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Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers
Journal of Biotechnology 144 (2009) 113–119
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Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers Rafael C. Rodrigues, Juan M. Bolivar, Giandra Volpato, Marco Filice, Cesar Godoy, Roberto Fernandez-Lafuente ∗ , Jose M. Guisan ∗ Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain
a r t i c l e
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Article history: Received 23 February 2009 Received in revised form 27 August 2009 Accepted 1 September 2009 Keywords: Chemical modification Enzyme reactivation Hydrophilic polymers Solid phase Lipase Enzyme stabilization
a b s t r a c t Immobilized-stabilized aminated lipase from Thermomyces lanuginosus (TLL-A) is not easily reactivated after inactivation by incubation in the presence of organic solvents or chaotropic reagents. To improve the recovered activity of this biocatalyst, immobilized TLL-A has been submitted to different modifications. The best results were obtained when the enzyme was coated with a very hydrophilic and inert polymer: dextran modified with glycine (Dx-Gly). This modification did not reduce enzymatic activity while it increased the stability of this already very stable preparation, in thermal and organic solvent induced inactivation (by a 4-fold factor). Simple incubation in aqueous medium at pH 7 and 25 ◦ C permitted to fully recover the activity of the immobilized and modified TLL-A enzyme inactivated by incubation in organic solvents or saturated guanidine during 3 cycles, while the non-modified enzyme only recover some activity. When the inactivation was caused by exposition at high temperatures, the reactivation was higher using the modified biocatalyst, but was far for complete (40% after 3 inactivation–reactivation cycles). The determination of the TLL-A activity in the presence of detergents (that helps the opening of active site of the lipase) allowed, in this case, to significantly improve the results, now near to 90% of the initial activity was recovered (using the non-modified enzyme the recovered activity was around 60%). This very hydrophilic and inert polymer, coating the enzyme surface, seems to help the correct positioning of the hydrophilic and hydrophobic groups of the enzyme, and that way improve both the stability and possibility of reactivation of the enzyme. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are very interesting biocatalysts, potentially useful as catalyst in many biotransformations (Bornscheuer and Kazlauskas, 2006). One of the key problems of enzymes as industrial biocatalysts is their moderate stability under industrially relevant conditions (Iyer and Ananthanarayan, 2008), such as moderately high temperatures, or presence of solvents, used to increase the solubility of substrates or products (Kim et al., 2007; Yeates et al., 2007), to shift thermodynamic equilibrium (Rosell et al., 1998), to improve enzyme properties (Fernández-Lafuente et al., 1998), or even to prevent microbial contamination. Thus, much effort has been paid to the development of strategies that may increase the stability of the enzymes by diverse techniques (Iyer and Ananthanarayan, 2008; Polizzi et al., 2007). Immobilization is a requisite for many industrial applications of enzymes (Cao, 2005; Sheldon, 2007), and in many cases may be coupled to the stabi-
∗ Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760. E-mail addresses: rfl@icp.csic.es, [email protected] (R. Fernandez-Lafuente), [email protected] (J.M. Guisan). 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.09.002
lization of the enzyme if a multipoint and multisubunit covalent attachment is achieved (Mateo et al., 2007). On the other hand, if the enzyme may be partially or fully reactivated after its inactivation, this may enlarge the operational half-life of the catalyst and, this way; it may improve the profitability of the process. In many cases, enzyme activity is lost just by the promotion of wrong enzyme conformations (Sarmento et al., 2009), in some other cases some chemical modification of different protein groups may occur (Daniel et al., 1996). It has been shown that multipoint covalent attachment of enzymes on finally inert supports (e.g., glyoxyl agarose) (Mateo et al., 2005) may help the reactivation of partially inactivated enzymes, because these multiple enzyme-support bonds may behave as reference points, easing the recovery of enzyme activity (Romero et al., 2009). In some instances, a significant enzyme activity recovery may be achieved just by incubating the enzyme under mild conditions after their inactivation (Rodrigues et al., 2009c,d; Romero et al., 2009). However, in other examples, enzyme reactivation requires the full unfolding of the protein structure to eliminate incorrect structures, and its further refolding under controlled conditions (Rehaber and Jaenicke, 1992; Zhi et al., 1992).
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Lipase from Thermomyces lanuginosus (TLL) is the enzyme responsible for the lipolytic activity of Lipolase® , a commercial lipase preparation supplied by Novozymes. This enzyme has been broadly used in many biotransformations (Rodrigues et al., 2009b; Turan et al., 2006). Its structure has been solved at 1.8 Å (Derewenda et al., 1994). In homogenous medium, lipases present a large flap or lid that isolates the active site from the reaction medium (Brzozowski et al., 1991). In the presence of a hydrophobic interface (e.g., an oil drop (Miled et al., 2001), a hydrophobic support (Fernandez-Lafuente et al., 1998), a hydrophobic protein (Palomo et al., 2003)), the lid moves, and the active site of the lipase is exposed to the medium. TLL has been recently stabilized by coupling the chemical amination of the enzyme surface to the multipoint covalent attachment of the modified enzyme on glyoxyl-agarose beads (Rodrigues et al., 2009a). The amination of the enzyme was necessary to highly stabilize TLL by its immobilization to glyoxyl support, and did not affect either the enzyme stability or its activity. This multipoint immobilized preparation also recovered a higher percentage of activity, when submitted to different reactivation strategies, compared to a derivative of non-modified TLL immobilized on cyanogen bromomide activated agarose (Rodrigues et al., 2009c). If the enzyme activity was measured in the presence of detergents (during inactivation or reactivation experiments) the enzyme inactivation rate was slower and the observed enzyme reactivation was higher. Detergents may help to stabilize the open form of lipases (Fernández-Lorente et al., 2006; Guncheva et al., 2007; Mogensen et al., 2005). In this paper, we try to advance some further steps on the reactivation of this TLL biocatalyst, using chemical modification on solid phase of the immobilized-stabilized enzyme (see Scheme 1) to help the recovering of activity after partial inactivation of the preparation. The modification of the primary amino groups of the protein was performed with formaldehyde; this will yield stable secondary amino groups. The enzyme surface has been also modified with very hydrophilic and inert polymers (dextran–aldehyde or dextran–aldehyde–glycine). These polymers are random coils and can hardly increase the rigidity of the enzyme, even if an intense cross-link is achieved. Nevertheless, they can produce several positive effects: (i) generate steric hindrances to the movements of the enzyme groups placed near to the protein surface; (ii) help to position hydrophobic groups and hydrophilic groups by generation a kind of “partition” by the high hydrophilic polymer (FernandezLafuente et al., 1999). The very open structure of these polymers permits that even when a multilayer of similar polymers is used to
fully coat the enzyme surface, the hindrances to the movements of small substrates are minimal (Fernandez-Lafuente et al., 1999). 2. Materials and methods 2.1. Materials Lipase from T. lanuginosus (TLL) was obtained from Novozymes (Denmark). 1-Ethyl-3-(dimethylaminopropyl) carbodiimide, pnitrophenyl butyrate (p-NPB) and hexadecyltrimethylammonium bromide (CTAB) were from Sigma. 1,4-Dioxane, guanidine hydrochloride, 2,4,6-trinitrobenzensulfonic acid (TNBS), formaldehyde (commercial 35% formaldehyde in water solution), and 1,2-ethylenediamine, were from Fluka. Octyl-Sepharose CL-4B was purchased from GE Healthcare (Uppsala, Sweden). Cross-linked agarose (10 BCL) was kindly donated by Hispanagar S.A. (Burgos, Spain) and its modification to glyoxyl agarose (activated with 200 mol/g of support) was performed as described elsewhere (Mateo et al., 2005). Other reagents and solvents used were of analytical or HPLC grade. 2.2. Methods The experiments were carried out at least by triplicate and the standard error was always under 5% represented by error bars in the figures. 2.2.1. Enzymatic activity assay Lipolytic activity assay was performed by measuring the increase in the absorbance at 348 nm produced by the released pnitrophenol in the hydrolysis of 0.4 mM pNPB in 25 mM sodium phosphate at pH 7 and 25 ◦ C, using a spectrophotometer with a thermostatized cell and with continuous magnetic stirring. To initialize the reaction, 0.05 mL of lipase solution or suspension were added to 2.5 mL of substrate solution. One international unit of pNPB activity was defined as the amount of enzyme necessary to hydrolyze 1 mol of pNPB/min (IU) under the conditions described above. In some instances, 0.01% of CTAB was added to the substrate solution. CTAB is able to hyperactivate Gx-TLL-A (see below), multiplying the enzyme activity by a 7-fold factor when used at 0.01%. 2.2.2. Purification of TLL TLL was purified by interfacial adsorption on hydrophobic supports. The enzyme was adsorbed on octyl-Sepharose beads under
Scheme 1. Chemical modifications of immobilized-stabilized TLL (Gx-TLL-A): (a) modification with formaldeyde and (b) modification with hydrophilic polymers dextran–aldehyde or dextran–aldehyde–Gly. In black amines from Lys residues, and in grey amines obtained by modification with 1,2-ethylenediamine (EDA).
