Commercial Candida Rugosa Lipase Research Articles

Protein-Coated Microcrystals from Candida rugosa Lipase: Its Immobilization, Characterization, and Application in Resolution of Racemic Ibuprofen.

In this study, an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs), was prepared from a commercial Candida rugosa lipase (CRL) and used for catalyzing esterification of (R, S)-ibuprofen enantiomers with isooctanol in isooctane. The main variables controlling the process (precipitating solvents, pH, saturated K2SO4 solution, and water content) were optimized via single-factorial experiments. Under optimum conditions, the enantiomeric excess of active S(+)-ibuprofen and total conversion rate were 97.34 and 49.83 %, respectively, and the corresponding enzyme (PCMC-CRL) activity attained 387.29 μmol/min/g protein, a 5.78-fold enhancement over the free lipase powder. Additionally, the thermostability, organic-solvent tolerance, and operational stability of PCMC-CRL were greatly improved as compared to the free enzyme. Fourier transform infrared (FTIR) spectroscopy was employed to reveal the correlation between conformation and enzyme activity enhancement. Moreover, the PCMC-CRL retained most of its original activity following use in more than 15 successive batches, suggesting that it exhibits adequate operational stability. These results indicate that PCMC-CRL is of great potential use in industrial applications.

Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes

The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1–CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50wt% water, an initial 1equivalent of methanol feeding, and an additional 0.5equivalents of methanol feeding at 24h for a total reaction time of 48h at 37°C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.

Megaporous poly(hydroxy ethylmethacrylate) based poly(glycidylmethacrylate-N-methacryloly-(l)-tryptophan) embedded composite cryogel

One-step activation, purification, and stabilization of lipase enzyme were performed by using composite hydrophobic support at low ionic strength with increased surface area during embedding process. A novel hydrophobic poly(hydroxyethylmethacrylate) [PHEMA] based, poly(glycidyl methacrylate-N-methacryloly-(l)-tryptophan) [PGMATrp] bead embedded composite cryogel membrane having specific surface area of 195m2/g was used as hydrophobic matrix for adsorption of commercial Candida Rugosa lipase in a continuous system. PGMATrp embedded PHEMA cryogel membrane with 60–100μm pore size was obtained by dispersion polymerization of GMA and MATrp to form PGMATrp beads followed by embedding of PGMATrp to HEMA via APS and TEMED redox pair. The introduction of hydrophobic MATrp monomer into bead structure aiming to increase interaction between lipase and composite membrane was estimated using nitrogen stoichiometry of elemental analysis and found to be 239μmol/g of polymer. Hydophobicity increment due to embedding process was confirmed by measuring contact angle, it was found 42° and 48.4° for the PHEMA and PHEMA/PGMATrp composite cryogel respectively. Some parameters i.e. pH, flow-rate, protein concentration, temperature, salt type and ionic intensity were evaluated on the adsorption capacity in a continuous system. Fast protein liquid chromatography (FPLC) studies were performed for specific adsorption of lipase onto the PHEMA/PGMATrp embedded composite cryogel membrane.

Enzymatic synthesis of phytosterol esters catalyzed by Candida rugosa lipase in water-in-[Bmim]PF6 microemulsion.

A water-in-ionic liquid microemulsion ([Bmim]PF6/Tween20/H2O) was applied as reusable reaction medium to esterify phytosterols with fatty acid by Candida rugosa lipase (CRL) successfully. Two kinds of commercial CRLs, AY30 and AYS which cannot effectively catalyze esterification in conventional reaction system were found effective in the microemulsion system. Effects of reaction parameters on esterification were investigated; results showed that the conversion rate of 87.9 and 95.1% was obtained in 24 and 48h of reaction, respectively, under the optimized condition: the molar ratio of water to Tween 20 (w 0 value) at 5.4, Tween 20 at a concentration of 305mM, 50°C,pH 7.4, 10% of enzyme loading (w/w, with respect to total reactants), and phytosterols/lauric acid molar ratio of 1:2. Moreover, by using n-hexane as the extraction agent, the lipase-encapsulated microemulsion could be reused at least seven times (>168h) without significant changes in the conversion rate, while achieving a purpose of simple separation and purification.

Bridging racemic lactate esters with stereoselective polylactic acid using commercial lipase catalysis

A productive and enantioselective hydrolysis of racemic mixtures of lactate esters with commercial Candida rugosa lipase was performed. This step contributes to a novel envisioned route for stereoselective PLA production by combining recent chemocatalytic developments with this biocatalytic contribution, foreseeing two separate L- and D-lactate enantiomer streams. A study of the hydrolysis kinetics identified an unexpected rate determining step at the origin of an unprecedented ester reactivity order.

Synthesis of n-amyl isobutyrate catalyzed by Candida rugosa lipase immobilized into poly(N-isopropylacrylamide-co-itaconic acid) hydrogels

A commercial Candida rugosa lipase was immobilized into poly(N-isopropylacrylamide-co-itaconic acid) hydrogel under different pH immobilization conditions, characterized and evaluated for both hydrolytic activity in an aqueous medium and esterolytic activity in an organic medium. Although the apparent enzyme activity in the hydrolytic reaction was acceptable, the immobilized lipase appeared to be more suitable for application in ester synthesis in low aqueous system based on n-hexane. The esterification reaction parameters, such as temperature, biocatalyst amount, added water content and substrate concentration was evaluated and optimized by response surface analysis. An adequate statistical quadratic model was obtained, making it possible to predict ester yields from known values of the four main factors. Under optimized conditions, the yield of ester>90% could be achieved after 48h of reaction, corresponding to the volumetric activity of 4.75mmolL−1h−1. The operational stability of the immobilized system in esterification reaction proved to be highly attractive with 15 consecutive 48h uses with a residual activity of 67.4%, implying that the developed hydrogel and immobilized system could provide a promising solution for the flavor ester synthesis at the industrial scale.

