Immobilization Of Candida Rugosa Lipase Research Articles

Utilization of bentonite as a support material for immobilization of Candida rugosa lipase

Bentonite is an inexpensive matrix for enzyme immobilization and has been frequently utilized for this purpose. In addition to its low cost, bentonite has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, Candida rugosa lipase was non-covalently immobilized on bentonite. The properties of immobilized enzyme were defined. Optimum pH and water content were determined as 7.5 and 100 μL, respectively. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Half-lives of the immobilized enzyme at 50, 60, and 70 °C were 45, 15, and 4 min, respectively, whereas for the soluble free lipase it was 17 min at 50 °C. Kinetics were tested at 37 °C following the hydrolysis of olive oil and obeys the Lineweaver–Burk type of rate equation. The K m was 4 × 10 −2 mM and the V max was 15 mmol h −1 mg −1.

Preparation of enantiopure ( S)-ketoprofen by immobilized Candidarugosa lipase in packed bed reactor

Various materials have been used as supports for the immobilization of a Candida rugosa lipase (Lipase OF). The enzyme adsorbed on silica gel showed the maximum activity recovery (37%), and was thus used to construct a packed bed reactor for the continuous production of ( S)-ketoprofen. The performance of the bioreactor containing 4.0 g immobilized biocatalyst was evaluated as a function of parameters such as flow rate, the ratio of height to diameter (H/D) and substrate concentration. The space velocity (SV) of the reactor showed a linear increase when the flow rate was increased in the range of 5.9 (SV = 0.9 h −1)–25 ml h −1 (SV = 3.9 h −1) and leveled out at values for flow rate between 25 (SV = 3.9 h −1) and 64 ml h −1 (SV = 10.2 h −1). The initial rate remained the same when the H/D was varied from 2.9 to 38.6. The initial rate rose in a linear fashion as the substrate concentration was increased from 5 to 20 mg ml −1 and remained almost at the same level when the substrate concentration was between 20 and 50 mg ml −1. Using a packed bed bioreactor, optically pure ( S)-ketoprofen (>99% ee (enantiomeric excess)) was produced with a conversion of 30% and productivity of 1.5 mg g −1 biocatalyst h −1.

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation ( Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity ( E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.

Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups

A method for immobilization of Candida rugosa lipase to two types of chitosan beads by activating the hydroxyl groups of chitosan using carbodiimide coupling agent has been successfully developed. The ability of carbodiimide to activate the hydroxyl groups of chitosan was confirmed using the electron spectroscopy for chemical analysis (ESCA) technique. The properties of lipase immobilized using dry and wet chitosan beads were also investigated and compared. Immobilization enhanced the enzyme stability against changes of pH and temperature. High storage stability of 30 days and an increased enzyme activity of 2,110% were observed in wet immobilized lipase. Immobilized lipase using dry and wet chitosan beads retained 78% and 85% of its initial activity after 10 batch hydrolytic cycles. The kinetic parameters K m and V max were determined for the free and immobilized lipase. The activation energy ( E a) was found to decrease for immobilization of lipase on chitosan beads.

Stereoselective esterification of 2,6-dimethyl-1,7-heptanedioic acid, catalysed by Candida rugosa lipase

The immobilised Candida rugosa lipase (CRL) displayed S-preference for both stereogenic centres in this sequential esterification of 2,6-dimethyl-1,7-heptanedioic acid ( 1) (pure meso and meso: (±) mixture, 53/47) with n-butanol in cyclohexane at a w=0.8. The reaction was faster when short-chain primary n-alcohols was used and very slow, or even none reactive, when a long-chain alcohol was used.

Lipases for biocatalysis: development of a chromatographic bioreactor

The development of a new chromatographic reactor based on immobilized Candida rugosa lipase (CRL) is described. The chromatographic system has been used to evaluate the rate differences by which the product enantiomers of esterolytic reactions catalyzed by immobilized CRL are obtained. The method has been applied to a series of racemic 2-aryloxyalkanoic acids and isosteric analogous methyl esters and to some non-steroidal antiinflammatory drugs 2-arylpropanoic acids methyl esters in order to study the structure effects on reaction rate and enantioselectivity. Lipase from C. rugosa has been non-covalently and covalently immobilized on HPLC chromatographic silica supports to develop an immobilized enzyme reactor (IMER). The reactor was connected through a switching valve to an analytical reversed-phase column, which was used for the on-line determination of the hydrolysis rate by peak area integration. The enantiomeric excess of the hydrolytic reaction products was determined off-line on a CSP utilizing immobilized penicillin G acylase (PGA-CSP).

Do enzymes recognise remotely located stereocentres? Highly enantioselective Candida rugosa lipase-catalysed esterification of the 2- to 8-methyldecanoic acids

Several racemic methyl decanoic acids have been synthesised and successfully resolved in esterification with 1-hexadecanol at a w=0.8 in cyclohexane using immobilised Candida rugosa lipase (CRL) as the catalyst. The enantiomeric ratios ( E=2.8–68) obtained were surprisingly high even when the methyl group was as remotely located as in 8-methyldecanoic acid ( E=25). Interestingly, the lipase shows enantiopreference for the S-enantiomer when the methyl group is located on even numbered carbons i.e. for the 2-,4-,6- and 8-methyldecanoic acids and to the R-enantiomer when the methyl group is located on uneven numbered carbons i.e. for the 3-,5- and 7-methyldecanoic acids.