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continuous stirring in 10 mM sodium phosphate at pH 7.0, according to a previously described procedure (Fernandez-Lafuente et al., 1998). The activities of suspensions and supernatants were periodically measured by using the pNPB assay. After enzyme adsorption, the lipase preparation was vacuum filtered using a sintered glass funnel and abundantly washed with distilled water. TLL was desorbed from octyl-Sepharose by suspending the immobilized enzyme in a 1/10 (w/v) ratio in 25 mM sodium phosphate at pH 7.0 containing 0.6% (v/v) of CTAB during 1 h at room temperature. Only one protein band was detected by SDS-PAGE (results not shown). 2.2.3. Chemical amination of immobilized TLL Chemical amination was performed on the TLL immobilized on octyl-Sepharose by using 1-ethyl-3-(dimethylaminopropyl) carbodiimide as carboxy activating reagent and 1,2-ethylenediamine at pH 4.75 as previously described (Rodrigues et al., 2009a). These reaction conditions have been reported as capable of fully modifying all exposed carboxylic groups of the protein (Hoare et al., 1968). The aminated TLL, termed TLL-A, immobilized on octyl-Sepharose was stored at 4 ◦ C. 2.2.4. Immobilization of TLL-A on glyoxyl-agarose beads Immobilized derivatives of TLL-A were prepared by using glyoxyl agarose (Gx-TLL-A), first immobilization was performed at pH 9 and further incubation at pH 10 to get an intense multipoint covalent attachment (using 60 IU of TLL/g of support). The full protocol is described in Rodrigues et al. (2009a). The final immobilized derivative, having around 2.5 mg of protein/g of support, had 50 IU/g of support, this moderate enzyme loading avoided the risks of diffusion limitations that could alter the enzyme performance. 2.2.5. Chemical modification of the amino groups of Gx-TLL-A 2.2.5.1. Chemical modification of Gx-TLL-A with formaldehyde. Modification with formaldehyde was performed according to a previously described methodology (Fernandez-Lafuente et al., 1992). One gram of immobilized derivative was suspended in 10 mL of 25 mM sodium phosphate at pH 7.0. Then 0.1% (v/v) of formaldehyde was added to the suspension and this was very gently stirred for 3 h at 25 ◦ C. The end point of this chemical modification was carried out by filtering the derivatives and by a further reduction made by using sodium borohydride at a concentration of 1 mg/mL in a solution of 100 mM sodium bicarbonate at pH 10. Modified and reduced derivatives were filtered and washed abundantly with 0.1 M sodium phosphate at pH 7.0 and then with distilled water. This preparation was termed as Gx-TLL-A-CH2 O. 2.2.5.2. Modification of Gx-TLL-A with dextran–aldehyde. Dextran (MW = 70,000 Da) with a 50% oxidation degree was used (Fuentes et al., 2004a). One gram of Gx-TLL-A was incubated with 10 mL of 30 mg/mL of dextran in 0.2 M sodium phosphate buffer at pH 7.0 and 4 ◦ C for 12 h. As an end point to the cross-linking reaction, the suspension was filtered, re-suspended and reduced by adding 100 mM sodium bicarbonate containing 1 mg/mL of sodium borohydride at pH 10.05 and 4 ◦ C. After reduction, the preparations were modified with formaldehyde as previously described. The biocatalysts modified with dextran–aldehyde were termed as Gx-TLL-A-Dx. 2.2.5.3. Modification of Gx-TLL-A with dextran–aldehyde–glycine. Preparation of dextran–aldehyde–glycine: Dextran (MW = 70,000 Da) with a 50% oxidation degree was used (Fuentes et al., 2004a). The protocol for dextran modification was similar to that previously described to prepare dextran–aspartic (Fuentes et al., 2004b).
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The dextran–aldehyde was mixed with an equal volume of 3 M glycine at pH 7.5, and solid trimethylaminoborane was added to a concentration of 200 mM. The amino groups of the glycine reacted with the aldehyde groups in the dextran. After 15 h, the reaction mixture was reduced (to stabilize the Schiff’s bases formed and destroy any remaining aldehyde) by the addition of 500 mM sodium carbonate buffer pH 10.5 containing 100 mg/mL of sodium borohydride. This mixture was incubated for 30 min at room temperature and pH of the mixture was lowered to pH 6 using hydrochloric acid to destroy the sodium borohydride. The dextran was modified with glycine and reduced, then it was dialyzed against ultrapure water. The dextran–glycine was oxidized once again at 50% with periodate, and it was dialyzed 10 times against distilled water with a 1:10 ratio to produce dextran–aldehyde–glycine. Coating with dextran–aldehyde–glycine: One gram of Gx-TLL-A was incubated with 10 mL of a solution containing 30 mg/mL of dextran–aldehyde–glycine in 0.2 M sodium phosphate at pH 7.0 and 4 ◦ C for 12 h. As an end point to the cross-linking reaction, the suspension was filtered and re-suspended in 100 mM sodium bicarbonate pH 10.05 at 4 ◦ C containing 1 mg/mL of sodium borohydride. After reduction, the preparations were modified with formaldehyde as previously described. The preparation was termed as Gx-TLL-A-Dx-Gly. 2.2.6. Titration of the amino groups using picrylsulfonic acid Primary amines residues were titrated using the picrylsulfonic acid methodology (Snyder and Sobocinski, 1975). One hundred milligrams of the desired TLL immobilized preparation was suspended in 0.4 mL of 100 mM sodium bicarbonate at pH 9. The suspension was incubated at 25 ◦ C, and 0.1 mL of picrylsulfonic acid (5%, w/w solution) was added; after 10 min, the coloured derivatives were filtered and washed with a saturated NaCl solution, distilled water, and with 100 mM sodium bicarbonate at pH 9. A total of 50 mg of the coloured preparations were then re-suspended in 2 mL of 100 mM sodium bicarbonate, pH 9, and their spectra were determined. As control, the enzyme-free support was treated in a similar way. 2.2.7. Inactivation of the different TLL preparations For all inactivation/reactivation steps a relation of 1:10 (g of support/mL of solution) was used. 2.2.7.1. Incubation of TLL preparations on chaotropic agent solutions. The different TLL preparations were incubated in 50 mM sodium phosphate containing 8 M of guanidine hydrochloride at pH 7.0 and 25 ◦ C for different times. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension, and the remaining activity of the immobilized enzyme was determined using the full inactivating suspension. When the enzyme activity did not decrease for a further 30 min period of incubation, the immobilized enzyme was filtered and washed with buffer to eliminate the chaotropic reagent and incubated in buffer solutions as described below. 2.2.7.2. Inactivation of different TLL immobilized preparations in the presence of organic cosolvent. TLL preparations were washed two times with 10 volumes of 95% dioxane/5% 50 mM Tris–HCl at pH 7 and 4 ◦ C. Subsequently, the enzyme derivatives were re-suspended in the same solution and incubated at 25 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension. The activity was measured using the pNPB assay described above in the presence or absence of CTAB by adding to the reaction mixture all the components of the inactivating suspension. Initial activity was determined in the absence or the presence of CTAB.
