant BTL2-C64S introducing one fairly exposed cysteine at position 93 (close to N-terminal), creating so far a double mutant BTL2-(C64S/T93C) (Supplementary Fig. 1). When the double mutant BTL2-(C64S/T93C) was immobilized on CNBr-agarose and undergone the unfolding/refolding process under oxidizing conditions, the derivative was poorly reactivated with values as low as the wild type (around 30% of reactivation). 3.4. Effect of pH on reactivation efficiency pH may play an important role during the refolding of enzyme tertiary structure. To examine this issue, refolding of different
immobilized BTL2 derivatives was carried out at different pH conditions (Fig. 5). When unfolding/refolding experiments were carried out at different pH conditions, different activities recoveries could be observed for each derivative. CNBr-BTL2 derivative was more efficiently reactivated by refolding in a non reducing environment at alkaline pH values unlike the Gx-BTL2 derivative that exhibited an opposite effect, being more efficiently refolded under the same conditions but at acid pH values. This pattern was even more pronounced when the unfolding/refolding process was carried out in the presence of DTT to avoid cysteine oxidations. In these conditions CNBr-BTL2 recovered full initial activity after unfolding/refolding at pH 9, whereas the equal recovery for Gx-BTL2 derivative took place at pH 5. 3.5. Effect of additives on the rate and yield of reactivation Effects of some additives on the refolding of the Gx-BTL2 derivative were studied at neutral pH regarding to both reactivation rate and yield (see Section 2). These experiments were carried out under reducing conditions to only study additive effects on refolding behaviour discarding any negative contribution by oxidation. Table 1 shows that Tris buffer is preferred to sodium phosphate. It has been described that Tris–HCl increased the intrinsic BTL2 stability regarding to phosphate buffer at neutral conditions [33]. Hence, this aminated buffer would confer a more rigid structure to the protein, which would avoid complete unfolding in the presence of guanidine, yielding higher and more rapidly reactivation efficiencies. On the other hand, Triton X-100 did not drive to better and faster refolding unlike those experimental evidences for
Fig. 5. Effect of the pH value during reactivation of inactivated BTL2 immobilized preparations. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated under different pH values at 25 ◦ C. The activity was monitored in both inactivation and reactivation stages. Black circles: Gx-BTL2; white rhombus: CNBr-BTL2. The unfolding/refolding was carried out either in the presence (dashed lines) or absence of DTT (solid lines). DTT concentration used in this experiment was 25 mM.
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Table 1 Effect of additives on the recovery rate and maximum recovered activity values (see Section 2). Additive
Extrapolated maximum recovered activity value (%)
25 mM Tris–HCl (control) 0.001% Triton X-100 0.01% Triton X-100 0.1% Triton X-100 0.5 M TMAO 1.0 M TMAO 2.0 M TMAO 10% glycerol 20% glycerol 30% glycerol 10% PEG 1500 20% PEG 1500 30% PEG 1500 10% dextran 1500 20% dextran 1500 30% dextran 1500 10% trehalose 20% trehalose 40% trehalose 25 mM NaH2 PO4 buffer
67 67 70 65 48 69 38 61 66 55 77 97 82 69 99 46 66 60 65 48
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3 5 6 3 3 3 4 5 3 3 6 4 3 4 5 2 3 4 4 3
Time to reach 50% of maximum recovery (s) 29 27 26 39 27 43 28 24 24 17 42 83 91 14 19 9 10 91 135 42
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3 3 4 3 4 5 4 2 5 3 2 6 6 3 2 2 5 3 6 2
The biocatalyst was inactivated by incubation in saturated guanidine solutions followed by the addition of 25 mM DTT. Aliquots of 50 L of the mixture were diluted in activity assay medium containing the respective additive in 25 mM Tris–HCl (pH 7). The best results are bolded.
lipase from Thermomyces lanuginose, where optimal refolding was achieved in the presence of detergents. Here, we used a battery of compounds to help protein folding, observing different effects with each one. Trehalose is stabilizing compound for enzymes [29,33,34] that slowed-down the reactivation rate while maintaining the final recovered activity. Glycerine and TMAO (a “chemical chaperone” used to reinforce hydrophobic interactions) [35,36] reduced the reactivation rate and the maximum value obtained. Finally, polymeric additives as polyethylene glycols and dextrans were also added during refolding stage. The best results were obtained for polymers such as dextran that improves both recovered activity and rate of reactivation. This positive effect observed in the presence of polymeric substances may be related to the preferential hydration during folding, stabilizing crucial folding intermediates [36]. 3.6. Effect of the support surface on reactivation efficiency Protein’s surrounding environment can also play an important role in the refolding process. The immobilization protocol using CNBr-agarose matrixes would allow us to control this environment by modifying the support surface. In this way, two different derivatives having the same number of enzyme–support bonds and the same enzyme orientation on the support were blocked with different molecules later on immobilization, obtaining different enzyme surroundings. The chosen molecules to achieve those two different derivatives were ethanolamine and ethylenediamine. When the refolding was carried out with these two differently blocked derivatives at pH 9 (optimal pH for refolding of CNBr-BTL2 derivative), refolding efficiencies were found to be quite different depending on the nature of the molecule used during the blocking. Ethanolamine blocked derivatives yielded less reactive surfaces that allowed much more efficient refolding (100% of recovered activity) than when blocking was carried out with ethylenediamine. The latter ones presented more charged surfaces that only recovered 55% of derivative’s initial activity (Fig. 6). This fact introduced a new variable on the unfolding/refolding process at solid phase.
Fig. 6. Reactivation of CNBr-BTL2 derivatives having an hydroxylated surface (blocked with ethanolamine) (circles) or an positively charged aminated surface (blocked with ethylenediamine) (rhombus). Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium carbonate at pH 9 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stages.