Molecular weight and degree of deacetylation effects on lipase-loaded chitosan bead characteristics

The effects of the molecular weight (MW) and degree of deacetylation (DD) of chitosan on chitosan hydrogel beads were characterized, and the entrapment efficiency, release of entrapped lipase, and activity of immobilized Candida rugosa lipase were investigated. Fresh and freeze-dried beads were characterized. A solution of lipase was prepared in a 1.5% (w/v) chitosan and 1% (v/v) acetic acid medium, and then dropped into a tripolyphosphate solution to prepare the beads. The release studies were performed over 36 h. The enzyme activity was assayed using the Sigma lipase activity method. Chitosan with high MW and DD resulted in a higher loading. A lower activity was observed for beads produced with high DD chitosan. MW did not have a marked effect on the activity. The release study revealed that enzyme release increased to a maximum when the bead was manufactured with a low MW and a moderate to high DD chitosan sample. Freeze drying did not affect the release or the activity of the lipase. Chitosan with a high MW and DD can thus improve loading and reduce the release of lipase in these beads. The choice of chitosan can affect the activity normalized for lipase loading, and beads with desirable qualities can be produced.

Immobilization of Candida rugosa lipase on colloidal gas aphrons (CGAs).

A novel technique for immobilization of Candida rugosa lipase onto anionic colloidal gas aphrons (CGAs) is described. CGAs are spherical microbubbles (10-100 microm) composed of an inner gas core surrounded by a surfactant shell. In this initial study, greater than 80% lipase (w/w) was effectively retained on the CGAs. Leakage of protein from the CGAs and the activity of the adsorbed lipase decreased with increasing enzyme loading; this indicates that multilayers of lipase may be adsorbing onto the CGAs. The CGA-immobilised lipase displayed normal Michaelis-Menten dependence on substrate concentration and also exhibited greater activity than the free enzyme.

Enrichment of polyunsaturated fatty acids from sardine cannery effluents by enzymatic selective esterification

AbstractThe sardine canning industry produces vast quantities of effluents that need expensive reprocessing. Their oily component contains valuable n−3 polyunsaturated fatty acids, namely EPA (5,8,11,14,17‐eicosapentaenoic acid) and DHA (7,10,13, 16,19‐docosahexaenoic acid), up to 10% each. Our aim was to develop a process allowing the recovery of these fatty acids. After removing solid particles, proteins, and peptides from the crude effluent, the obtained oil was hydrolyzed. EPA and DHA were enriched from the recovered free fatty acid fraction by selective enzymatic esterification. Lipases were used as biocatalysts: LipozymeTM allowed up to 80% DHA enrichment but gave no EPA enrichment. By immobilizing Candida rugosa lipase on Amberlite IRC50 cation‐exchange resin, a 30% EPA enrichment was obtained.

Enzymatic resolution to (−)‐ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (−)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel®, were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (−)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether β-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions. Chirality 12:568–573, 2000. © 2000 Wiley-Liss, Inc.

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase.

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.

γ‐Linolenic acid‐rich triacylglycerols derived from borage oil via lipase‐catalyzed reactions

Abstractγ‐Linolenic acid (GLA), a precursor of arachidonic acid, possesses physiological functions of modulating immune and inflammatory response. Highly purified GLA is desired both as a medicine and as an ingredient of cosmetics. In this work, urea fractionation and lipase‐catalyzed reactions were employed for the enrichment of GLA in borage oil. GLA content in free fatty acids from saponified borage oil can be increased from 23.6 to 94% by the method of urea fractionation. Partial hydrolysis of borage oil catalyzed by immobilized Candida rugosa lipase raises GLA content in the unhydrolyzed acylglycerols from 23.6 to 52.1%. The IM‐60 catalyzed acidolysis reaction between the GLA‐rich free fatty acid and the unhydrolyzed acylglycerols increases the GLA content in the acylglycerols from 52.1 to 75%. The acylglycerols in the reaction product contains ca. 90% triacylglycerol. The effects of temperature, water content, substrate weight ratio, and organic solvents on the GLA content in the acylglycerols were examined.

Candida rugosa lipase as an enantioselective catalyst in the esterification of methyl branched carboxylic acids: resolution of rac-3,7-dimethyl-6-octenoic acid (citronellic acid)

Some chiral methyl branched alkanoic and alkenoic acids were used as substrates in esterifications with 1-hexadecanol in cyclohexane at water activity a w=0.8 catalysed by immobilised Candida rugosa lipase (CRL). Citronellic acid was one of several chiral 2- and 3-methyl branched acids that were successfully resolved by the catalyst. With 3-methyl-branched substrates the R-enantiomers reacted fastest, whereas with 2-methyl acids the lipase generally displayed S-preference except in one case where it displayed R-preference. When a double bond was present in the chain, the selectivity of CRL ( E=24–51) was greater compared with the corresponding saturated acid ( E=17–23). Attempted transesterifications of vinyl acetate with some chiral 2- or 3-methyl branched primary alcohols using crude CRL gave very low E-values ( E=1–2).