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R.C. Rodrigues et al. / Journal of Biotechnology 144 (2009) 113–119 Table 1 Colorimetric determination of primary amino groups by titration with TNBS. Derivatives
Amino groups (%)
Gx-TLL-A Gx-TLL-A-CH2 O Gx-TLL-A-Dx Gx-TLL-A-Dx-Gly
100 ± 5 0 55 ± 3 48 ± 3
For details, see Section 2.
2.2.7.3. Thermal inactivation of different TLL immobilized preparations. TLL preparations were incubated in 50 mM sodium phosphate at pH 7.0 and 70 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension. The activity was measured immediately using the pNPB assay described above in the presence or absence of CTAB. Initial activity was determined in the absence or the presence of CTAB. 2.2.8. Reactivation experiments Fully or partially inactivated TLL preparations were washed with buffer and re-suspended in 50 mM sodium phosphate at pH 7 and their activities were determined over the time. When a constant value of residual activity was observed, this was considered the maximum recovered activity. In some cases, several consecutive cycles of inactivation/reactivation of immobilized TLL were performed. The activity was measured using the pNPB assay described above in the presence or the absence of CTAB. Initial activity was determined in the absence or the presence of CTAB, and compared to the activity obtained in the presence or the absence of this detergent. 3. Results and discussion 3.1. Modification of Gx-TLL-A The modification of Gx-TLL-A with formaldehyde, dextran–aldehyde or dextran–aldehyde–glycine under the conditions described in Section 2 did not present any effect on the enzyme activity (recovered activity was over 95% in all cases), perhaps due to the high activity and stability of this immobilized enzyme. This lack of effect on the enzyme activity when the enzyme was modified with the polymer suggested that it did not cause any diffusion problems. In fact, the activity/substrate profiles of the modified and non-modified enzymes were identical, although the solubility of pBPB did not permit to determine the Km and Vmax (results not shown). This may be related to the small size of the substrate used and the very open structure of the polymer. In fact, even when modifying some enzymes with a shell formed by several layers of polymers, trying to fully coat the whole enzyme surface, the substrate diffusion problems were not so significantly affected as to altering the enzyme kinetics (Fernandez-Lafuente et al., 1999). The quantification of the remaining primary amino groups of the enzyme confirms that the modification of the enzyme with formaldehyde allowed the transformation of all the exposed primary amino groups of the enzyme into secondary amino groups (Table 1). Modification with dextran produced a decrease of the content of primary amino groups by around 50%. The remaining primary amino groups were eliminated by incubation with formaldehyde. 3.2. Effect of the chemical modifications on Gx-TLL-A stability Fig. 1 shows the inactivation course of the different TLL preparations at pH 7 and 70 ◦ C. The chemical modification with
Fig. 1. Inactivation courses of different TLL immobilized preparations by incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-CH2 O; () Gx-TLL-A-Dx; () Gx-TLL-A-DxGly. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts.
formaldehyde allowed to marginally improving the enzyme stability. The modification with dextran–aldehyde increased the stabilization (half-life is extended by a 2-fold factor) and dextran–aldehyde–glycine presented the most significant enzyme stabilization (by a 4-fold factor). Fig. 2 shows the inactivation in the presence of 95% dioxane. Again, all modifications seemed to improve the enzyme stability, being Gx-TLL-A-Dx-Gly the most stable preparation. The improvement in the enzyme stability could be due to the generation of some steric hindrances to the enzyme chain movements produced by the polymer, or to the prevention of chemical modification of the primary amino groups of the enzyme (Daniel et al., 1996). In organic solvents, the hydrophilic polymer may also produce some partition of the organic solvents away from the enzyme surface, thus improving enzyme stability, although usu-
Fig. 2. Inactivation courses of different TLL immobilized preparations by incubation in the presence of 95% of dioxane at pH 7 and 25 ◦ C. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-CH2 O; () Gx-TLL-A-Dx; () Gx-TLL-A-DxGly. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts.
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Fig. 3. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations by successive incubation in guanidine and 50 mM sodium phosphate at pH 7 and 25 ◦ C. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-Dx-Gly. The arrows show the times when the enzyme preparations were incubated in guanidine or aqueous buffer.
ally for this effect it is necessary to fully coat the enzyme surface (Fernandez-Lafuente et al., 1999). As the Gx-TLL-A-Dx-Gly presented the best prospects after these studies, this TLL immobilized preparation was used in all further analysis. 3.3. Recovering of the activity of different preparations of TLL after incubation in saturated guanidine The incubation of the immobilized TLL in saturated guanidine is expected to destroy all the interactions that keep the enzyme conformation (Rehaber and Jaenicke, 1992; Zhi et al., 1992). If this unfolded protein is re-incubated in aqueous buffer, it is likely to reobtain the active structure of the enzyme (Rodrigues et al., 2009c,d). Thus, Gx-TLL-A incubated in 8 M guanidine lost most of its activity in a few minutes. After re-incubation in aqueous buffer, it recovered around 80% of the initial enzyme activity during 3 consecutive cycles of inactivation–reactivation (Fig. 3). As discussed in Section 1, using CTAB during activity determination, the recovered activity was 100% (Rodrigues et al., 2009c). This suggested that to regain the correct opening mechanism of the lipase lid could be the main obstacle in the TLL reactivation process. Gx-TLL-A-Dx-Gly also became almost fully inactivated by incubation in saturated guanidine solutions. However, Gx-TLL-ADx-Gly preparation recovered 100% of the initial activity during 3 consecutive cycles even in the absence of CTAB. This result suggested that, effectively, the modification with this very hydrophilic polymer can help to recover the enzyme native conformation, even in the case of these extremely complex enzymes, where there are large movements between open and closed forms during catalysis. One likely explanation of this better enzyme reactivation may be the generation of steric hindrances to the movements of the enzyme groups near the surface, which makes that the inactivated enzyme structure had to be fairly similar to the native one. Other possible hypothesis to explain these results may be that the very high hydrophilicity of the polymer near to the surface of the enzyme may help to fix the positions of the hydrophobic (inside) and hydrophilic (outside) groups of
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Fig. 4. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations by incubating in organic solvents or 50 mM sodium phosphate at pH 7 and 25 ◦ C. Other specifications are described in Section 2. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Dashed line: activity changes in time caused by the incubation in the presence of 95% of dioxane at pH 7 and 25 ◦ C. Solid line: activity evolution of the previously inactivated TL after washing with buffer and re-incubation in 50 mM sodium phosphate at pH 7 and 25 ◦ C. () GxTLL-A; () Gx-TLL-A-Dx-Gly. The arrows indicate the times when the immobilized enzyme was passed to aqueous buffer.