4. Discussion 4.1. Solid-phase BTL2 refolding depends on reducing media conditions When BTL2 insoluble derivatives were incubated with chaotropic reagent as guanidine, they were inactivated. At those conditions a minimal fraction was not apparently inactivated (15%), it may be due to a small fraction of protein that is refolded during the kinetic enzymatic assay in aqueous media. Here, we consider that guanidine may promote either partial or total unfolding of enzyme 3D structure even in the insoluble derivatives. Unexpectedly, structural cations did not help to successful BLT2 reactivations, meaning that low refolding efficiencies did not rely on cation location on enzyme tertiary structure. Surprisingly, here, unlike in other published studies [10], more intense covalent multipoint attachment between BTL2 and the support surface did not result in better refolding efficiencies regarding to those derivatives with fewer bonds enzyme–support (Fig. 2). Since covalent attachment was not a determining factor for achieving high refolding efficiencies for BTL2, different factors may be leading to the enzyme inactivation rather than unfolded conformations. BTL2 3D structure revealed the presence of two cysteines that are located on enzyme surface, both of which are susceptible to be oxidized possibly resulting in the activity loss. The implication of both cysteines in the success of unfolding/refolding processes was demonstrated because activity recovery was much higher in the presence of DTT. Therefore, a reducing agent as DTT can play two roles: (1) keeping the cysteines reduced during the unfolding process, enabling successful refolding and (2) reverting during refolding process those oxidations occurred in the protein while the unfolding process under oxidizing conditions. 4.2. BTL2’s cysteines play a key role on unfolding/refolding events The two cysteines presented on BTL2 primary sequence could be forming a disulfide bond during either unfolding or refolding process. This internal covalent bond that would not be present at wild type conformation may drive a wrong enzyme folding, resulting in inactive enzyme conformations. Moreover, in oxidizing conditions (DTT absence), the two cysteines may be oxidized to different groups such as sulfinic, sulfonic acids, and even to sulfenyl-amides formed between the oxidized thiol and the peptide bond [37]. These chemical modifications may also explain low refolding efficien-
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cies obtained with both BTL2 insoluble derivatives. On the other hand, cysteine oxidation seemed to be totally independent on covalent attachment intensity, explaining so similar behaviours for both insoluble derivatives. In order to shed light on the relationship between cysteines and reactivation efficiency during unfolding/refolding processes, a double BTL2 mutant BTL2-(C64S/C295S) with both native cysteines residues replaced by serine was prepared. In the cysteine free BTL2 variant, DTT hardly affected to its refolding efficiency, being this efficiently reactivated under both reducing and oxidizing conditions. In first term, these data may be suggesting a wrong disulfide bond formation between C295 and C64 during the unfolding, in other words an intramolecular cross-linking mediated by a disulfide bond may be taking place. Those cysteines are quite far away in the tertiary structure but likely in the presence of guanidine they may be found next to each other in the space, forming a disulfide bond, difficulting the optimal protein refolding. However, these experiments did not give us full understanding about whether two or only one cysteine is needed to lead wrong folding in oxidizing conditions. For this reason, an additional pair of cysteines in BTL2 primary sequence (BTL2-(C64S/T93C)) was made. It was also susceptible to be oxidized, possibly triggering disulfide formation during unfolding step regardless the position of each cysteine from the pair. In this variant as well as the wild type, refolding was very inefficient likely because the wrong disulfide bond would drive to a suboptimal folded BTL2, avoiding the successful enzyme reactivation after refolding. On the contrary, BTL2 variants with only one cysteine (BTL2-C64S and BTL2-C295S) were much better reactivated than the BTL2 variant with two cysteines, nevertheless the reactivation behaviour was different between the two different mono-cysteine BTL2 variants. Depending on the cysteine position, reactivation was higher or lower in the absence of DTT. Likely the variant with Cys-295 may undergo a single point oxidation of the thiol group that is negative for BTL2 folding, while Cys-64 may be less susceptible to be oxidized explaining so the lower impact of that cysteine in the reactivation efficiency. 4.3. pH and support surface nature are important parameters to successfully reactivated BTL2 through solid-phase unfolding/refolding processes The pH had different effects during the unfolding/refolding of either CNBr-BTL2 or Gx-BTL2. These differences can be mainly explained by the different immobilization protocols carried out to obtain each derivative. In the case of CNBr-BLT2, the enzyme is covalently attached to the support through few protein–support interactions, and thus this insoluble version of the enzyme would behave similarly to the soluble one during unfolding/refolding process. Contrarily, when the enzyme was immobilized onto glyoxyl-agarose supports, the immobilization took place via the richest lysine region on enzyme surface. Herein, enzyme was very intensely covalently attached to the support surface with a different orientation than the one adopted by the enzyme on CNBragarose matrixes (Fig. 7). This different orientation, along with the more intense covalent attachment, may result in different starting points for the refolding process. Those different starting points may lead refolding through different interaction networks that can be strongly affected by the pH. In the same context, the oxidation/reduction of Cys during unfolding/refolding of BTL2 might also be affected by the pH. For example, Cys reduction mediated by DTT would be favoured at basic pH since thiolate group in this reducer is predominant [38], thus explaining the higher reactivation efficiency of CNBr-BTL2 derivative under alkaline conditions. Surprisingly, for Gx-BTL2 derivative was found just the opposite effect, acid pH values promoted higher reactivation efficiencies. In this latter case, formation of thiolate ion of DTT would not be the
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Fig. 7. Surface representation of BTL2 open structure. The representation was displayed using Pymol 0.99 v. Lid is depicted in light grey and in cartoon representation. N-terminal was represented in black. (A) Region from where enzyme would be immobilized on CNBr-agarose supports at pH 7. (B) Region from where enzyme would be immobilized on glyoxyl-agarose supports at pH 10. Lysines are represented in dark grey. The two BTL2 representations show the different orientations that BTL2 would be immobilized for in the two derivatives studied in this research.