The incorporation of n‐3 polyunsaturated fatty acids into acylglycerols of borage oil via lipase‐catalyzed reactions

AbstractThe purpose of this work was to add n‐3 polyunsaturated fatty acids (PUFA) into the acylglycerols of borage oil. The acidolysis reaction between borage oil and n‐3 PUFA was carried out with lipase (Lipozyme IM‐60) in organic solvent. The effects of temperature, solvent, and water content on the reaction product were investigated. For the acidolysis reaction between acylglycerols (product of the selective hydrolysis of borage oil, catalyzed by immobilized Candida rugosa lipase) and n‐3 PUFA, the total content of n‐3 and n‐6 PUFA in acylglycerols was 72.8% after a reaction time of 18 h. The contents of γ‐linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were 26.5, 19.8, and 18.1%, respectively. By properly controlling the reaction time, acylglycerols with ca. 70–72% PUFA and a ratio of n‐3 PUFA to n‐6 PUFA from 0–1.09 can be obtained.

Selection of hydrophobic membranes in the lipase-catalyzed hydrolysis of olive oil

A hydrophobic membrane (HVHP, polyvinylidene difluoride) was selected out of HVHP, PTHK and PTGC (polysulfone) membranes to immobilize Candida rugosa lipase by physical adsorption in the hydrolysis of olive oil in a stirred diffusion cell. A previous model that assumed the Michaelis–Menten kinetics and Langmuir adsorption isotherm for the adsorbed lipase was used to interpret the variation of initial hydrolysis rates with enzyme and substrate concentrations. Replacing the aqueous phase by a fresh buffer, with or without containing partially deactivated lipases, during the reaction did not affect the enzyme activity for the adsorbed lipase. Moreover, the same enzyme performance was obtained when a fresh and a regenerated membrane was used as the carrier in the membrane reactor.

Synthesis of lovastatin with immobilizedCandida rugosa lipase in organic solvents: Effects of reaction conditions on initial rates

Lipase from Candida rugosa immobilized on a nylon support has been used to synthesize lovastatin, a drug which lowers serum cholesterol levels, by the regioselective acylation of a diol lactone precursor with 2-methylbutyric acid in mixtures of organic solvents. Analogs of lovastatin having a different side chain were also obtained through this method by reacting the diol substrate with different carboxylic acids. The selection of reaction conditions that maximize the initial reaction rate is investigated. Since the diol substrate has very low solubility in non-polar solvents, reaction solvents consisting of mixtures of hexane with a different, more polar cosolvent are considered. For each of the cosolvent mixtures studied, the reaction rate is maximum for an intermediate percentage of cosolvent in hexane. With total concentrations of the diol lactone in the range 6.25-12.5 mM, maximum initial rates correspond approximately to those cosolvent concentrations that permit a complete solubilization of the substrate. At higher cosolvent concentrations, lower rates are obtained. When considering the same dissolved substrate concentration, the reaction rate was found to increase with increasing values of logP(mix) and decreasing values of the dielectric constant, when varying the composition of a binary solvent mixture. However, when comparing different cosolvents, no general trend with respect to these properties was observed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56:671-680, 1997.

Immobilization ofCandida rugosa lipase to nylon fibers using its carbohydrate groups as the chemical link

The aim of this study was to evaluate the immobilization of lipase from Candida rugosa on a nylon support by methods used to attach biomolecules to solid supports through their carbohydrate moieties. The carbohydrate groups were converted to dialdehydes by treatment with sodium periodate. The length of exposure and the periodate amount were optimized to the point where almost total activity retention was obtained. Tests of the immobilized enzyme showed the expressed activity to be significantly higher than the activity obtained with the unimmobilized enzyme. The use of reverse micelles as a way of delivering water to the enzyme was tested and found to give significantly higher activities. The immobilized enzyme activity was also tested with other substrates, one of which was a chiral ester. The immobilized enzyme was found to have high stereoselective efficiency and activity toward racemic methyl methoxyphenyl glycidate, a chiral intermediate used in the manufacture of the drug diltiazem.

Immobilization of Candida rugosa lipase in lyotropic liquid crystals and some properties of the immobilized enzyme

Lipase from Candida rugosa has been immobilized in lyotropic liquid crystals consisting of a nonionic surfactant, hexane, and aqueous buffer with the enzyme. The kinetics of butyl butyrate synthesis, diffusion effects, and enzyme stability were investigated. Some basic rules have been formulated for a rational medium design in liquid-crystalline matrices.

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of ImmobilizedCandida rugosaLipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.In the case of the esterification reaction, no similar correlation was found.