the enzyme by strong positive or negative interactions with the polymer. 3.4. Recovering of the activity of different TLL preparations partially inactivated by incubation in organic solvents Gx-TLL-A incubated in 95% dioxane may be expected to reach some incorrect structure that leads to enzyme inactivation (thus, inactivation is different than that produced by guanidine). In fact, it has been described that the incubation of these partially inactivated preparations by incubation in dioxane, when further incubated in 8 M guanidine to destroy the incorrect structures of the enzyme, allowed to recover 80% activity for several cycles, even when determined in the absence of CTAB (Rodrigues et al., 2009c). However, direct incubation in aqueous buffer yielded lower activity recoveries (Fig. 4), that decreased in each reactivation cycle (from 75% in the first cycle to 65% in the third cycle). Using Gx-TLL-A-Dx-Gly, the activity was fully and rapidly recovered. During 3 cycles the biocatalyst recovered 100% activity after re-incubation in aqueous buffer (Fig. 4). This result shows the very good prospects of this biocatalyst to be used in organic solvents medium: not only it is very stable, but may also recover all its activity after partial inactivation by incubation during just 5 h in aqueous buffer. 3.5. Recovering of the activity of different TLL preparations after partial inactivation by incubation at high temperatures Fig. 5 shows that, Gx-TLL-A-Dx-Gly recovered some more activity than Gx-TLL-A after thermal inactivation but the difference was not very significant. The recovered enzyme activity decreased after each cycle; in fact only 40% of the activity was recovered in the last cycle. The incubation of this partially inactivated derivative in 8 M guanidine did not improve the recovered activity (results not shown). To study the possibilities of enzyme reactivation, we decided to measure the activity in the presence of CTAB, compared to the ini-
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opening mechanism of the lipase lid, and, if this could be achieved, reactivation was almost complete. 4. Conclusions
Fig. 5. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations caused by incubation at high temperature/incubation at 25 ◦ C. Other specifications are described in Section 2. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Dashed line: TLL-activity evolution caused by incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Solid line: activity evolution during incubation in aqueous buffer at 25 ◦ C and pH 7. () Gx-TLL-A; () Gx-TLL-ADx-Gly. The arrows indicate the moment of incubation at 25 ◦ C.
tial activity also measured in CTAB (Fig. 6). This detergent produced a very significant hyperactivation of the immobilized enzyme, perhaps by stabilizing the open form of the enzyme (FernándezLorente et al., 2006), by around a 7-fold factor. Results were, in this case, significantly better using Gx-TLL-A-Dx-Gly than using GxTLL-A, with almost 100% activity recovering in the first cycle and near to 90% in the third cycle (versus 65 and 50% using the nonmodified preparation). Again, this suggested that the main problem of the reactivation of immobilized TLL was to regain the correct
Coating the immobilized TLL-A with a very hydrophilic dextran–aldehyde–glycine polymer (with the same amount of cationic and anionic groups) presented a very positive effect on the properties of the enzyme. The chemical modification of the immobilized enzyme has not effect on the enzyme activity. Moreover, the modification showed some very positive effects on the enzyme operational stability. First, the modified enzyme presented higher stability at high temperatures or in the presence of organic solvents. Second, the new derivative activity may be easy and fully recovered after inactivation by incubation in organic solvents or saturated guanidine by simple re-incubation in aqueous buffers for some hours, while the non-modified enzyme cannot be fully reactivated. In the case of inactivation by high temperature, the reactivation is more complex and only if CTAB is added to the assay buffer (conditions that help to the opening of the enzyme) the reactivation is really significant. This might be due to the existence of some chemical modifications of the enzyme (modifications that did not involve primary amino groups) (Daniel et al., 1996) or to the production of very stable but wrong structures that cannot spontaneously be reverted. The positive effects of this strategy may be related to the high hydrophilicity of the polymer that, together with some steric hindrances to the movement of the enzyme groups attached to the polymer, may help to keep in the enzyme surface its hydrophilic groups (that will be “partitioned” towards this hydrophilic shell) and to keep inside the protein the hydrophobic groups (that will be “partitioned” from this very hydrophilic environment). This should be similar to the salting out/salting in effects reported for soluble enzymes and soluble compounds for many proteins (Ikegaya, 2005). Acknowledgments The authors gratefully recognize the support from the Spanish CICYT (projects BIO-2005-8576 and CTQ2009-07568) and CAM (project S0505/PPQ/0344). The authors wish to thank CAPES (Brazil) for the scholarships of Dr. Rafael Costa Rodrigues and Dr. Giandra Volpato, the Spanish MEC for the PhD fellowship of Mr. Godoy and CAM for a PhD fellowship for Mr Bolivar. Dr. Ángel Berenguer (Universidad de Alicante) is kindly acknowledged for his help during the writing of this paper. References
Fig. 6. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations caused by incubation at high temperature/incubation at 25 ◦ C. All the measures were performed in the presence of CTAB. 100% activity corresponds to 350 IU/g of derivative for all biocatalysts. Dashed line: effect on the TLL activity of the incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Solid line: effect on the activity of thermally inactivated TLL preparations of their incubation in aqueous buffer at 25 ◦ C and pH 7. () Gx-TLL-A; () Gx-TLL-A-Dx-Gly. The arrows indicate the moment of incubation at 25 ◦ C.
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R.C. Rodrigues et al. / Journal of Biotechnology 144 (2009) 113–119 Fernandez-Lafuente, R., Rosell, C.M., Caanan-Haden, L., Rodes, L., Guisan, J.M., 1999. Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives: dramatic stabilization of penicillin acylase versus organic solvents. Enzyme Microb. Technol. 24, 96–103. Fernández-Lafuente, R., Rosell, C.M., Guisán, J.M., 1998. The presence of methanol exerts a strong and complex modulation of the synthesis of different antibiotics by immobilized Penicillin G acylase. Enzyme Microb. Technol. 23, 305–310. Fernández-Lorente, G., Palomo, J.M., Mateo, C., Munilla, R., Ortiz, C., Cabrera, Z., Guisán, J.M., Fernández-Lafuente, R., 2006. Glutaraldehyde cross-linking of lipases adsorbed on aminated supports in the presence of detergents leads to improved performance. Biomacromolecules 7, 2610–2615. Fuentes, M., Segura, R.L., Abian, O., Betancor, L., Hidalgo, A., Mateo, C., Fernandez-Lafuente, R., Guisan, J.M., 2004a. Determination of protein–protein interactions through aldehyde–dextran intermolecular cross-linking. Proteomics 4, 2602–2607. Fuentes, M., Mateo, C., García, L., Tercero, J.C., Guisán, J.M., Fernández-Lafuente, R., 2004b. Directed covalent immobilization of aminated DNA probes on aminated plates. Biomacromolecules 5, 883–888. Guncheva, M., Zhiryakova, D., Radchenkova, N., Kambourova, M., 2007. Effect of nonionic detergents on the activity of a thermostable lipase from Bacillus stearothermophilus MC7. J. Mol. Catal. B: Enzym. 49, 88–91. Hoare, D.G., Olson, A., Koshland Jr., D.E., 1968. The reaction of hydroxamic acids with water-soluble carbodiimides. A Lossen rearrangement. J. Am. Chem. Soc. 90, 1638–1643. Ikegaya, K., 2005. Kinetic analysis about the effects of neutral salts on the thermal stability of yeast alcohol dehydrogenase. J. Biochem. 137, 349–354. Iyer, P.V., Ananthanarayan, L., 2008. Enzyme stability and stabilization: aqueous and non-aqueous environment. Process Biochem. 43, 1019–1032. Kim, P.Y., Pollard, D.J., Woodley, J.M., 2007. Substrate supply for effective biocatalysis. Biotechnol. Prog. 23, 74–82. Mateo, C., Abian, O., Ernedo, M.B., Cuenca, E., Fuentes, M., Fernandez-Lorente, G., Palomo, J.M., Grazu, V., Pessela, B.C.C., Giacomini, C., Irazoqui, G., Villarino, A., Ovsejevi, K., Batista-Viera, F., Fernandez-Lafuente, R., Guisan, J.M., 2005. Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb. Technol. 37, 456–462. Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente, R., 2007. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 40, 1451–1463. Miled, N., Beisson, F., De Caro, J., De Caro, A., Arondel, V., Verger, R., 2001. Interfacial catalysis by lipases. J. Mol. Catal. B: Enzym. 11, 165–171. Mogensen, J.E., Sehgal, P., Otzen, D.E., 2005. Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents. Biochemistry 44, 1719–1730. Palomo, J.M., Fuentes, M., Fernandez-Lorente, G., Mateo, C., Guisan, J.M., FernandezLafuente, R., 2003. General trend of lipase to self-assemble giving bimolecular