Fine separation and characterization of Candida rugosa lipase isoenzymes.

Commercial Candida rugosa lipase has been separated into two distinct fractions (CRLA and CRLB) by anion-exchange chromatography. As analyzed on SDS-polyacrylamide gel electrophoresis, CRLA and CRLB are homogenous. At high ionic strength, CRLA and CRLB have similar hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pockets of CRLA and CRLB may be similar. At low ionic strength, using "hydrophobic interfacial affinity chromatography", both CRLA and CRLB have been separated into four isofractions. They have different hydrophobicity and UV spectra, suggesting that the open extent of the large hydrophobic pocket of the four forms may be different. Further, the conversion of CRL isoenzymes in the process of organic solvent treatment and ester hydrolysis were examined. The results clearly showed not only that CRLB had been converted to CRLA, but also that CRLA sub-fractions with different open extent of large hydrophobic pocket had been converted

Primary allenic alcohols of high optical purity via lipase catalyzed resolution

The lipase from Candida rugosa lipase (CRL) was used for the preparation of optically active primary allenic alcohols with axial chirality. The biocatalytic material used was the commercial CRL (C-CRL) and propan-2-ol-treated CRL (PT-CRL). The kinetic resolution of (±)- 1 and (±)- 3a– c in water using C-CRL resulted in either the absence of or low enantiomeric ratio, whereas PT-CRL increased the E-value. Under optimized conditions (temperature and medium used) (+)- S- 3a and (−)- R- 4 were isolated in excellent yields and high optical purity (o.p.). The resolution was carried out on multigram scale in water/ n-hexane at 4 °C.

Enantioselectivity of alcohol-treated Candidarugosa lipase in the kinetic resolution of racemic methyl 2-aryloxypropionates in water and aqueous organic media

Racemic methyl 2-aryloxypropionates (±)- 1 were subjected to hydrolysis in water and in a series of two-phase aqueous organic media in the presence of Candida rugosa lipase (CRL). The biocatalytic material used was the enzyme of commercial CRL purified by treatment with different alcohols. The purification of CRL and the reaction medium play an important role in the enantioselection of racemates (±)- 1. While it is not possible to use the same protocol for all substrates, by combining the different ways of purifying the enzyme with the various reaction media, it is possible to achieve high enantioselectivities of racemic esters.

Enzymatic resolution of 2-fluoro-2-arylacetic acid derivatives

Optical resolutions of 2-fluoro-2-arylacetic acids, ArC *FRCOOH (R=CN, H, and Me), were performed by enantioselective hydrolysis of the corresponding esters using Candida rugosa lipase (CRL). The enantioselectivity of commercial CRL toward the esters was greatly improved when commercial CRL was treated with 2-propanol solution. In the enantioselective hydrolysis of these esters, as represented by ArC * SMCOOR ( S and M are small and medium substituents, respectively), the active site of the 2-propanol-treated CRL recognized fluorine as S in both PhCF(CH 3)COOR and p-TolCF(CN)COOR but recognized fluorine as M in PhCHFCOOR.

Enantioselectivity of Pseudomonas cepacia and Candida rugosa lipases for the resolution of secondary alcohols: The effect of Candida rugosa isoenzymes

In this work, the lipase ( Pseudomonas cepacia) (PS)- and Candida rugosa lipase (CRL)-catalyzed asymmetric deacylations of the esters of structurally different secondary alcohols were studied. Enantioselectivity in the case of lipase PS was always easily predicted according to the sizes of the substituents at the alcoholic stereocenter whereas the favored enantiomer in the case of CRL strongly depended on the structure of the racemic alcohol moiety in an ester. In the next step, commercial CRL was fractionated into its isoenzymes (A and B). The two isoenzymes A and B always gave the same enantiopreferences. This observation clearly showed that the heterogeneity of the commerical CRL was not a reason for the reversed enantioselectivity of certain substrates such as cyanohydrin, α-hydroxycarboxylic acid, and 2,2,2-trihaloethanol derivatives when compared to the selectivity observed when lipase PS was used as a catalyst.

Comparative studies on the sugar esters synthesis catalysed by Candida antarctica and Candida rugosa lipases in hexane

The regioselective acylation of monosaccharides, such as glucose, fructose, mannose and arabinose with lauric acid (C12) has been investigated in hexane using commercial Candida rugosa lipase (EC 3.1.1.3) and Candida antarctica (NOVO-ZYME) lipase immobilised on anionic resin. In order to overcome sugars insolubility in hexane, sugars were introduced in the reaction mixture preabsorbed on silica gel. The chemical structure of the products has been determined by 1H NMR analysis. The two enzymes tested seemed to prefer hexoses rather than pentoses as the sugar substrate. They exhibited different water activity optimums as well as different optimum sugar/acyl donor molar ratios. Water activity control throughout the reaction and temperature increase from 30 to 50 °C enhanced sugar ester production using both lipases.