only reason that assures an optimal BTL2 refolding. A similar relation between pH and refolding efficiency has also been found for the murine prion mPrP(23-231) [39], where the authors suggested that pH-mediated structural changes may promote native-like conformations by increasing the proximity of properCys residues regardless the efficiency of Cys redox reaction mediated by agents like DTT. Another interesting parameter to take in account for solid-phase unfolding/refolding processes was the support surface chemical nature. BTL2 was much more efficient reactivated when it was immobilized on less reactive surface than when it was on more reactive ones. Less reactive surfaces would promote less undesirable interactions between protein and surface while unfolding/refolding process is taking place. On the contrary, more reactive (e.g. cationic) surfaces can ionically interact with the negative groups on the protein surfaces during either unfolding or refolding, yielding aberrant conformations with much lower activity than the optimal one. These non desirable interactions could thus lead to incorrect conformation, explaining the lower recovered activities in the ethylenediamine blocked CNBr-BTL2 derivative. 5. Conclusions The active refolding of wild type BTL2 is highly dependent on the oxidation state of the Cys residues presented in its primary sequence. In fact, refoldings in conditions where a potential disulfide bond might be formed yielded low reactivation efficiencies. Such issue was overcome using reducing environments that allowed actively refolding wild type BTL2 in insoluble preparations, CNBr-BTL2 and Gx-BTL2. Here, we proposed DTT as reducing agent for achieving 100% activity recovery after the unfolding/refolding process under optimal pH. Despite of only studying here the reactivation behaviour of BTL2 by unfolding/refolding, this methodology may be applicable to other proteins containing free and reduced cysteines in their sequences. Moreover, these techniques are the starting point for new and simpler reactivation protocols applied to enzyme derivatives that have been operationally inactivated. The use of different buffers, different pH values, and different additives strongly modifies the rate and yield of activity recovery. On the other hand, inactivation by guanidine should promote a total or partial unfolding of the enzyme and hence reactivation was strongly influenced by the enzyme surroundings on the support surface. Herein, it has been demonstrated that more reactive
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surfaces promote undesirable interactions between enzyme and support during refolding of the inactivated enzyme. Therefore, under optimal conditions, the best immobilized derivative, the best buffer and pH, the presence of additives and the use of support surfaces with low reactivity allow a very rapid and complete reactivation, achieving 100% of activity recovery in only 1 min. This result is even more relevant considering the very complex mechanism of activity of this thermostable lipase and its biotechnological interest in kinetic resolution of chiral compounds. Therefore, we have opened a new avenue to overcome bottlenecks for the reactivation in solid phase by unfolding/refolding approaches of enzymes containing cysteines in their primary sequence. Acknowledgments This work has been supported by Comunidad Autónoma de Madrid (CAM) (grant S0505/PPQ/0344) and CICYT (grants BIO2005-8576 and AGL-2009-07625). C. Godoy has a PhD fellowship from MCI. D. Bezbradica has postdoctoral fellowship of Serbian Ministry of Science and Technological Development for research on project bt20064. Fernandez-Lorente has a Ramon y Cajal contract from MCI. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.enzmictec.2011.06.018. References [1] Akoh CC, Chang SW, Lee GC, Shaw JF. Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce. J Agric Food Chem 2008;56:10445–51. [2] Brenna E, Fuganti C, Serra S. Applications of biocatalysis in fragrance chemistry: the enantiomers of alpha-, beta-, and gamma-irones. Chem Soc Rev 2008;37:2443–51. [3] Woodley JM. New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol 2008;26:321–7. [4] Iyer PV, Ananthanarayan L. Enzyme stability and stabilization—aqueous and non-aqueous environment. Process Biochem 2008;43:1019–32. [5] Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF. Stability of biocatalysts. Curr Opin Chem Biol 2007;11:220–5. [6] Mozhaev VV, Berezin IV, Martinek K. Reactivation of immobilized enzymes. Methods Enzymol 1987;135:586–96. [7] Guisan JM. Immobilization of enzymes as 21st century begins. In: Guisan JM, editor. Immobilization of enzymes and cells. Totowa, NJ: Humana Presss Inc.; 2006. p. 1–14. [8] Teshima T, Kondo A, Fukuda H. Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES. Appl Microbiol Biotechnol 1997;48:41–6. [9] Bolivar JM, Rocha-Martin J, Godoy C, Rodrigues RC, Guisan JM. Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus. Process Biochem 2010;45:107–13. [10] Rodrigues RC, Bolivar JM, Palau-Ors A, Volpato G, Ayub MAZ, FernandezLafuente R, et al. Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus. Enzyme Microb Technol 2009;44:386–93. [11] Rodrigues RC, Bolivar JM, Volpato G, Filice M, Godoy C, Fernandez-Lafuente R, et al. Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers. J Biotechnol 2009;144:113–9. [12] Rodrigues RC, Godoy CA, Filice M, Bolivar JM, Palau-Ors A, Garcia-Vargas JM, et al. Reactivation of covalently immobilized lipase from Thermomyces lanuginosus. Process Biochem 2009;44:641–6. [13] Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandstrom AG, Backvall JE, et al. X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation. J Mol Biol 2008;376:109–19. [14] Gotor-Fernández V, Busto E, Gotor V. Candida antarctica lipase B: an ideal biocatalyst for the preparation of nitrogenated organic compounds. Adv Synth Catal 2006;348:797–812.
[15] Reetz MT. Lipases as practical biocatalysts. Curr Opin Chem Biol 2002;6:145– 50. [16] Brzozowski AM, Savage H, Verma CS, Turkenburg JP, Lawson DM, Svendsen A, et al. Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase. Biochemistry 2000;39:15071–82. [17] Verger R. Interfacial activation’ of lipases: facts and artifacts. Trends Biotechnol 1997;15:32–8. [18] Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, et al. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus G. th. Int J Syst Evol Microbiol 2001;51:433–46. [19] Schmidt-Dannert C, Rua ML, Wahl S, Schmid RD. Bacillus thermocatenulatus lipase: a thermoalkalophilic lipase with interesting properties. Biochem Soc Trans 1997;25:178–82. [20] Schmidt-Dannert C, Sztajer H, Stocklein W, Menge U, Schmid RD. Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochim Biophys Acta 1994;1214:43–53. [21] Liu AMF, Somers NA, Kazlauskas RJ, Brush TS, Zocher F, Enzelberger MM, et al. Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes. Tetrahedron Asymmetry 2001;12:545–56. [22] Martín JR, Nus M, Gago JVS, Sánchez-Montero JM. Selective esterification of phthalic acids in two ionic liquids at high temperatures using a thermostable lipase of Bacillus thermocatenulatus: a comparative study. J Mol Catal B: Enzym 2008;52–53:162–7. [23] Palomo JM, Fernández-Lorente G, Rúa ML, Guisán JM, Fernández-Lafuente R. Evaluation of the lipase from Bacillus thermocatenulatus as an enantioselective biocatalyst. Tetrahedron Asymmetry 2003;14:3679–87. [24] Carrasco-Lopez C, Godoy C, de las Rivas B, Fernandez-Lorente G, Palomo JM, Guisan JM, et al. Crystallization and preliminary X-ray diffraction studies of the BTL2 lipase from the extremophilic microorganism Bacillus thermocatenulatus. Acta Crystallogr F 2008;64:1043–5. [25] Carrasco-Lopez C, Godoy C, de Las Rivas B, Fernandez-Lorente G, Palomo JM, Guisan JM, et al. Activation of bacterial thermoalkalophilic lipases is spurred by dramatic structural rearrangements. J Biol Chem 2009;284:4365–72. [26] Tyndall JD, Sinchaikul S, Fothergill-Gilmore LA, Taylor P, Walkinshaw MD. Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1. J Mol Biol 2002;323:859–69. [27] Fernandez-Lorente G, Godoy CA, Mendes AA, Lopez-Gallego F, Grazu V, de Las Rivas B, et al. 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Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Reactivation of a thermostable lipase by solid phase unfolding/refolding Effect of cysteine residues on refolding efficiency César A. Godoy a , Blanca de las Rivas c , Dejan Bezbradica b , Juan M. Bolivar a,b,c , Fernando López-Gallego a,b,c , Gloria Fernandez-Lorente c , Jose M. Guisan a,∗ a