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aggregates greatly modifies the enzyme functionality. Biomacromolecules 4, 1–6. Polizzi, K.M., Bommarius, A.S., Broering, J.M., Chaparro-Riggers, J.F., 2007. Stability of biocatalysts. Curr. Opin. Chem. Biol. 11, 220–225. Rehaber, V., Jaenicke, R., 1992. Stability and reconstitution of d-glyceraldehyde-3phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima. J. Biol. Chem. 267, 10999–11006. Rodrigues, R.C., Godoy, C.A., Volpato, G., Ayub, M.A.Z., Fernandez-Lafuente, R., Guisan, J.M., 2009a. Immobilization–stabilization of the lipase from Thermomyces lanuginosus: critical role of chemical amination. Process Biochem. 44, 963–968. Costa Rodrigues, R., Volpato, G., Wada, K., Záchia Ayub, M.A., 2009b. Improved enzyme stability in lipase-catalyzed synthesis of fatty acid ethyl ester from soybean oil. Appl. Biochem. Biotechnol. 152, 394–404. Rodrigues, R.C., Bolivar, J.M., Palau-Ors, A., Volpato, G., Ayub, M.A.Z., FernandezLafuente, R., Guisan, J.M., 2009c. Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus. Enzyme Microb. Technol. 44, 386– 393. Rodrigues, R.C., Godoy, C.A., Filice, M., Bolivar, J.M., Palau-Ors, A., Garcia-Vargas, J.M., Romero, O., Wilson, L., Ayub, M.A.Z., Fernandez-Lafuente, R., Guisan, J.M., 2009d. Reactivation of covalently immobilized lipase from Thermomyces lanuginosus. Process Biochem. 44, 641–646. Romero, O., Vergara, J., Fernández-Lafuente, R., Guisán, J.M., Illanes, A., Wilson, L., 2009. Simple strategy of reactivation of a partially inactivated penicillin G acylase biocatalyst in organic solvent and its impact on the synthesis of -lactam antibiotics. Biotechnol. Bioeng. 103, 472–479. Rosell, C.M., Terreni, M., Fernandez-Lafuente, R., Guisan, J.M., 1998. A criterion for the selection of monophasic solvents for enzymatic synthesis. Enzyme Microb. Technol. 23, 64–69. Sarmento, A.C., Oliveira, C.S., Pereira, A., Esteves, V.I., Moir, A.J.G., Saraiva, J., Pires, E., Barros, M., 2009. Unfolding of cardosin A in organic solvents and detection of intermediaries. J. Mol. Catal. B: Enzym. 57, 115–122. Sheldon, R.A., 2007. Enzyme immobilization: the quest for optimum performance. Adv. Synth. Catal. 349, 1289–1307. Snyder, S.L., Sobocinski, P.Z., 1975. An improved 2,4,6 trinitrobenzenesulfonic acid method for the determination of amines. Anal. Biochem. 64, 284–288. Turan, S., Karabulut, I., Vural, H., 2006. Effects of reaction parameters on the incorporation of caprylic acid into soybean oil for production of structured lipids. J. Food Lipids 13, 306–317. Yeates, C.A., Krieg, H.M., Breytenbach, J.C., 2007. Hydroxypropyl--cyclodextrin induced complexation for the biocatalytic resolution of a poorly soluble epoxide. Enzyme Microb. Technol. 40, 228–235. Zhi, W., Landry, S.J., Gierasch, L.M., Srere, P.A., 1992. Renaturation of citrate synthase: influence of denaturant and folding assistants. Protein Sci. 1, 522–529.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers Rafael C. Rodrigues, Juan M. Bolivar, Giandra Volpato, Marco Filice, Cesar Godoy, Roberto Fernandez-Lafuente ∗ , Jose M. Guisan ∗ Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 23 February 2009 Received in revised form 27 August 2009 Accepted 1 September 2009 Keywords: Chemical modification Enzyme reactivation Hydrophilic polymers Solid phase Lipase Enzyme stabilization
a b s t r a c t Immobilized-stabilized aminated lipase from Thermomyces lanuginosus (TLL-A) is not easily reactivated after inactivation by incubation in the presence of organic solvents or chaotropic reagents. To improve the recovered activity of this biocatalyst, immobilized TLL-A has been submitted to different modifications. The best results were obtained when the enzyme was coated with a very hydrophilic and inert polymer: dextran modified with glycine (Dx-Gly). This modification did not reduce enzymatic activity while it increased the stability of this already very stable preparation, in thermal and organic solvent induced inactivation (by a 4-fold factor). Simple incubation in aqueous medium at pH 7 and 25 ◦ C permitted to fully recover the activity of the immobilized and modified TLL-A enzyme inactivated by incubation in organic solvents or saturated guanidine during 3 cycles, while the non-modified enzyme only recover some activity. When the inactivation was caused by exposition at high temperatures, the reactivation was higher using the modified biocatalyst, but was far for complete (40% after 3 inactivation–reactivation cycles). The determination of the TLL-A activity in the presence of detergents (that helps the opening of active site of the lipase) allowed, in this case, to significantly improve the results, now near to 90% of the initial activity was recovered (using the non-modified enzyme the recovered activity was around 60%). This very hydrophilic and inert polymer, coating the enzyme surface, seems to help the correct positioning of the hydrophilic and hydrophobic groups of the enzyme, and that way improve both the stability and possibility of reactivation of the enzyme. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Enzymes are very interesting biocatalysts, potentially useful as catalyst in many biotransformations (Bornscheuer and Kazlauskas, 2006). One of the key problems of enzymes as industrial biocatalysts is their moderate stability under industrially relevant conditions (Iyer and Ananthanarayan, 2008), such as moderately high temperatures, or presence of solvents, used to increase the solubility of substrates or products (Kim et al., 2007; Yeates et al., 2007), to shift thermodynamic equilibrium (Rosell et al., 1998), to improve enzyme properties (Fernández-Lafuente et al., 1998), or even to prevent microbial contamination. Thus, much effort has been paid to the development of strategies that may increase the stability of the enzymes by diverse techniques (Iyer and Ananthanarayan, 2008; Polizzi et al., 2007). Immobilization is a requisite for many industrial applications of enzymes (Cao, 2005; Sheldon, 2007), and in many cases may be coupled to the stabi-
∗ Corresponding authors. Tel.: +34 91 585 4809; fax: +34 91 585 4760. E-mail addresses: rfl@icp.csic.es, [email protected] (R. Fernandez-Lafuente), [email protected] (J.M. Guisan). 0168-1656/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.09.002
lization of the enzyme if a multipoint and multisubunit covalent attachment is achieved (Mateo et al., 2007). On the other hand, if the enzyme may be partially or fully reactivated after its inactivation, this may enlarge the operational half-life of the catalyst and, this way; it may improve the profitability of the process. In many cases, enzyme activity is lost just by the promotion of wrong enzyme conformations (Sarmento et al., 2009), in some other cases some chemical modification of different protein groups may occur (Daniel et al., 1996). It has been shown that multipoint covalent attachment of enzymes on finally inert supports (e.g., glyoxyl agarose) (Mateo et al., 2005) may help the reactivation of partially inactivated enzymes, because these multiple enzyme-support bonds may behave as reference points, easing the recovery of enzyme activity (Romero et al., 2009). In some instances, a significant enzyme activity recovery may be achieved just by incubating the enzyme under mild conditions after their inactivation (Rodrigues et al., 2009c,d; Romero et al., 2009). However, in other examples, enzyme reactivation requires the full unfolding of the protein structure to eliminate incorrect structures, and its further refolding under controlled conditions (Rehaber and Jaenicke, 1992; Zhi et al., 1992).
