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Advanced Synthesis & Catalysis 11/2011; 353(16):2885 - 2904. · 6.05 Impact Factor
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Advanced Synthesis & Catalysis 07/2011; 353(13):2216 - 2238. · 6.05 Impact Factor
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ABSTRACT: Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.
Biotechnology Progress 05/2011; 27(3):717-23. · 2.34 Impact Factor
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ABSTRACT: 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 degrees 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.
Journal of biotechnology 10/2009; 144(2):113-9. · 2.88 Impact Factor
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ABSTRACT: The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17 under different oxygen volumetric mass transfer coefficients (kLa) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were
conducted at different kLa (26, 38, 50, and 83 h−1) and pH values (6.0, 7.0, and 8.0). The optimal kLa and pH were 38 h−1 and 7.0, respectively. Under these conditions, the maximal cell production obtained was 8.0 g/L, and the volumetric and specific
lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation.
This result was approximately five times higher than that obtained in the shake flask cultures. The relationship between cell
growth and lipase production was found to be associated with growth by the Luedeking-Piret model.
Biotechnology and Bioprocess Engineering 01/2009; 14(1):105-111. · 1.28 Impact Factor
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ABSTRACT: The lipase from Rhizomucor miehei (formerly Mucor miehei) (RML) is a commercially available enzyme in both soluble and immobilized form with very high activity and good stability under diverse conditions (anhydrous organic solvents, supercritical fluids, etc.). Although this lipase was initially produced to be used in food industry, in this review we will focus our attention on the application of this enzyme in organic chemistry, from biodiesel production to fine chemicals (mainly in enantio or regioselective or specific processes). After showing the enzyme features, some of the most efficient methods of RML immobilization will be commented (entrapping on reverse micelles, preparation of cross-linked RML aggregates or immobilization on pre-existing solids). Finally, the main uses of the enzyme in organic chemistry will be revised. The use of RML in the production of biodiesel will be analyzed, and compared to the performance of other lipases. The synthesis of esters of carboxylic acids as flavors is other example where RML has been successfully employed. Taking advantage of the wide specificity of the enzyme, mainly a high enantiospecificity, many examples of the use of RML in the resolution of racemic mixtures of chiral carboxylic acids, alcohols or esters will be presented. Special mention requires the use of the regioselectivity of RML, mainly the chemistry of sugars. Finally, more unusual uses of RML will be presented (anomalous substrates, novel uses, etc.). In general, this enzyme seems very adequate for esterification reactions due to its high stability in anhydrous media and good esterification activity.
Journal of Molecular Catalysis B: Enzymatic.
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ABSTRACT: First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 °C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity.
Process Biochemistry.
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ABSTRACT: In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.
Process Biochemistry.
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ABSTRACT: Highly activated glyoxyl-supports rapidly immobilize proteins at pH 10 (where the ɛ-amino groups of the Lys groups of the protein surface are very reactive), and stabilize them by multipoint covalent attachment. However, they do not immobilize proteins at pH 8. This paper shows that the enzyme immobilization at this mild pH value is possible by incubation of the enzymes in the presence of different thiolated compounds (dithiothreitol, DTT; was selected as optimal reagent). The thiolated compounds (even the not reducing ones) stabilized the imino bonds formed at pH 8 between the aldehydes in the support and the amino groups of the protein. However, thiolated compounds are unable to reduce the imino bonds or the aldehyde groups and a final reduction step (e.g., using sodium borohydride) was always necessary. After enzyme immobilization through the most reactive amino group of the protein, the further incubation of this immobilized enzyme at pH 10 would improve the reactivity of the ɛ-amino groups of the Lys residues of the protein surface. Then, an intense multipoint covalent reaction of the enzyme with the dense layer of glyoxyl groups in the support could be obtained, increasing the stability of the immobilized enzyme. Using three different industrially relevant enzymes (penicillin G acylase from Escherichia coli (PGA), lipase from Bacillus thermocatenulatus (BTL2) and glutaryl acylase from Pseudomonas sp. (GA)), new immobilized-stabilized biocatalysts of the enzymes were produced. After reduction, the preparations incubated at pH 10 were more stable than those that were only immobilized and reduced at pH 8. In the case of the PGA, this preparation was even 4–5-fold more stable than those obtained by direct immobilization at pH 10 (around 40,000–50,000-fold more stable than the soluble enzyme).
Enzyme and Microbial Technology.
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ABSTRACT: This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.
Process Biochemistry. 44(9):963-968.
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ABSTRACT: Different immobilized preparations of lipase from Thermomyces lanuginosus (TLL) have been inactivated by exposure to high temperatures, guanidine or 95% of dioxane. The studied preparations were: non-stabilized cyanogen bromide (CNBr-TLL), aminated CNBr-TLL (CNBr-TLL-A), and two stabilized preparations of aminated TLL by immobilization on glyoxyl support, Gx(9/10)-TLL-A (TLL-A immobilized at pH 9 and later incubated at pH 10) or Gx(10)-TLL-A (directly immobilized at pH 10). The reactivation of the partially inactivated immobilized enzymes under mild conditions by incubation in aqueous buffer, allowed recovery of some of the original activity, which was improved when it was pre-incubated in guanidine. Amination produced a fairly negative effect on the reactivation of the enzyme, but the multipoint covalent attachment of this aminated enzyme reversed the effect (e.g., recovered activity increased from 20% for CNBr-TLL to 80% for Gx(9/10)-TLL-A). The negative effect of the amination was clearer when the inactivation was caused by exposure to high temperatures, although the multipoint attachment of aminated enzyme was able to improve the recovered activity. The determination of enzyme activity in the presence of hexadecyltrimethylammonium bromide slowed the inactivation rates of all preparations and improved the recovery of activity after incubation under mild conditions, suggesting that the opening mechanism of the lipase could be a critical step in the TLL inactivation/reactivation. The use of multipoint attached TLL preparations did not only improve enzyme stability, but it also increased activity recovery when the preparation was incubated under mild conditions.
Enzyme and Microbial Technology.
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Rafael C. Rodrigues,
Cesar A. Godoy,
Marco Filice,
Juan M. Bolivar,
Armand Palau-Ors,
Jesus M. Garcia-Vargas,
Oscar Romero,
Lorena Wilson,
Marco A.Z. Ayub,
Roberto Fernandez-Lafuente,
Jose M. Guisan
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ABSTRACT: Lipase from Thermomyces lanuginosus (TLL) immobilized on cyanogen bromide agarose (CNBr) may be fully inactivated when incubated in saturated solutions of guanidine. When this inactivated enzyme is re-incubated in aqueous medium, 20% of the activity may be recovered for several cycles. However, if the activity was determined in the presence of a detergent (CTAB, an activator of this enzyme), 100% of the initial activity in the presence of detergent was recovered. The enzyme was also inactivated in the presence of organic solvents and at high temperatures. Inactivations were more rapid when the activity was determined in absence of detergent. In both cases, some activity could be recovered just by incubation under mild conditions, and this increase was higher if the activity measurements were performed in the presence of CTAB. These results suggested that the opening of the lipase could be a critical step in the inactivation or reactivation of immobilized TLL. In inactivations in the presence of solvents, 100% of activity could be recovered during several cycles, while in thermal inactivations, the recovered activity decreased in each inactivation–reactivation cycle. The incubation of the enzyme inactivated by temperature in guanidine improved the results, but still 100% could not be achieved during several cycles even measured in the presence of CTAB.Thus, the simple incubation of the partially or fully inactivated enzyme under mild conditions permitted to recover some activity (enhancing the half life of the biocatalysts), even in thermal inactivations.
Process Biochemistry.