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Serbia c Departamento de Microbiologia, Instituto de Fermentaciones Industriales, CSIC, C/Juan de la Cierva 3, 2006 CSIC, Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 4 January 2011 Received in revised form 17 June 2011 Accepted 23 June 2011 Keywords: Additives Enzyme reactivation Refolding Lipase Cysteine oxidation Immobilization
a b s t r a c t Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25–30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface’s chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Enzymes are useful catalysts for many industrially relevant processes [1–3], but their moderate stability under industrial conditions hampers their widespread implementation as cost-effective biocatalysts [4,5]. Preparation of very robust enzyme derivatives would greatly improve their industrial applicability. However, immobilized enzymes on solid carriers are used under different operational conditions (e.g., at neutral pH values and mild temperatures in the presence of inert organic solvents) that can lead to enzyme inactivation through deleterious conformational changes [6]. In this way, after full enzyme inactivation, the enzyme may be completely unfolded and further refolded into its initial conformation, thus recovering quantitatively the catalytic activity. Thereby,
Abbreviations: BTL2, Lipase 2 from Geobacillus thermocathenolatus; CNBR-BTL2, BTL2 immobilized on agarose activated with cyanogen bromide groups; Gx-BTL2, BTL2 immobilized on agarose activated with glyoxylgroups. ∗ Corresponding author. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses: [email protected], [email protected] (J.M. Guisan). 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.06.018
the recovery of enzymatic activity after operational inactivation is a breakthrough for facilitating the implementation of enzymes on industrial scale [7]. In this context, enzyme unfolding/refolding has been applied to reactivate those enzymes that have been inactivated over long periods of operation [6,8]. Nevertheless, most protocols for enzyme reactivation by unfolding/refolding have been carried out with soluble enzymes; there are only a few examples of this process taking place in the solid phase. Recently, different immobilized enzymes were reactivated after their inactivation under different operational conditions (temperature, pH, organic solvents, etc.) [9–12]. The refolding was carried out in the solid phase under aqueous conditions and resulted that those enzymes immobilized by intense covalent attachment to the solid supports were refolded much better than those immobilized through a few covalent bonds. Hence, intense covalent attachments may prevent complete unfolding and preserve “residual” 3D conformations that may act as starting points for guiding the protein toward successful refolding as judged by the recovered activity. Since the lipases have broad substrate specificity accompanied in many instances by a high regio- and/or enantioselectivity [13–15], they have been broadly used in biocatalysis. This family
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of enzymes undergoes interfacial activation mechanisms, and they are active in the presence of hydrophobic surfaces such as drops of fat. When lipases are in aqueous media their active sites are completely shielded by normally one ␣-helix called “lid” [12,16,17]. However, in hydrophobic environments, normally in the surroundings of hydrophobic substrates, this “lid” is opened to allow substrate molecules get into the lipase binding pocket for catalysis to take place. This interfacial activation mechanism implies thereby two lipase conformations, open and close, and the equilibrium between them is shifted to favour either the open or closed conformation depending on media conditions [16,17]. Hence, reactivation of lipases by unfolding/refolding process should restore this complex movement to recover initial activity once the enzyme is refolded. Lipase from Geobacillus thermocatenulatus [18] (BTL2) has been heterologously overexpressed in Escherichia coli [19,20]. This is a thermo-alkalophilic enzyme able to perform some interesting reactions, such as resolution of esters yielding enantiomerically pures alcohols or acids [21–23]. Open structure of BTL2 has been recently crystallized and determined [24,25]. Hence, details of transition between the closed and the open form of BTL2 are now well-known [25]. The close-open transition involves a huge conformational change and such change induced by interfacial activation is so far quite complex in terms of structural reorganization of the protein. Furthermore, in the 3D structure, two metal binding domains (one for Ca2+ and one for Zn2+ ) [25,26] have been found playing important roles for both activity and stability (Fig. 1). Therefore, this protein requires organizing many structural domains to be folded in an active form which would mean a bigger challenge in order to successfully refold its tertiary structure. In previous studies, BTL2 stability was increased by multipoint covalent immobilization on glyoxyl-agarose (Gx-BTL2), while it was not when it was immobilized on CNBr-agarose (CNBr-BTL2) [27]. So, enzyme stabilization relied on the higher 3D rigidification achieved by multipoint covalent attachment.
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In the current research, the enzyme reactivation parameters of both insoluble derivatives described above have been studied after unfolding/refolding. Here we present a solid-phase unfolding/refolding protocol to reactivate BTL2 under reducing conditions. We have shed light on inactivation mechanism during unfolding that relies mainly on oxidation state of the protein. The refolding/unfolding process recovered both initial specific activity and enantioselectivity for both achiral and chiral substrates. Finally, we studied different variables that affected to refolding efficiencies, such as additives during the refolding, nature of support surface of insoluble derivatives, and refolding pH. 2. Materials and methods 2.1. Materials and conventional protocols All the materials were of analytical grade. Lipase expression, purification and lipase colorimetric assay proceeded as described [27]. Sited directed mutagenesis was performed according to conventional proceedings. For more details see supporting information. 2.2. Incubation of BTL2 catalyst in 8 M guanidine Experiments were performed as previously described by Soler et al. [28]. Two wet grams of each immobilized preparation were washed 10 times with 10 volumes of 8 M guanidine at 25 ◦ C and the desired pH value (0.1 M sodium acetate was used at pH 5, 0.1 M sodium phosphate at pH 7, or 0.1 sodium bicarbonate at pH 9). In some instances, 25 mM DTT was added to the guanidine solution. Then, the immobilized enzyme was suspended in 20 mL of the same solution. Periodically, samples of 50 L were withdrawn and the activity was determined as stated above. We also checked that the presence of diluted guanidine did not affect the enzyme activity. 2.3. Reincubation of the unfolded BTL2 catalyst in aqueous medium Insoluble enzyme preparations previously unfolded by incubation in guanidine solutions were washed 10 times with 10 volumes of aqueous buffer (with or without DTT) to eliminate guanidine and were then suspended in the same buffer at the desired temperature. Periodically, samples were withdrawn and the activity was determined as stated above. The buffer in some cases contained different additives such as DTT and trehalose. 2.4. Determination of the short-term reactivation rate Derivatives were unfolded by incubation in 8 M guanidine and 25 mM DTT. Later on enzyme inactivation, unfolding solution was removed by filtration without washing. Those derivatives were resuspended on the media where the activity assay was carried out. In situ activities of the derivatives were determined at different time intervals (0–25 s, 25–50 s, 50–75 s, 75–100 s, 100–150 s, 150–200 s, 200–300 s and 300–400 s) in the presence or absence of different additives. The maximum of activity to be recovered in the presence of one additive was that observed with CNBr-BTL2 derivative without being unfolded in the presence of the corresponding additive. Activity values at different times are referred to that maximum activity defined above. The activity grew in asymptote way up to a maximum, thereby activities were empirically adjusted to a second order equation to calculate the maximum recovered activity and the refolding rate (time to reach 50% of the maximum recovered activity).
