Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads

Two commercial porous styrene-divinylbenzene beads (Diaion HP20LX and MCI GEL CHP20P) have been evaluated as supports to immobilize lipase B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immobilized the enzyme, permitting a very high loading capacity: around 110 mg CALB/wet g of support compared to the 50 mg obtained using decaoctyl Sepabeads. Although enzyme specificity of the enzyme immobilized on different supports was quite altered by the support used in the immobilization, specific activity of the enzyme immobilized on MCI GEL CHP20P was always higher than those found using decaoctyl Sepabeads for all assayed substrates. Thus, a CALB biocatalyst having 3-8 folds (depending on the substrate) higher activity/wet gram of support than the commercial Novozym 435 was obtained. Half-live of CAL-Diaion HP20LX at 60 °C was 2-3 higher than the one of Novozym 435, it was 30-40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10 M hydrogen peroxide.Results indicate that styrene-divinylbenzene supports may be promising alternatives as supports to immobilize CALB.     

Biomimetic affinity purification of Candida antarctica lipase B

Candida antarctica lipase B (CalB) is one of the most widely used biocatalysts in organic synthesis. The traditional method for purification of CalB is a multi-step, high cost and low recovery procedure. Biomimetic affinity purification had high efficiency purification. We selected 298 ligand columns from a 700-member library of synthetic ligands to screen Pichia pastoris protein extract. Of the 298, three columns (named as A9-14, A9-10, and A11-33) had one-step purification effect, and A9-14 of these affinity ligands, had both high purification and recovery. The one-step recovery of CalB reached 73% and the purification reached 91% upon purification. The active groups of A9-14 were cyclohexylamine and propenylamine. Furthermore, both A9-14 and A9-10 had the same R1 active group of cyclohexylamine which might act the main binding role for CalB. The synthetic ligand A9-14 had a binding capacity of 0.4 mg/mL and had no negative effects on its hydrolytic activity. Unlike a natural affinity ligand, this synthetic ligand is highly stable to resist 1M NaOH, and thus has great potential for industrial scale production of CalB.     

Solvent as a competitive inhibitor for Candida antarctica lipase B

In enzyme-catalyzed reactions, the choice of solvent often has a marked effect on the reaction outcome. In this paper, it is shown that solvent effects could be explained by the ability of the solvent to act as a competitive inhibitor to the substrate. Experimentally, the effect of six solvents, 2-pentanone, 3-pentanone, 2-methyl-2-pentanol, 3-methyl-3-pentanol, 2-methylpentane and 3-methylpentane, was studied in a solid/gas reactor. As a model reaction, the CALB-catalyzed transacylation between methyl propanoate and 1-propanol, was studied. It was shown that both ketones inhibited the enzyme activity whereas the tertiary alcohols and the hydrocarbons did not. Alcohol inhibition constants, K(i)(I) were changed to "K(i)", determined in presence of 2-pentanone, 3-pentanone, and 3-methyl-3-pentanol, confirmed the marked inhibitory character of the ketones and an absence of inhibition of 3-methyl-3-pentanol. The molecular modeling study was performed on three solvents, 2-pentanone, 2-methyl-2-pentanol and 2-methyl pentane. It showed a clear inhibitory effect for the ketone and the tertiary alcohol, but no effect for the hydrocarbon. No change in enzyme conformation was seen during the simulations. The study led to the conclusion that the effect of added organic component on lipase catalyzed transacylation could be explained by the competitive inhibitory character of solvents towards the first binding substrate methyl propanoate.     

Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations

Adsorption onto solid supports has proven to be an easy and effective way to improve the mechanical and catalytic properties of lipases. Covalent binding of lipases onto the support surface enhances the active lifetime of the immobilized biocatalysts. Our study indicates that mesoporous silica gels grafted with various functions are ideal supports for both adsorptive and covalent binding for lipase B from Candida antarctica (CaLB). Adsorption of CaLB on phenyl-functionalized silica gels improved in particular its specific activity, whereas adsorption on aminoalkyl-modified silica gels enabling covalent binding with the proper reagents resulted in only moderate specific activity. In addition, adsorption on silica gels modified by mixtures of phenyl- and aminoalkyl silanes significantly increased the productivity of CaLB. Furthermore, CaLB adsorbed onto a phenyl/aminoalkyl-modified surface and then treated with glutardialdehyde (GDA) as cross-linking agent provided a biocatalyst of enhanced durability. Adsorbed and cross-linked CaLB was resistant to detergent washing that would otherwise physically deactivate adsorbed CaLB preparations. The catalytic properties of our best immobilized CaLB variants, including temperature-dependent behavior were compared between 0 and 70 °C with those of two commercial CaLB biocatalysts in the continuous-flow kinetic resolutions of racemic 1-phenylethanol rac-1a and 1-phenylethanamine rac-1b.     