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Lipase from Thermomyces lanuginosus (TLL) is the enzyme responsible for the lipolytic activity of Lipolase® , a commercial lipase preparation supplied by Novozymes. This enzyme has been broadly used in many biotransformations (Rodrigues et al., 2009b; Turan et al., 2006). Its structure has been solved at 1.8 Å (Derewenda et al., 1994). In homogenous medium, lipases present a large flap or lid that isolates the active site from the reaction medium (Brzozowski et al., 1991). In the presence of a hydrophobic interface (e.g., an oil drop (Miled et al., 2001), a hydrophobic support (Fernandez-Lafuente et al., 1998), a hydrophobic protein (Palomo et al., 2003)), the lid moves, and the active site of the lipase is exposed to the medium. TLL has been recently stabilized by coupling the chemical amination of the enzyme surface to the multipoint covalent attachment of the modified enzyme on glyoxyl-agarose beads (Rodrigues et al., 2009a). The amination of the enzyme was necessary to highly stabilize TLL by its immobilization to glyoxyl support, and did not affect either the enzyme stability or its activity. This multipoint immobilized preparation also recovered a higher percentage of activity, when submitted to different reactivation strategies, compared to a derivative of non-modified TLL immobilized on cyanogen bromomide activated agarose (Rodrigues et al., 2009c). If the enzyme activity was measured in the presence of detergents (during inactivation or reactivation experiments) the enzyme inactivation rate was slower and the observed enzyme reactivation was higher. Detergents may help to stabilize the open form of lipases (Fernández-Lorente et al., 2006; Guncheva et al., 2007; Mogensen et al., 2005). In this paper, we try to advance some further steps on the reactivation of this TLL biocatalyst, using chemical modification on solid phase of the immobilized-stabilized enzyme (see Scheme 1) to help the recovering of activity after partial inactivation of the preparation. The modification of the primary amino groups of the protein was performed with formaldehyde; this will yield stable secondary amino groups. The enzyme surface has been also modified with very hydrophilic and inert polymers (dextran–aldehyde or dextran–aldehyde–glycine). These polymers are random coils and can hardly increase the rigidity of the enzyme, even if an intense cross-link is achieved. Nevertheless, they can produce several positive effects: (i) generate steric hindrances to the movements of the enzyme groups placed near to the protein surface; (ii) help to position hydrophobic groups and hydrophilic groups by generation a kind of “partition” by the high hydrophilic polymer (FernandezLafuente et al., 1999). The very open structure of these polymers permits that even when a multilayer of similar polymers is used to
fully coat the enzyme surface, the hindrances to the movements of small substrates are minimal (Fernandez-Lafuente et al., 1999). 2. Materials and methods 2.1. Materials Lipase from T. lanuginosus (TLL) was obtained from Novozymes (Denmark). 1-Ethyl-3-(dimethylaminopropyl) carbodiimide, pnitrophenyl butyrate (p-NPB) and hexadecyltrimethylammonium bromide (CTAB) were from Sigma. 1,4-Dioxane, guanidine hydrochloride, 2,4,6-trinitrobenzensulfonic acid (TNBS), formaldehyde (commercial 35% formaldehyde in water solution), and 1,2-ethylenediamine, were from Fluka. Octyl-Sepharose CL-4B was purchased from GE Healthcare (Uppsala, Sweden). Cross-linked agarose (10 BCL) was kindly donated by Hispanagar S.A. (Burgos, Spain) and its modification to glyoxyl agarose (activated with 200 mol/g of support) was performed as described elsewhere (Mateo et al., 2005). Other reagents and solvents used were of analytical or HPLC grade. 2.2. Methods The experiments were carried out at least by triplicate and the standard error was always under 5% represented by error bars in the figures. 2.2.1. Enzymatic activity assay Lipolytic activity assay was performed by measuring the increase in the absorbance at 348 nm produced by the released pnitrophenol in the hydrolysis of 0.4 mM pNPB in 25 mM sodium phosphate at pH 7 and 25 ◦ C, using a spectrophotometer with a thermostatized cell and with continuous magnetic stirring. To initialize the reaction, 0.05 mL of lipase solution or suspension were added to 2.5 mL of substrate solution. One international unit of pNPB activity was defined as the amount of enzyme necessary to hydrolyze 1 mol of pNPB/min (IU) under the conditions described above. In some instances, 0.01% of CTAB was added to the substrate solution. CTAB is able to hyperactivate Gx-TLL-A (see below), multiplying the enzyme activity by a 7-fold factor when used at 0.01%. 2.2.2. Purification of TLL TLL was purified by interfacial adsorption on hydrophobic supports. The enzyme was adsorbed on octyl-Sepharose beads under
Scheme 1. Chemical modifications of immobilized-stabilized TLL (Gx-TLL-A): (a) modification with formaldeyde and (b) modification with hydrophilic polymers dextran–aldehyde or dextran–aldehyde–Gly. In black amines from Lys residues, and in grey amines obtained by modification with 1,2-ethylenediamine (EDA).
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continuous stirring in 10 mM sodium phosphate at pH 7.0, according to a previously described procedure (Fernandez-Lafuente et al., 1998). The activities of suspensions and supernatants were periodically measured by using the pNPB assay. After enzyme adsorption, the lipase preparation was vacuum filtered using a sintered glass funnel and abundantly washed with distilled water. TLL was desorbed from octyl-Sepharose by suspending the immobilized enzyme in a 1/10 (w/v) ratio in 25 mM sodium phosphate at pH 7.0 containing 0.6% (v/v) of CTAB during 1 h at room temperature. Only one protein band was detected by SDS-PAGE (results not shown). 2.2.3. Chemical amination of immobilized TLL Chemical amination was performed on the TLL immobilized on octyl-Sepharose by using 1-ethyl-3-(dimethylaminopropyl) carbodiimide as carboxy activating reagent and 1,2-ethylenediamine at pH 4.75 as previously described (Rodrigues et al., 2009a). These reaction conditions have been reported as capable of fully modifying all exposed carboxylic groups of the protein (Hoare et al., 1968). The aminated TLL, termed TLL-A, immobilized on octyl-Sepharose was stored at 4 ◦ C. 2.2.4. Immobilization of TLL-A on glyoxyl-agarose beads Immobilized derivatives of TLL-A were prepared by using glyoxyl agarose (Gx-TLL-A), first immobilization was performed at pH 9 and further incubation at pH 10 to get an intense multipoint covalent attachment (using 60 IU of TLL/g of support). The full protocol is described in Rodrigues et al. (2009a). The final immobilized derivative, having around 2.5 mg of protein/g of support, had 50 IU/g of support, this moderate enzyme loading avoided the risks of diffusion limitations that could alter the enzyme performance. 2.2.5. Chemical modification of the amino groups of Gx-TLL-A 2.2.5.1. Chemical modification of Gx-TLL-A with formaldehyde. Modification with formaldehyde was performed according to a previously described methodology (Fernandez-Lafuente et al., 1992). One gram of immobilized derivative was suspended in 10 mL of 25 mM sodium phosphate at pH 7.0. Then 0.1% (v/v) of formaldehyde was added to the suspension and this was very gently stirred for 3 h at 25 ◦ C. The end point of this chemical modification was carried out by filtering the derivatives and by a further reduction made by using sodium borohydride at a concentration of 1 mg/mL in a solution of 100 mM sodium bicarbonate at pH 10. Modified and reduced derivatives were filtered and washed abundantly with 0.1 M sodium phosphate at pH 7.0 and then with distilled water. This preparation was termed as Gx-TLL-A-CH2 O. 2.2.5.2. Modification of Gx-TLL-A with dextran–aldehyde. Dextran (MW = 70,000 Da) with a 50% oxidation degree was used (Fuentes et al., 2004a). One gram of Gx-TLL-A was incubated with 10 mL of 30 mg/mL of dextran in 0.2 M sodium phosphate buffer at pH 7.0 and 4 ◦ C for 12 h. As an end point to the cross-linking reaction, the suspension was filtered, re-suspended and reduced by adding 100 mM sodium bicarbonate containing 1 mg/mL of sodium borohydride at pH 10.05 and 4 ◦ C. After reduction, the preparations were modified with formaldehyde as previously described. The biocatalysts modified with dextran–aldehyde were termed as Gx-TLL-A-Dx. 2.2.5.3. Modification of Gx-TLL-A with dextran–aldehyde–glycine. Preparation of dextran–aldehyde–glycine: Dextran (MW = 70,000 Da) with a 50% oxidation degree was used (Fuentes et al., 2004a). The protocol for dextran modification was similar to that previously described to prepare dextran–aspartic (Fuentes et al., 2004b).