3. Results 3.1. Unfolding of immobilized BTL2 derivatives in guanidine and further refolding in aqueous media
Fig. 1. Structure of the open form of BTL2 (see Ref. [25]). BTL2 is shown in cartoon representation where lid, C94, C294 and cations (Zn2+ and Ca2+ ), are depicted accordingly labelled. Both side chain from C64 and C294 are shown in sticks. Both metals are represented as spheres.
Chaotropic agents such as concentrated guanidine solutions have been extensively used to promote unfolding of aggregated or misfolded free enzymes [29–31]. In the context of operationally inactivated enzymes, their unfolding by such reagents would bring them to a suitable starting point for further enzyme reactivation proceedings. Equivalent amounts of BTL2 were immobilized per unit weight of both supports. As it had been previously described [27], immobilization on both Gx-agarose and CNBr-agarose matrixes resulted in approximately 60–65% of BTL2 expressed activity, meaning that both derivatives presented equivalent activity and protein load.
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3.2. Effect of DTT on reactivation efficiency
Fig. 2. Reactivation courses of different immobilized BTL2 derivatives by incubation in aqueous solution. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, afterwards the derivatives were incubated with 0.1 M sodium phosphate at pH 7 under the same conditions. The activity was monitored in both inactivation and reactivation stage. Black circles: Gx-BTL2; white rhombus: CNBrBTL2.
Gx-BTL2 and CNBr-BTL2 derivatives were inactivated by incubation in saturated guanidine solutions. When enzymes are incubated with chaotropic reagents as guanidine, they are unfolded because water is sequestered by such reagent [30]. Thus, enzyme inactivation is due to protein unfolding in the presence of these molecules. In this way, both immobilized derivatives lost rapidly their activities, expressing less than 15% of activity after 1 h. After unfolding, insoluble derivatives were extensively washed in order to completely eliminate guanidine and then incubated in aqueous buffer at pH 7 and 25 ◦ C (Fig. 2). Both lipase derivatives slowly recovered about 30% of their initial activity in 2.5 h and longer incubation times did not promote higher reactivation percentages. These values are much lower than those observed in unfolding/refolding of others enzymes [9,12]. Moreover, different loads of derivatives had negligible effect on reactivation efficiencies; thereby unfolding/refolding showed no dependence on protein load for both Gx-agarose and CNBr-agarose matrixes. Because of the cation dependence of this lipase, EDTA or TPEN were added to remove the structural cations during the unfolding process. Afterwards, the refolding process was carried out either in the presence or absence of both cations (Zn2+ and Ca2+ ) to study their effects on the enzyme folding. None of these two metals had measurable effect on the final refolding efficiency because improvement of neither recovered activity nor the refolding rate was detected (data not shown).
In addition to conformational changes, chemical modifications may also inactivate enzymes during their operational performance. Furthermore, those modifications can directly affect protein folding avoiding optimal refolding and thereby resulting in low reactivation efficiencies. In order to determine the cysteines’ role in enzyme refolding, we carried out the complete unfolding/refolding process in the presence of mild reducing agents such as dithiothreitol (DTT). This reducing compound was chosen to address this issue because it has been reported as very good reducing agent to prevent cysteine oxidation or disulfide bond formation [32]. When both unfolding and refolding steps were carried out at neutral pH under reducing conditions (DTT added to the media), Gx-BTL2 recovered over 70% of its initial catalytic activity whereas CNBr-BTL2 recovered around 60% (Fig. 3A). The effect of DTT also preserved enzyme activity because when BTL2 was unfolded by guanidine, the catalyst inactivation proceeded more slowly. Moreover, the addition of DTT seemed to be important during the unfolding step to prevent cysteine oxidation because when unfolding was carried out with DTT, this reducing agent was not needed during refolding to achieve higher reactivation efficiencies (Fig. 3B). On the contrary, the addition of DTT to the refolding step also led to high reactivation efficiencies albeit unfolding was carried in the absence of DTT (Fig. 3B). Oxidation events involving Cys residues were thus demonstrated to play a crucial role in enzyme reactivation. They are prevented to be oxidized in reducing environments during the unfolding step or enzyme oxidation state is reverted by reducing agent (DTT) addition to the refolding step. Expectedly, the addition of metals to the unfolding/refolding process in reducing conditions did have no effect on refolding efficiency, just as it was observed in non reducing conditions. In this way, the enzyme, after unfolding/refolding process in reducing conditions, kept unaffected in terms of specific activity (26.6 U/mg vs 27.8 U/mg from the wild type), stability against organic solvents and enantioselectivity (E > 100 toward S isomer as well as wild type) for the hydrolysis of rac-␣-methylbenzylacetate [27]. The optimal reactivation was kept after at least five unfolding/refolding cycles. This fact further supports the use of reactivation as another alternative to make longer the biocatalysts life-time. 3.3. The role of Cys during unfolding/refolding In the light of the observed insights, we studied which role was played by each cysteine in the deleterious oxidation process during
Fig. 3. Effect of the presence of 25 mM DTT on the reactivation of different immobilized BTL2 derivatives. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium phosphate at pH 7 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stage. (A) Reactivation course of insoluble BTL2 preparation in the presence of DTT after unfolding with guanidine. Refolding was performed in 25 mM sodium phosphate at pH 7 and 25 ◦ C as described in Section 2. Black circles: Gx-BTL2 derivative; white rhombus: CNBr-BTL2 derivative. Solid lines: the absence of DTT. Dashed lines: the presence of DTT. (B) Dynamic unfolding/refolding of Gx-BTL2 derivative in the presence and absence of DTT at pH 5. The Gx-BTL2 derivative was incubated with guanidine 8 M either in the presence (grey dash line) or absence (black dash line) of DTT. Afterwards, the derivatives were incubated in aqueous media with DTT (grey solid line) or without DTT (dark solid line).
