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.     

Immobilized lipases from Candida antarctica for producing tyrosyl oleate in solvent-free medium

Abstract

Two immobilized lipases from Candida antarctica have been compared for the direct esterification of tyrosol with oleic acid in equimolar conditions and in the absence of organic solvent. Candida antarctica lipase B was immobilized on octyl-silica agglomerates and compared with commercial Novozym 435. Reduction of tyrosol particle size to 0.1 mm significantly increased the reaction rate with both immobilized lipases, and reduced pressure improved the final tyrosyl oleate yield up to 95% (w/w) in both cases. Immobilized lipases were recovered and reutilized in three consecutive trials with negligible inactivation. Under optimum conditions, a product mixture comprising more than 95% of tyrosyl oleate (w/w) was attained in less than 2 hours. Finally, the index of antioxidant activity obtained, according to the Rancimat method, indicated that tyrosyl oleate was slightly more effective than tyrosol as an antioxidant in a low polar matrix.     

Enzymatic resolutions of α- and β-hydroxypropionic acid esters

The synthesis of some acyloxy-methoxy-cinnamic acid derivatives, azidohydroxy butanoates, and azidohydroxy butanedioates in enantiomerically pure form is presented. Racemic diastereomerically pure educts were prepared in few steps. These racemates are resolved with lipases from Candida cylindracea (CC) and Pseudomonas fluorescens (P).     

Fourier‐transform infrared spectroscopy study of dehydrated lipases from Candida antarctica B and Pseudomonas cepacia

Fourier‐transform infrared (FT‐IR) spectroscopy was employed to investigate potential lyophilization‐induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the α‐helix and increased the β‐sheet content. However, FT‐IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the β‐sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT‐IR for analysis of the structure of solid proteins, at least as far as the β‐sheet content is concerned, because it is possible that the method overestimates the β‐sheets by measuring other hydrogen‐bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 545–551, 1999.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Enhancement of Candida rugosa lipase activity in non-aqueous media by co-lyophilization with amphiphiles from aqueous solution

Modified Candida rugosa lipase was co-lyophilized with two gemini-type amphiphiles, l- and d-2-(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionylamino)-pentanedioic acid didodecyl ester or dodecanoic acid 2-[(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionyl)-(2-dodecanoyloxy-ethyl)-amino]-ethyl ester. Enzymatic activities of the modified lipases in the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in cyclohexane were enhanced as much as by 37-78, 1.5–5- and 41–83-fold of magnitude relative to that of native enzyme, respectively. The lack of significant enhancement of the enzymatic activity, only in the case of the d-isomeric amphiphile-modified lipase, was considered from the topological view of the amphiphile.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Lipase-catalyzed synthesis of medium- and long-chain diesters of 2-oxoglutaric acid

Abstract

The influence of various reaction parameters, such as alcohol-to-substrate ratio, enzyme-to-substrate ratio, solvent and temperature, on the enzymatic preparation of a series of novel medium- and long-chain esters of 2-oxoglutaric acid has been evaluated. Among the tested lipases, those from Candida antarctica and Carica papaya appeared to be the best catalysts. Mild reaction conditions and low environmental impact make the biocatalytic procedure a convenient way to prepare the reported products, which are potential fat substitutes in the food industry.     

Purification and characterization of two isoforms from Candida rugosa lipase B

Two isoforms of Candida rugosalipase B (LB1 and LB2) were purified by anionic exchange chromatography. The lipases had the same N-terminal sequence, carbohydrate content and pH and thermal stability but different pIs and significant differences in their activities against different p-nitrophenol esters and triacylglycerides.     

Lipase‐Catalyzed Kinetic Resolution of (±)‐1‐(2‐Furyl) Ethanol in Nonaqueous Media

S‐1‐(2‐Furyl) ethanol serves as an important chiral building block for the preparation of various natural products, fine chemicals, and is widely used in the chemical and pharmaceutical industries. In this work, lipase‐catalyzed kinetic resolution of (R/S)‐1‐(2‐furyl) ethanol using different acyl donors was investigated. Vinyl esters are good acyl donors vis‐à‐vis alkyl esters for kinetic resolution. Among them, vinyl acetate was found to be the best acyl donor. Different immobilized lipases such as Rhizomucor miehei lipase, Thermomyces lanuginosus lipase, and Candida antarctica lipase B were evaluated for this reaction, among which C. antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n‐heptane as solvent. The effect of various parameters was studied in a systematic manner. Maximum conversion of 47% and enantiomeric excess of the substrate (ee_s) of 89% were obtained in 2 h using 5 mg of enzyme loading with an equimolar ratio of alcohol to vinyl acetate at 60°C at a speed of 300 rpm in a batch reactor. From the analysis of progress curve and initial rate data, it was concluded that the reaction followed the ordered bi–bi mechanism with dead‐end ester inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is more economical, green, and easily scalable than the chemical processes. Chirality 26:286–292, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris , was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g^?1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi‐Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L^?1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.     

Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Enzymatic Hydrolysis of Esters Containing a Tetrazole Ring

The lipase‐catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4‐(5‐aryl‐2H‐tetrazol‐2yl)butan‐2‐ol and 1‐(5‐aryl‐2H‐tetrazol‐2yl)‐propan‐2‐ol and their acetates with the highest optical purities (ee = 95%‐99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4‐[5‐(4‐chlorophenyl)‐2H‐tetrazol‐2‐yl)butan‐2‐ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger. Chirality 26: 811–816, 2014. © 2014 Wiley Periodicals, Inc.     

Meetings & Symposia

Abstract

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.     

Enhanced conjugation of Candida rugosa lipase onto multiwalled carbon nanotubes using reverse micelles as attachment medium and application in nonaqueous biocatalysis

Three liquid phases (viz. aqueous, nonaqueous, and reverse micelles) were scrutinized as medium for attachment of the enzyme Candida rugosa lipase (CRL) onto multiwalled carbon nanotubes (CNTs). The nanotubes were functionalized to attain carboxyl and amino groups on their surfaces before enzyme conjugation. Transmission electron microscopy and Fourier transformation infrared spectroscopic studies were used for characterization of the nanotubes during the course of functionalization. High enzyme loadings associated with the functionalized CNTs were observed when reverse micelles were used as the attachment medium. In addition, high activity in terms of ester synthesis in organic solvents was also observed while using those preparations. The nanobioconjugates prepared using reverse micelles were found to be highly sturdy and exhibited appreciable operational stability of around 95 ± 3% at 20th cycle (in case of carboxylated nanotubes) and 90 ± 5% at 10th cycle (in case of aminated nanotubes) for esterification. This shows the potential application of reverse micelles as the attachment medium for surface active enzymes such as CRL onto CNTs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:828–836, 2014     

Improved stability of the lipase from Candida rugosa in different purification degrees by chemical modification

Summary Semipurified lipase of Candida rugosa and pure isoforms (lipase A and lipase B) have been chemically modified using two methodologies based on polyethyleneglycol (PEG). The activation of PEG with p-NO₂-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG in the enzymatic activity of the lipase. The chemical modification increases the stability of pure lipases in isooctane at 50 °C.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

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.     

High-yield synthesis of wax esters catalysed by modified <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained in nearly 100% yield for 10 h at 50°C when immobilized PEG₂₀₀₀-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic preparation with 83% of its activity conserved after the seventh run of repeated batch reactions.