Cross-linked enzyme aggregates of recombinant Candida antarctica lipase B for the efficient synthesis of olvanil,a nonpungent capsaicin analogue

Despite the proven therapeutic role of capsaicin in human health, its usage is still hampered by its high pungency. In this sense, nonpungent capsaicin analogues as olvanil are a feasible alternative to the unpleasant sensations produced by capsaicin while maintaining a similar pharmacological profile. Olvanil can be obtained by a lipase-catalyzed chemoenzymatic process. In the present work, recombinant Candida antarctica lipase B (CALB) was expressed in Pichia pastoris and subsequently immobilized by cross-linked enzyme aggregate (CLEA) methodology for the synthesis of olvanil. The CALB-CLEAs were obtained directly from the fermentation broth of P. pastoris without any purification step in order to assess the role of the contaminant proteins of the crude extract as co-feeders. The CALB-CLEAs were also bioimprinted to enhance the catalytic performance in olvanil synthesis. When CALB was precipitated with isopropanol, the obtained CALB-CLEAs exhibited the highest activity in the synthesis of olvanil, regardless of the glutaraldehyde concentration. The maximum product synthesis was found at 72 hr obtaining 6.8 g L⁻¹ of olvanil with a reaction yield of 16%. When CALB was bioimprinted with olvanil, the synthesis was enhanced 1.3 times, reaching 10.7 g L⁻¹ of olvanil at 72 hr of reaction with a reaction yield of 25%. Scanning electron microscopy images indicated different morphologies of the CLEAs depending on the precipitating agent and the template used for bioimprinting. Recombinant CALB-CLEAs obtained directly from the fermentation broth are a suitable alternative to commercial enzymatic preparations for the synthesis of olvanil in organic medium.     

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.     

The atypical lipase B from Candida antarctica is better adapted for organic media than the typical lipase from Thermomyces lanuginosa

Candida antarctica lipase B (CALB) and Thermomyces lanuginosa lipase (TLL) were evaluated as catalysts in different reaction media using hydrolysis of tributyrin as model reaction. In o/w emulsions, the enzymes were used in the free form and for use in monophasic organic media, the lipases were adsorbed on porous polypropylene (Accurel EP-100). In monophasic organic media, the highest specific activity of both lipases was obtained in pure tributyrin at a water activity of >0.5 and at an enzyme loading of 10 mg/g support. With tributyrin emulsified in water, the specific activities were 2780 micromol min(-1) mg(-1) for TLL and 535 micromol min(-1) mg(-1) for CALB. Under optimal conditions in pure tributyrin, CALB expressed 49% of the activity in emulsion (264 micromol min(-1) mg(-1)) while TLL expressed only 9.2% (256 micromol min(-1) mg(-1)) of its activity in emulsion. This large decrease is probably due to the structure of TLL, which is a typical lipase with a large lid domain. Conversion between open and closed conformers of TLL involves large internal movements and catalysis probably requires more protein mobility in TLL than in CALB, which does not have a typical lid region. Furthermore, TLL lost more activity than CALB when the water activity was reduced below 0.5, which could be due to further reduction in protein mobility.     

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.     

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.     

New application of depth filters for the immobilization of Candida antarctica lipase B

Applied Microbiology and Biotechnology - The objective of this study was to use for the first time depth filters, which are usually intended for clarification of cell culture broth, as a direct...     

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.     

Remarkable stability of Candida antarctica lipase B immobilized via cross-linking aggregates (CLEA) in deep eutectic solvents