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Fig. 4. Reactivation of different mutants of BTL2 immobilized on CNBr-agarose beads after inactivation by incubation in guanidine. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium phosphate at pH 7 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stages. (A) Full circles: wild type BTL2; empty rhombus: BTL2(C64S/C295S) and (B) full circles: wild type BTL2, empty triangles: BTL2(C64S) and empty squares: BTL2(C295S).
BTL2 unfolding, causing low refolding efficiencies. A double BTL2 mutant with both native cysteines residues replaced by serine was prepared. Such double mutant showed similar biochemical properties in terms of activity and stability than the wild type variant. This double mutant was immobilized on CNBr-agarose (see Section 2), and then unfolded in guanidine solution. Finally, the unfolded derivative was incubated in aqueous solution in order to allow protein refolding. At no reducing conditions (no DTT), 94% of initial activity was recovered for refolded BTL2-(C64S/C295S) unlike wild type that only recovered 30% of its activity under the same conditions (Fig. 4A). Therefore, in the cysteine free BTL2 variant, DTT hardly affected to its refolding efficiency, supporting once more the negative effect of cysteine oxidation on BTL2 reactivation during unfolding/refolding processes. The optimal reactivation of the double mutant lacking cysteines was observed under at least five cycles of unfolding/refolding. Moreover, the enzyme properties of the mutant lacking the two cysteines residues were exactly the same before and after the unfolding/refolding process (data not shown). In addition to the double mutant BTL2-(C64S/C295S), two single mutants of each cysteine were prepared. In BTL2-C295S and BTL2C64S, cysteines were again substituted by serines. As well as in the case of the double mutant the substitution of one single cysteine at position either 295 or 64 negligible affected to the biochemical behaviour of BTL2. They were finally immobilized on CNBr-agarose support, and later on the insoluble derivates of each BTL2 single mutant underwent the unfolding/refolding process. BTL2-C295S variant recovered more than 90% of its initial activity in the absence of DTT (non reducing conditions). Contrarily BTL-C64S could recovered 65% of its initial activity, but reactivation was 2-fold more efficient than the wild type variant (Fig. 4B). Nevertheless, a cysteine in that position still promoted a suboptimal BTL2 refolding. It means that C295 individually had a more negative effect on BTL2 refolding than C64 but the combination of both (wild type enzyme) led to a much worse reactivation yield (35%). In order to discover whether this effect was specific for these two cysteines, the cysteine pair was restored in the single mutant BTL2-C64S introducing one fairly exposed cysteine at position 93 (close to N-terminal), creating so far a double mutant BTL2-(C64S/T93C) (Supplementary Fig. 1). When the double mutant BTL2-(C64S/T93C) was immobilized on CNBr-agarose and undergone the unfolding/refolding process under oxidizing conditions, the derivative was poorly reactivated with values as low as the wild type (around 30% of reactivation). 3.4. Effect of pH on reactivation efficiency pH may play an important role during the refolding of enzyme tertiary structure. To examine this issue, refolding of different
immobilized BTL2 derivatives was carried out at different pH conditions (Fig. 5). When unfolding/refolding experiments were carried out at different pH conditions, different activities recoveries could be observed for each derivative. CNBr-BTL2 derivative was more efficiently reactivated by refolding in a non reducing environment at alkaline pH values unlike the Gx-BTL2 derivative that exhibited an opposite effect, being more efficiently refolded under the same conditions but at acid pH values. This pattern was even more pronounced when the unfolding/refolding process was carried out in the presence of DTT to avoid cysteine oxidations. In these conditions CNBr-BTL2 recovered full initial activity after unfolding/refolding at pH 9, whereas the equal recovery for Gx-BTL2 derivative took place at pH 5. 3.5. Effect of additives on the rate and yield of reactivation Effects of some additives on the refolding of the Gx-BTL2 derivative were studied at neutral pH regarding to both reactivation rate and yield (see Section 2). These experiments were carried out under reducing conditions to only study additive effects on refolding behaviour discarding any negative contribution by oxidation. Table 1 shows that Tris buffer is preferred to sodium phosphate. It has been described that Tris–HCl increased the intrinsic BTL2 stability regarding to phosphate buffer at neutral conditions [33]. Hence, this aminated buffer would confer a more rigid structure to the protein, which would avoid complete unfolding in the presence of guanidine, yielding higher and more rapidly reactivation efficiencies. On the other hand, Triton X-100 did not drive to better and faster refolding unlike those experimental evidences for
Fig. 5. Effect of the pH value during reactivation of inactivated BTL2 immobilized preparations. Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated under different pH values at 25 ◦ C. The activity was monitored in both inactivation and reactivation stages. Black circles: Gx-BTL2; white rhombus: CNBr-BTL2. The unfolding/refolding was carried out either in the presence (dashed lines) or absence of DTT (solid lines). DTT concentration used in this experiment was 25 mM.
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Table 1 Effect of additives on the recovery rate and maximum recovered activity values (see Section 2). Additive
Extrapolated maximum recovered activity value (%)
25 mM Tris–HCl (control) 0.001% Triton X-100 0.01% Triton X-100 0.1% Triton X-100 0.5 M TMAO 1.0 M TMAO 2.0 M TMAO 10% glycerol 20% glycerol 30% glycerol 10% PEG 1500 20% PEG 1500 30% PEG 1500 10% dextran 1500 20% dextran 1500 30% dextran 1500 10% trehalose 20% trehalose 40% trehalose 25 mM NaH2 PO4 buffer
67 67 70 65 48 69 38 61 66 55 77 97 82 69 99 46 66 60 65 48
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3 5 6 3 3 3 4 5 3 3 6 4 3 4 5 2 3 4 4 3
Time to reach 50% of maximum recovery (s) 29 27 26 39 27 43 28 24 24 17 42 83 91 14 19 9 10 91 135 42
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3 3 4 3 4 5 4 2 5 3 2 6 6 3 2 2 5 3 6 2
The biocatalyst was inactivated by incubation in saturated guanidine solutions followed by the addition of 25 mM DTT. Aliquots of 50 L of the mixture were diluted in activity assay medium containing the respective additive in 25 mM Tris–HCl (pH 7). The best results are bolded.