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The dextran–aldehyde was mixed with an equal volume of 3 M glycine at pH 7.5, and solid trimethylaminoborane was added to a concentration of 200 mM. The amino groups of the glycine reacted with the aldehyde groups in the dextran. After 15 h, the reaction mixture was reduced (to stabilize the Schiff’s bases formed and destroy any remaining aldehyde) by the addition of 500 mM sodium carbonate buffer pH 10.5 containing 100 mg/mL of sodium borohydride. This mixture was incubated for 30 min at room temperature and pH of the mixture was lowered to pH 6 using hydrochloric acid to destroy the sodium borohydride. The dextran was modified with glycine and reduced, then it was dialyzed against ultrapure water. The dextran–glycine was oxidized once again at 50% with periodate, and it was dialyzed 10 times against distilled water with a 1:10 ratio to produce dextran–aldehyde–glycine. Coating with dextran–aldehyde–glycine: One gram of Gx-TLL-A was incubated with 10 mL of a solution containing 30 mg/mL of dextran–aldehyde–glycine in 0.2 M sodium phosphate at pH 7.0 and 4 ◦ C for 12 h. As an end point to the cross-linking reaction, the suspension was filtered and re-suspended in 100 mM sodium bicarbonate pH 10.05 at 4 ◦ C containing 1 mg/mL of sodium borohydride. After reduction, the preparations were modified with formaldehyde as previously described. The preparation was termed as Gx-TLL-A-Dx-Gly. 2.2.6. Titration of the amino groups using picrylsulfonic acid Primary amines residues were titrated using the picrylsulfonic acid methodology (Snyder and Sobocinski, 1975). One hundred milligrams of the desired TLL immobilized preparation was suspended in 0.4 mL of 100 mM sodium bicarbonate at pH 9. The suspension was incubated at 25 ◦ C, and 0.1 mL of picrylsulfonic acid (5%, w/w solution) was added; after 10 min, the coloured derivatives were filtered and washed with a saturated NaCl solution, distilled water, and with 100 mM sodium bicarbonate at pH 9. A total of 50 mg of the coloured preparations were then re-suspended in 2 mL of 100 mM sodium bicarbonate, pH 9, and their spectra were determined. As control, the enzyme-free support was treated in a similar way. 2.2.7. Inactivation of the different TLL preparations For all inactivation/reactivation steps a relation of 1:10 (g of support/mL of solution) was used. 2.2.7.1. Incubation of TLL preparations on chaotropic agent solutions. The different TLL preparations were incubated in 50 mM sodium phosphate containing 8 M of guanidine hydrochloride at pH 7.0 and 25 ◦ C for different times. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension, and the remaining activity of the immobilized enzyme was determined using the full inactivating suspension. When the enzyme activity did not decrease for a further 30 min period of incubation, the immobilized enzyme was filtered and washed with buffer to eliminate the chaotropic reagent and incubated in buffer solutions as described below. 2.2.7.2. Inactivation of different TLL immobilized preparations in the presence of organic cosolvent. TLL preparations were washed two times with 10 volumes of 95% dioxane/5% 50 mM Tris–HCl at pH 7 and 4 ◦ C. Subsequently, the enzyme derivatives were re-suspended in the same solution and incubated at 25 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension. The activity was measured using the pNPB assay described above in the presence or absence of CTAB by adding to the reaction mixture all the components of the inactivating suspension. Initial activity was determined in the absence or the presence of CTAB.
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R.C. Rodrigues et al. / Journal of Biotechnology 144 (2009) 113–119 Table 1 Colorimetric determination of primary amino groups by titration with TNBS. Derivatives
Amino groups (%)
Gx-TLL-A Gx-TLL-A-CH2 O Gx-TLL-A-Dx Gx-TLL-A-Dx-Gly
100 ± 5 0 55 ± 3 48 ± 3
For details, see Section 2.
2.2.7.3. Thermal inactivation of different TLL immobilized preparations. TLL preparations were incubated in 50 mM sodium phosphate at pH 7.0 and 70 ◦ C. Samples were periodically withdrawn using a pipette with a cut-tip and under vigorous stirring to have a homogeneous biocatalyst suspension. The activity was measured immediately using the pNPB assay described above in the presence or absence of CTAB. Initial activity was determined in the absence or the presence of CTAB. 2.2.8. Reactivation experiments Fully or partially inactivated TLL preparations were washed with buffer and re-suspended in 50 mM sodium phosphate at pH 7 and their activities were determined over the time. When a constant value of residual activity was observed, this was considered the maximum recovered activity. In some cases, several consecutive cycles of inactivation/reactivation of immobilized TLL were performed. The activity was measured using the pNPB assay described above in the presence or the absence of CTAB. Initial activity was determined in the absence or the presence of CTAB, and compared to the activity obtained in the presence or the absence of this detergent. 3. Results and discussion 3.1. Modification of Gx-TLL-A The modification of Gx-TLL-A with formaldehyde, dextran–aldehyde or dextran–aldehyde–glycine under the conditions described in Section 2 did not present any effect on the enzyme activity (recovered activity was over 95% in all cases), perhaps due to the high activity and stability of this immobilized enzyme. This lack of effect on the enzyme activity when the enzyme was modified with the polymer suggested that it did not cause any diffusion problems. In fact, the activity/substrate profiles of the modified and non-modified enzymes were identical, although the solubility of pBPB did not permit to determine the Km and Vmax (results not shown). This may be related to the small size of the substrate used and the very open structure of the polymer. In fact, even when modifying some enzymes with a shell formed by several layers of polymers, trying to fully coat the whole enzyme surface, the substrate diffusion problems were not so significantly affected as to altering the enzyme kinetics (Fernandez-Lafuente et al., 1999). The quantification of the remaining primary amino groups of the enzyme confirms that the modification of the enzyme with formaldehyde allowed the transformation of all the exposed primary amino groups of the enzyme into secondary amino groups (Table 1). Modification with dextran produced a decrease of the content of primary amino groups by around 50%. The remaining primary amino groups were eliminated by incubation with formaldehyde. 3.2. Effect of the chemical modifications on Gx-TLL-A stability Fig. 1 shows the inactivation course of the different TLL preparations at pH 7 and 70 ◦ C. The chemical modification with
Fig. 1. Inactivation courses of different TLL immobilized preparations by incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-CH2 O; () Gx-TLL-A-Dx; () Gx-TLL-A-DxGly. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts.
formaldehyde allowed to marginally improving the enzyme stability. The modification with dextran–aldehyde increased the stabilization (half-life is extended by a 2-fold factor) and dextran–aldehyde–glycine presented the most significant enzyme stabilization (by a 4-fold factor). Fig. 2 shows the inactivation in the presence of 95% dioxane. Again, all modifications seemed to improve the enzyme stability, being Gx-TLL-A-Dx-Gly the most stable preparation. The improvement in the enzyme stability could be due to the generation of some steric hindrances to the enzyme chain movements produced by the polymer, or to the prevention of chemical modification of the primary amino groups of the enzyme (Daniel et al., 1996). In organic solvents, the hydrophilic polymer may also produce some partition of the organic solvents away from the enzyme surface, thus improving enzyme stability, although usu-
Fig. 2. Inactivation courses of different TLL immobilized preparations by incubation in the presence of 95% of dioxane at pH 7 and 25 ◦ C. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-CH2 O; () Gx-TLL-A-Dx; () Gx-TLL-A-DxGly. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts.