The combination of Deep-eutectic-solvents (DES) with water as “co-solvent” enables a low-viscous reaction medium that keeps its “non-conventional” nature and thus enables synthetic lyophilization reactions (e.g. esterification) catalyzed by hydrolases. Substrates with different polarity may be employed. This paper shows how the enzyme immobilization with cross-linking aggregates (CLEA) leads to highly stable and active immobilized catalysts in different DES. As a remarkable case, when choline chloride-glycerol DES is used, CLEA derivatives of Candida antarctica lipase B (CLEA-CALB) are stable for at least 14?days without any loss of activity. The immobilized biocatalysts are applied in non-viscous DES-water blends (8% v/v) to catalyze the esterification of benzoic acid and glycerol to furnish glyceryl monobenzoate (α-MBG) in productivities of ～35?g α-MBG L^?1d^?1. Compared to other commercial immobilized CALB, the CLEA-CALB derivatives rendered more product (higher conversions by 30%). Moreover, CLEA derivatives were successfully reused for six times without any loss of activity. Given the ease of immobilization (CLEA), their excellent performance in DES and the low viscosity of the DES-water blends, the reported approach may be useful for many synthetic procedures and even for continuous processes with largely optimized outcomes.     

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.     

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.     

Enantiopure derivatives of 1,2-alkanediols: substrate requirements of lipase B from Candida antarctica

Efficient methods for kinetic resolution of 1-phenoxy-2-butanol, 1-phenylmethoxy-2-butanol, and 1-phenoxy-2-pentanol were developed using lipase B from Candida antarctica as catalyst. Resolutions were performed in order to investigate the substrate requirements needed to obtain a high E-value. The effect of the substrate structure on E is different for transesterifications in organic media as compared to hydrolysis. The influence of different acyl donors on the E-value was also investigated.     

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.     

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.     

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.     

Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

Lipase B from Candida antarctica (CALB) has been modified using succinic polyethyleneglycol via the carbodiimide route. Immobilized enzyme (on octyl Sepharose or Eupergit C) has been used, to take advantage of the solid phase. Modification of immobilized CALB's native amino groups did not produce a significant alteration of CALB. However, if the enzyme was previously aminated, around 14–15 PEG molecules could be introduced per enzyme molecule. Also, it has been found that succinic groups are far more reactive than acetic acid following this strategy.Even after this drastic double modification, the functional properties of the enzyme have not been impoverished to a large extent: stability decreased only to some extent (by a 5–6 fold factor), activity versus some substrates even increased (e.g., around 60% using p-nitrophenyl butyrate). It has been found that both modifications (amination and pegylation) have very different effects on enzyme properties when performed on CALB immobilized on Eupergit C or octyl Sepharose. For example, activity versus pNPP increased using CALB-octyl Sepharose while it decreased when using Eupergit C following amination and PEGylation. The effects also depend on the reaction and substrate, for example in hydrolysis of methyl mandelate, the activity decreased by 50% using CALB-octyl Sepharose after PEGylation of the aminated enzyme, while using CALB-Eupergit C had no effect. In this last case, enantioselecitvity in this hydrolysis significantly improved after both chemical modifications (from 7.5 to 20), while using CALB-octyl Sepharose almost had no effect.     

Immobilization on octyl-agarose beads and some catalytic features of commercial preparations of lipase a from Candida antarctica (Novocor ADL): Comparison with immobilized lipase B from Candida antarctica

Lipase A from Candida antarctica (CALA, commercialized as Novocor ADL) was immobilized on octyl-agarose, which is a very useful support for lipase immobilization, and coated with polyethylenimine to improve the stability. The performance was compared to that of the form B of the enzyme (CALB) immobilized on the same support, as both enzymes are among the most popular ones used in biocatalysis. CALA immobilization produced a significant increase in enzyme activity vs. p-nitrophenyl butyrate (pNPB) (by a factor of seven), and the coating with PEI did not have a significant effect on enzyme activity. CALB reduced its activity slightly after enzyme immobilization. Octyl-CALA was less stable than octyl-CALB at pH 9 and more stable at pH 5 and, more clearly, at pH 7. PEI coating only increased octyl-CALA stability at pH 9. In organic solvents, CALB had much better stability in methanol and was similarly stable in acetonitrile or dioxane. In these systems, the PEI coating of octyl-CALA permitted some stabilization. While octyl-CALA was more active vs. pNPB, octyl-CALB was much more active vs. mandelic esters or triacetin. Thus, depending on the specific reaction and the conditions, CALA or CALB may offer different advantages and drawbacks. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2735, 2019