Transgenic rice as bioreactor for production of the Candida antarctica lipase B

Candida antarctica lipase B (CALB) is a versatile biocatalyst used for a wide range of biotransformation. Methods for low cost production of this enzyme are highly desirable. Here, we report a mass production method of CALB using transgenic rice seeds as the bioreactor. The transgenic rice transformed with the CALB gene under the control of the promoter of the rice seed storage protein GT1 was found to have accumulated a large quantity of CALB in seeds. The transgenic line with the highest lipolytic activity reached to 85 units per gram of dry seeds. One unit is defined as the amount of lipase necessary to liberate 1 μmol p‐nitrophenol from p‐nitrophenyl butyrate in 1 min. The rice recombinant lipase (rOsCALB) from this line represents 40% of the total soluble proteins in the crude seed extracts. The enzyme purified from the rice seeds had an optimal temperature of 40 °C, and optimal pH of 8.5, similar to that of the fermentation products. Test of its conversion ability as a biocatalyst for biodiesel production suggested that rOsCALB is functionally identical to the fermentation products in its industrial application.     

Kinetic model of biodiesel production using immobilized lipase Candida antarctica lipase B

We have designed a kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst. The scheme assumed reversibility of all reaction steps and imitated phase effects by introducing various molecular species of water and methanol. The global model was assembled from separate reaction blocks analyzed independently. Computer simulations helped to explore behavior of the reaction system under different conditions. It was found that methanolysis of refined oil by CALB is slow, because triglycerides (T) are the least reactive substrates. Conversion to 95% requires 1.5–6 days of incubation depending on the temperature, enzyme concentration, glycerol inhibition, etc. Other substrates, free fatty acids (F), diglycerides (D) and monoglycerides (M), are utilized much faster (1–2 h). This means that waste oil is a better feedstock for CALB. Residual enzymatic activity in biodiesel of standard quality causes increase of D above its specification level because of the reaction 2M ↔ D + G. Filtration or alkaline treatment of the product prior to storage resolves this problem. The optimal field of Nz435 application appears to be decrease of F, M, D in waste oil before the conventional alkaline conversion. Up to 30-fold reduction of F-content can be achieved in 1–2 h, and the residual enzyme (if any) does not survive the following alkaline treatment.     

Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

Functional expression of Candida antarctica lipase B in Eschericha coli

Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants k(M), v(max) and k(cat) were determined using the substrates p-NPB and p-NPL. Almost identical k(cat)/k(M) values (0.423-0.466 min(-1) microM(-1) for p-NPB and 0.068-0.071 min(-1) microM(-1) for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.     

Improved triglyceride transesterification by circular permuted Candida antarctica lipase B

Lipases represent a versatile class of biocatalysts with numerous potential applications in industry including the production of biodiesel via enzyme‐catalyzed transesterification. In this article, we have investigated the performance of cp283, a variant of Candida antarctica lipase B (CALB) engineered by circular permutation, with a series of esters, as well as pure and complex triglycerides. In comparison with wild‐type CALB, the permutated enzyme showed consistently higher catalytic activity (2.6‐ to 9‐fold) for trans and interesterification of the different substrates with 1‐butanol and ethyl acetate as acyl acceptors. Differences in the observed rates for wild‐type CALB and cp283 are believe to be related to changes in the rate‐determining step of the catalytic cycle as a result of circular permutation. Biotechnol. Bioeng. 2010;105: 44–50. © 2009 Wiley Periodicals, Inc.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Capsaicin hydrolysis by Candida antarctica lipase

Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase.     