lipase from Thermomyces lanuginose, where optimal refolding was achieved in the presence of detergents. Here, we used a battery of compounds to help protein folding, observing different effects with each one. Trehalose is stabilizing compound for enzymes [29,33,34] that slowed-down the reactivation rate while maintaining the final recovered activity. Glycerine and TMAO (a “chemical chaperone” used to reinforce hydrophobic interactions) [35,36] reduced the reactivation rate and the maximum value obtained. Finally, polymeric additives as polyethylene glycols and dextrans were also added during refolding stage. The best results were obtained for polymers such as dextran that improves both recovered activity and rate of reactivation. This positive effect observed in the presence of polymeric substances may be related to the preferential hydration during folding, stabilizing crucial folding intermediates [36]. 3.6. Effect of the support surface on reactivation efficiency Protein’s surrounding environment can also play an important role in the refolding process. The immobilization protocol using CNBr-agarose matrixes would allow us to control this environment by modifying the support surface. In this way, two different derivatives having the same number of enzyme–support bonds and the same enzyme orientation on the support were blocked with different molecules later on immobilization, obtaining different enzyme surroundings. The chosen molecules to achieve those two different derivatives were ethanolamine and ethylenediamine. When the refolding was carried out with these two differently blocked derivatives at pH 9 (optimal pH for refolding of CNBr-BTL2 derivative), refolding efficiencies were found to be quite different depending on the nature of the molecule used during the blocking. Ethanolamine blocked derivatives yielded less reactive surfaces that allowed much more efficient refolding (100% of recovered activity) than when blocking was carried out with ethylenediamine. The latter ones presented more charged surfaces that only recovered 55% of derivative’s initial activity (Fig. 6). This fact introduced a new variable on the unfolding/refolding process at solid phase.
Fig. 6. Reactivation of CNBr-BTL2 derivatives having an hydroxylated surface (blocked with ethanolamine) (circles) or an positively charged aminated surface (blocked with ethylenediamine) (rhombus). Immobilized derivatives were inactivated with 8 M guanidine at pH 7 and 25 ◦ C, and then the derivatives were incubated with 0.1 M sodium carbonate at pH 9 and 25 mM DTT. The activity was monitored in both inactivation and reactivation stages.
4. Discussion 4.1. Solid-phase BTL2 refolding depends on reducing media conditions When BTL2 insoluble derivatives were incubated with chaotropic reagent as guanidine, they were inactivated. At those conditions a minimal fraction was not apparently inactivated (15%), it may be due to a small fraction of protein that is refolded during the kinetic enzymatic assay in aqueous media. Here, we consider that guanidine may promote either partial or total unfolding of enzyme 3D structure even in the insoluble derivatives. Unexpectedly, structural cations did not help to successful BLT2 reactivations, meaning that low refolding efficiencies did not rely on cation location on enzyme tertiary structure. Surprisingly, here, unlike in other published studies [10], more intense covalent multipoint attachment between BTL2 and the support surface did not result in better refolding efficiencies regarding to those derivatives with fewer bonds enzyme–support (Fig. 2). Since covalent attachment was not a determining factor for achieving high refolding efficiencies for BTL2, different factors may be leading to the enzyme inactivation rather than unfolded conformations. BTL2 3D structure revealed the presence of two cysteines that are located on enzyme surface, both of which are susceptible to be oxidized possibly resulting in the activity loss. The implication of both cysteines in the success of unfolding/refolding processes was demonstrated because activity recovery was much higher in the presence of DTT. Therefore, a reducing agent as DTT can play two roles: (1) keeping the cysteines reduced during the unfolding process, enabling successful refolding and (2) reverting during refolding process those oxidations occurred in the protein while the unfolding process under oxidizing conditions. 4.2. BTL2’s cysteines play a key role on unfolding/refolding events The two cysteines presented on BTL2 primary sequence could be forming a disulfide bond during either unfolding or refolding process. This internal covalent bond that would not be present at wild type conformation may drive a wrong enzyme folding, resulting in inactive enzyme conformations. Moreover, in oxidizing conditions (DTT absence), the two cysteines may be oxidized to different groups such as sulfinic, sulfonic acids, and even to sulfenyl-amides formed between the oxidized thiol and the peptide bond [37]. These chemical modifications may also explain low refolding efficien-
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cies obtained with both BTL2 insoluble derivatives. On the other hand, cysteine oxidation seemed to be totally independent on covalent attachment intensity, explaining so similar behaviours for both insoluble derivatives. In order to shed light on the relationship between cysteines and reactivation efficiency during unfolding/refolding processes, a double BTL2 mutant BTL2-(C64S/C295S) with both native cysteines residues replaced by serine was prepared. In the cysteine free BTL2 variant, DTT hardly affected to its refolding efficiency, being this efficiently reactivated under both reducing and oxidizing conditions. In first term, these data may be suggesting a wrong disulfide bond formation between C295 and C64 during the unfolding, in other words an intramolecular cross-linking mediated by a disulfide bond may be taking place. Those cysteines are quite far away in the tertiary structure but likely in the presence of guanidine they may be found next to each other in the space, forming a disulfide bond, difficulting the optimal protein refolding. However, these experiments did not give us full understanding about whether two or only one cysteine is needed to lead wrong folding in oxidizing conditions. For this reason, an additional pair of cysteines in BTL2 primary sequence (BTL2-(C64S/T93C)) was made. It was also susceptible to be oxidized, possibly triggering disulfide formation during unfolding step regardless the position of each cysteine from the pair. In this variant as well as the wild type, refolding was very inefficient likely because the wrong disulfide bond would drive to a suboptimal folded BTL2, avoiding the successful enzyme reactivation after refolding. On the contrary, BTL2 variants with only one cysteine (BTL2-C64S and BTL2-C295S) were much better reactivated than the BTL2 variant with two cysteines, nevertheless the reactivation behaviour was different between the two different mono-cysteine BTL2 variants. Depending on the cysteine position, reactivation was higher or lower in the absence of DTT. Likely the variant with Cys-295 may undergo a single point oxidation of the thiol group that is negative for BTL2 folding, while Cys-64 may be less susceptible to be oxidized explaining so the lower impact of that cysteine in the reactivation efficiency. 4.3. pH and support surface nature are important parameters to successfully reactivated BTL2 through solid-phase unfolding/refolding processes The pH had different effects during the unfolding/refolding of either CNBr-BTL2 or Gx-BTL2. These differences can be mainly explained by the different immobilization protocols carried out to obtain each derivative. In the case of CNBr-BLT2, the enzyme is covalently attached to the support through few protein–support interactions, and thus this insoluble version of the enzyme would behave similarly to the soluble one during unfolding/refolding process. Contrarily, when the enzyme was immobilized onto glyoxyl-agarose supports, the immobilization took place via the richest lysine region on enzyme surface. Herein, enzyme was very intensely covalently attached to the support surface with a different orientation than the one adopted by the enzyme on CNBragarose matrixes (Fig. 7). This different orientation, along with the more intense covalent attachment, may result in different starting points for the refolding process. Those different starting points may lead refolding through different interaction networks that can be strongly affected by the pH. In the same context, the oxidation/reduction of Cys during unfolding/refolding of BTL2 might also be affected by the pH. For example, Cys reduction mediated by DTT would be favoured at basic pH since thiolate group in this reducer is predominant [38], thus explaining the higher reactivation efficiency of CNBr-BTL2 derivative under alkaline conditions. Surprisingly, for Gx-BTL2 derivative was found just the opposite effect, acid pH values promoted higher reactivation efficiencies. In this latter case, formation of thiolate ion of DTT would not be the
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Fig. 7. Surface representation of BTL2 open structure. The representation was displayed using Pymol 0.99 v. Lid is depicted in light grey and in cartoon representation. N-terminal was represented in black. (A) Region from where enzyme would be immobilized on CNBr-agarose supports at pH 7. (B) Region from where enzyme would be immobilized on glyoxyl-agarose supports at pH 10. Lysines are represented in dark grey. The two BTL2 representations show the different orientations that BTL2 would be immobilized for in the two derivatives studied in this research.