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Fig. 3. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations by successive incubation in guanidine and 50 mM sodium phosphate at pH 7 and 25 ◦ C. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Other specifications are described in Section 2. () Gx-TLL-A; () Gx-TLL-A-Dx-Gly. The arrows show the times when the enzyme preparations were incubated in guanidine or aqueous buffer.
ally for this effect it is necessary to fully coat the enzyme surface (Fernandez-Lafuente et al., 1999). As the Gx-TLL-A-Dx-Gly presented the best prospects after these studies, this TLL immobilized preparation was used in all further analysis. 3.3. Recovering of the activity of different preparations of TLL after incubation in saturated guanidine The incubation of the immobilized TLL in saturated guanidine is expected to destroy all the interactions that keep the enzyme conformation (Rehaber and Jaenicke, 1992; Zhi et al., 1992). If this unfolded protein is re-incubated in aqueous buffer, it is likely to reobtain the active structure of the enzyme (Rodrigues et al., 2009c,d). Thus, Gx-TLL-A incubated in 8 M guanidine lost most of its activity in a few minutes. After re-incubation in aqueous buffer, it recovered around 80% of the initial enzyme activity during 3 consecutive cycles of inactivation–reactivation (Fig. 3). As discussed in Section 1, using CTAB during activity determination, the recovered activity was 100% (Rodrigues et al., 2009c). This suggested that to regain the correct opening mechanism of the lipase lid could be the main obstacle in the TLL reactivation process. Gx-TLL-A-Dx-Gly also became almost fully inactivated by incubation in saturated guanidine solutions. However, Gx-TLL-ADx-Gly preparation recovered 100% of the initial activity during 3 consecutive cycles even in the absence of CTAB. This result suggested that, effectively, the modification with this very hydrophilic polymer can help to recover the enzyme native conformation, even in the case of these extremely complex enzymes, where there are large movements between open and closed forms during catalysis. One likely explanation of this better enzyme reactivation may be the generation of steric hindrances to the movements of the enzyme groups near the surface, which makes that the inactivated enzyme structure had to be fairly similar to the native one. Other possible hypothesis to explain these results may be that the very high hydrophilicity of the polymer near to the surface of the enzyme may help to fix the positions of the hydrophobic (inside) and hydrophilic (outside) groups of
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Fig. 4. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations by incubating in organic solvents or 50 mM sodium phosphate at pH 7 and 25 ◦ C. Other specifications are described in Section 2. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Dashed line: activity changes in time caused by the incubation in the presence of 95% of dioxane at pH 7 and 25 ◦ C. Solid line: activity evolution of the previously inactivated TL after washing with buffer and re-incubation in 50 mM sodium phosphate at pH 7 and 25 ◦ C. () GxTLL-A; () Gx-TLL-A-Dx-Gly. The arrows indicate the times when the immobilized enzyme was passed to aqueous buffer.
the enzyme by strong positive or negative interactions with the polymer. 3.4. Recovering of the activity of different TLL preparations partially inactivated by incubation in organic solvents Gx-TLL-A incubated in 95% dioxane may be expected to reach some incorrect structure that leads to enzyme inactivation (thus, inactivation is different than that produced by guanidine). In fact, it has been described that the incubation of these partially inactivated preparations by incubation in dioxane, when further incubated in 8 M guanidine to destroy the incorrect structures of the enzyme, allowed to recover 80% activity for several cycles, even when determined in the absence of CTAB (Rodrigues et al., 2009c). However, direct incubation in aqueous buffer yielded lower activity recoveries (Fig. 4), that decreased in each reactivation cycle (from 75% in the first cycle to 65% in the third cycle). Using Gx-TLL-A-Dx-Gly, the activity was fully and rapidly recovered. During 3 cycles the biocatalyst recovered 100% activity after re-incubation in aqueous buffer (Fig. 4). This result shows the very good prospects of this biocatalyst to be used in organic solvents medium: not only it is very stable, but may also recover all its activity after partial inactivation by incubation during just 5 h in aqueous buffer. 3.5. Recovering of the activity of different TLL preparations after partial inactivation by incubation at high temperatures Fig. 5 shows that, Gx-TLL-A-Dx-Gly recovered some more activity than Gx-TLL-A after thermal inactivation but the difference was not very significant. The recovered enzyme activity decreased after each cycle; in fact only 40% of the activity was recovered in the last cycle. The incubation of this partially inactivated derivative in 8 M guanidine did not improve the recovered activity (results not shown). To study the possibilities of enzyme reactivation, we decided to measure the activity in the presence of CTAB, compared to the ini-
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opening mechanism of the lipase lid, and, if this could be achieved, reactivation was almost complete. 4. Conclusions
Fig. 5. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations caused by incubation at high temperature/incubation at 25 ◦ C. Other specifications are described in Section 2. 100% activity corresponds to 50 IU/g of derivative for all biocatalysts. Dashed line: TLL-activity evolution caused by incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Solid line: activity evolution during incubation in aqueous buffer at 25 ◦ C and pH 7. () Gx-TLL-A; () Gx-TLL-ADx-Gly. The arrows indicate the moment of incubation at 25 ◦ C.
tial activity also measured in CTAB (Fig. 6). This detergent produced a very significant hyperactivation of the immobilized enzyme, perhaps by stabilizing the open form of the enzyme (FernándezLorente et al., 2006), by around a 7-fold factor. Results were, in this case, significantly better using Gx-TLL-A-Dx-Gly than using GxTLL-A, with almost 100% activity recovering in the first cycle and near to 90% in the third cycle (versus 65 and 50% using the nonmodified preparation). Again, this suggested that the main problem of the reactivation of immobilized TLL was to regain the correct
Coating the immobilized TLL-A with a very hydrophilic dextran–aldehyde–glycine polymer (with the same amount of cationic and anionic groups) presented a very positive effect on the properties of the enzyme. The chemical modification of the immobilized enzyme has not effect on the enzyme activity. Moreover, the modification showed some very positive effects on the enzyme operational stability. First, the modified enzyme presented higher stability at high temperatures or in the presence of organic solvents. Second, the new derivative activity may be easy and fully recovered after inactivation by incubation in organic solvents or saturated guanidine by simple re-incubation in aqueous buffers for some hours, while the non-modified enzyme cannot be fully reactivated. In the case of inactivation by high temperature, the reactivation is more complex and only if CTAB is added to the assay buffer (conditions that help to the opening of the enzyme) the reactivation is really significant. This might be due to the existence of some chemical modifications of the enzyme (modifications that did not involve primary amino groups) (Daniel et al., 1996) or to the production of very stable but wrong structures that cannot spontaneously be reverted. The positive effects of this strategy may be related to the high hydrophilicity of the polymer that, together with some steric hindrances to the movement of the enzyme groups attached to the polymer, may help to keep in the enzyme surface its hydrophilic groups (that will be “partitioned” towards this hydrophilic shell) and to keep inside the protein the hydrophobic groups (that will be “partitioned” from this very hydrophilic environment). This should be similar to the salting out/salting in effects reported for soluble enzymes and soluble compounds for many proteins (Ikegaya, 2005). Acknowledgments The authors gratefully recognize the support from the Spanish CICYT (projects BIO-2005-8576 and CTQ2009-07568) and CAM (project S0505/PPQ/0344). The authors wish to thank CAPES (Brazil) for the scholarships of Dr. Rafael Costa Rodrigues and Dr. Giandra Volpato, the Spanish MEC for the PhD fellowship of Mr. Godoy and CAM for a PhD fellowship for Mr Bolivar. Dr. Ángel Berenguer (Universidad de Alicante) is kindly acknowledged for his help during the writing of this paper. References
Fig. 6. Three consecutive inactivation/reactivation cycles of different TLL immobilized preparations caused by incubation at high temperature/incubation at 25 ◦ C. All the measures were performed in the presence of CTAB. 100% activity corresponds to 350 IU/g of derivative for all biocatalysts. Dashed line: effect on the TLL activity of the incubation in 50 mM sodium phosphate at 70 ◦ C and pH 7. Solid line: effect on the activity of thermally inactivated TLL preparations of their incubation in aqueous buffer at 25 ◦ C and pH 7. () Gx-TLL-A; () Gx-TLL-A-Dx-Gly. The arrows indicate the moment of incubation at 25 ◦ C.
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