Catalytic properties of Candida antarctica lipase B clusters solubilized in hexane

The objective of this work was to investigate the particle size and determine the catalytic competency of a solubilized lipase in hexane. Purified Candida antarctica lipase B (CALB) was solubilized in hexane using the non-ionic surfactant Span 60. The amount of surfactant was chosen so that complete coverage of the individual enzyme molecules with surfactant was not possible. Dynamic Light Scattering (DLS) was used to directly investigate the particle size of the solubilized entities. The enzyme was found to be solubilized in the form of clusters of lipase molecules with a radius of 37±5 nm at 42°C, which we estimate to correspond to about 1200 CALB molecules. The solubilized enzyme clusters showed lower catalytic activity in a model esterification reaction in hexane compared with a commercial immobilizate of the same enzyme (Novozym 435). Further gains in catalytic activity may be possible by striving for true molecular-level dispersion of the enzyme in hexane.     

Glycerol acyl-transfer kinetics of a circular permutated Candida antarctica lipase B

Triacylglycerols containing a high abundance of unusual fatty acids, such as γ-linolenic acid, or novel arylaliphatic acids, such as ferulic acid, are useful in pharmaceutical and cosmeceutical applications. Candida antarctica lipase B (CALB) is quite often used for non-aqueous synthesis, although the wild-type enzyme can be rather slow with bulky and sterically hindered acyl donor substrates. The catalytic performance of a circularly permutated variant of CALB, cp283, with various acyl donors and glycerol was examined. In comparison to wild-type CALB, butyl oleate and ethyl γ-linolenate glycerolysis rates were 2.2- and 4.0-fold greater, respectively. Cp283 showed substrate inhibition by glycerol, which was not the case with the wild-type version. With either ethyl ferulate or vinyl ferulate acyl donors, cp283 matched the performance of wild-type CALB. Changes in active site accessibility resulting from circular permutation led to increased catalytic rates for bulky fatty acid esters but did not overcome the steric hindrance or energetic limitations experienced by arylaliphatic esters.     

Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica

Glutaraldehyde chemistry has been used to immobilize lipase B from Candida antarctica (CALB) under different situations. Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support via interfacial activation. Using non-ionic detergents (e.g., Triton X-100), the enzyme becomes ionically adsorbed on the activated support. If detergent and salt are simultaneously present during immobilization, a covalent attachment to the support is first produced. In absence of detergent or high ionic strength, a mixture of all of the previous immobilization reasons should coexist. Thus, 5 different CALB biocatalysts were prepared following the previous described protocols, and its stability and activity, pH/activity profile and specificity versus R and S methyl mandelate were analyzed. The existence of covalent attachment of more than 95% of the enzyme molecules was confirmed by washing the biocatalysts in salt and detergent solutions. The glutaraldehyde treatment of the enzyme adsorbed on aminated supports did not produce a significant improvement on the activity of the enzyme versus p-nitrophenylpropinate (pNPB) nor a high stabilization of the enzyme. This differed from the effects of a similar treatment of CAL adsorbed on octyl agarose. However, they were similar to the effects of this treatment on covalently immobilized CALB, suggesting that the immobilization protocol may greatly affect the final effect of a chemical modification on the enzyme properties.Dramatic changes in the enzyme features were observed comparing the different preparations, mainly in the specificity of CALB versus p-NPB and R-methyl mandelate (from 2.5 to 20), or in the enantiospecificity versus R/S methyl mandelate (from 1.8 to 16), confirming that these different immobilization protocols produced biocatalysts with different features. Moreover, changes in experimental conditions produced very different effects on the properties of the different CALB preparations.     

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.     

Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity

Changes in solvent type were shown to yield significant improvement of enzyme enantioselectivity. The resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B, CALB, was studied in eight liquid organic solvents and supercritical carbon dioxide, SCCO(2). Studies of the temperature dependence of the enantiomeric ratio allowed determination of the enthalpic (Delta(R-S)Delta H(++)) as well as the entropic (Delta(R-S)Delta S(++)) contribution to the overall enantioselectivity (Delta(R-S)Delta G(++)= -RTlnE). A correlation of the enantiomeric ratio, E, to the van der Waals volume of the solvent molecules was observed and suggested as one of the parameters that govern solvent effects on enzyme catalysis. An enthalpy-entropy compensation relationship was indicated between the studied liquid solvents. The enzymatic mechanism must be of a somewhat different nature in SCCO(2), as this reaction in this medium did not follow the enthalpy-entropy compensation relation.     

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.