only reason that assures an optimal BTL2 refolding. A similar relation between pH and refolding efficiency has also been found for the murine prion mPrP(23-231) [39], where the authors suggested that pH-mediated structural changes may promote native-like conformations by increasing the proximity of properCys residues regardless the efficiency of Cys redox reaction mediated by agents like DTT. Another interesting parameter to take in account for solid-phase unfolding/refolding processes was the support surface chemical nature. BTL2 was much more efficient reactivated when it was immobilized on less reactive surface than when it was on more reactive ones. Less reactive surfaces would promote less undesirable interactions between protein and surface while unfolding/refolding process is taking place. On the contrary, more reactive (e.g. cationic) surfaces can ionically interact with the negative groups on the protein surfaces during either unfolding or refolding, yielding aberrant conformations with much lower activity than the optimal one. These non desirable interactions could thus lead to incorrect conformation, explaining the lower recovered activities in the ethylenediamine blocked CNBr-BTL2 derivative. 5. Conclusions The active refolding of wild type BTL2 is highly dependent on the oxidation state of the Cys residues presented in its primary sequence. In fact, refoldings in conditions where a potential disulfide bond might be formed yielded low reactivation efficiencies. Such issue was overcome using reducing environments that allowed actively refolding wild type BTL2 in insoluble preparations, CNBr-BTL2 and Gx-BTL2. Here, we proposed DTT as reducing agent for achieving 100% activity recovery after the unfolding/refolding process under optimal pH. Despite of only studying here the reactivation behaviour of BTL2 by unfolding/refolding, this methodology may be applicable to other proteins containing free and reduced cysteines in their sequences. Moreover, these techniques are the starting point for new and simpler reactivation protocols applied to enzyme derivatives that have been operationally inactivated. The use of different buffers, different pH values, and different additives strongly modifies the rate and yield of activity recovery. On the other hand, inactivation by guanidine should promote a total or partial unfolding of the enzyme and hence reactivation was strongly influenced by the enzyme surroundings on the support surface. Herein, it has been demonstrated that more reactive
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surfaces promote undesirable interactions between enzyme and support during refolding of the inactivated enzyme. Therefore, under optimal conditions, the best immobilized derivative, the best buffer and pH, the presence of additives and the use of support surfaces with low reactivity allow a very rapid and complete reactivation, achieving 100% of activity recovery in only 1 min. This result is even more relevant considering the very complex mechanism of activity of this thermostable lipase and its biotechnological interest in kinetic resolution of chiral compounds. Therefore, we have opened a new avenue to overcome bottlenecks for the reactivation in solid phase by unfolding/refolding approaches of enzymes containing cysteines in their primary sequence. Acknowledgments This work has been supported by Comunidad Autónoma de Madrid (CAM) (grant S0505/PPQ/0344) and CICYT (grants BIO2005-8576 and AGL-2009-07625). C. Godoy has a PhD fellowship from MCI. D. Bezbradica has postdoctoral fellowship of Serbian Ministry of Science and Technological Development for research on project bt20064. Fernandez-Lorente has a Ramon y Cajal contract from MCI. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.enzmictec.2011.06.018. References [1] Akoh CC, Chang SW, Lee GC, Shaw JF. Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce. J Agric Food Chem 2008;56:10445–51. [2] Brenna E, Fuganti C, Serra S. Applications of biocatalysis in fragrance chemistry: the enantiomers of alpha-, beta-, and gamma-irones. Chem Soc Rev 2008;37:2443–51. [3] Woodley JM. New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol 2008;26:321–7. [4] Iyer PV, Ananthanarayan L. Enzyme stability and stabilization—aqueous and non-aqueous environment. Process Biochem 2008;43:1019–32. [5] Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF. Stability of biocatalysts. Curr Opin Chem Biol 2007;11:220–5. [6] Mozhaev VV, Berezin IV, Martinek K. Reactivation of immobilized enzymes. Methods Enzymol 1987;135:586–96. [7] Guisan JM. Immobilization of enzymes as 21st century begins. In: Guisan JM, editor. Immobilization of enzymes and cells. Totowa, NJ: Humana Presss Inc.; 2006. p. 1–14. [8] Teshima T, Kondo A, Fukuda H. Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES. Appl Microbiol Biotechnol 1997;48:41–6. [9] Bolivar JM, Rocha-Martin J, Godoy C, Rodrigues RC, Guisan JM. Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus. Process Biochem 2010;45:107–13. [10] Rodrigues RC, Bolivar JM, Palau-Ors A, Volpato G, Ayub MAZ, FernandezLafuente R, et al. Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus. Enzyme Microb Technol 2009;44:386–93. [11] Rodrigues RC, Bolivar JM, Volpato G, Filice M, Godoy C, Fernandez-Lafuente R, et al. Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers. J Biotechnol 2009;144:113–9. [12] Rodrigues RC, Godoy CA, Filice M, Bolivar JM, Palau-Ors A, Garcia-Vargas JM, et al. Reactivation of covalently immobilized lipase from Thermomyces lanuginosus. Process Biochem 2009;44:641–6. [13] Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandstrom AG, Backvall JE, et al. X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation. J Mol Biol 2008;376:109–19. [14] Gotor-Fernández V, Busto E, Gotor V. Candida antarctica lipase B: an ideal biocatalyst for the preparation of nitrogenated organic compounds. Adv Synth Catal 2006;348:797–812.
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