Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Immobilization of Candida rugosa lipase on poly(3-hydroxybutyrate-co-hydroxyvalerate): a new eco-friendly support

The overall objective of this study is to evaluate the morphological [scanning electron microscopy (SEM)], physicochemical [differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), chemical composition analysis, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)], and biochemical properties of Candida rugosa lipase (CRL) immobilized on a natural biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. CRL was immobilized by physical adsorption with efficiency of 30%. Compared with free CRL enzyme, there were slight changes in immobilized CRL activity as a function of temperature (from 37°C to 45°C), but a similar optimal pH value of 7.0. Inactivation rate constants for immobilized CRL enzyme were 0.009 and 0.334 h⁻¹, and half-lives were 77 and 2 h at 40°C and 60°C, respectively. Kinetic parameters obtained for immobilized CRL include the Michaelis–Menten constant of K _m = 213.18 mM and maximum reaction velocity of V _max = 318.62 U/g. The operational stability of immobilized CRL was tested repeatedly, and after 12 cycles of reuse, the enzyme retained 50% activity. Based on our results, we propose that PHBV-immobilized CRL could serve as a promising biocatalyst in several industrial applications.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase

An interesting observation was found during our continued studies on the hydrolysis of ibuprofen esters by Candida rugosa lipase (CRL). An important role is played by pH in the stereospecific hydrolysis of these esters. The flap region of CRL plays a significant role in the access of the substrate to the active site of the enzyme. At pH 5.6, 48% of the methyl ester and 5% of the butyl ester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2, 9% of methyl ester and 45% of the butyl ester of ibuprofen was hydrolysed in a identical reaction time using CRL. This lead us to assume that CRL prefers the methyl ester of ibuprofen as a substrate at an acidic pH and the butyl ester of ibuprofen at a neutral pH. Therefore, in order to understand the role of pH in the substrate selection by CRL for the esters of ibuprofen we used the crystallographic coordinates of the open form of the CRL (1CRL) for molecular dynamics (MD) simulations under acidic and neutral conditions for 2 ns using GROMACS. The final structures obtained after simulation in acidic and neutral conditions were compared with the energy-minimized structure, and the root-mean-square deviations (r.m.s.ds) were calculated. The r.m.s.d. of the CRL flap at neutral pH was found to be greater than that of the CRL flap at acidic pH. The extent to which the flap opens at neutral pH allowed the bulkier substrate, the butyl ester of ibuprofen, to diffuse into the active site and provides the best enzyme-substrate fit for this specific substrate. At acidic pH there is a decreased opening of the flap thereby accommodating a more compact substrate, namely the methyl ester of ibuprofen. Thus, simulation experiments using MD provide reasonable insight for the pH-dependent substrate selectivity of CRL in aqueous environments.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Candida rugosa lipase encapsulated with magnetic sporopollenin: design and enantioselective hydrolysis of racemic arylpropanoic acid esters

Abstract

The aim of this study was to prepare the encapsulation of Candida rugosa lipase (CRL) with magnetic sporopollenin. The sporopollenin was covalent immobilized onto magnetic nanoparticles (Fe₃O₄), grafted amino (APTES), or epoxy groups (EPPTMS). CRL was sol-gel encapsulated in the presence of magnetic sporopollenin/Fe₃O₄ nanoparticles. The influence of activation agents ([3-(2,3-epoxypropoxy) propyl] trimethoxysilane (EPPTMS), (3-Aminopropyl)triethoxysilane (APTES) and pH and thermal stabilities of the biocatalyst were assessed. Experimental data showed the improved catalytic activity at different pH and temperature values. At 60?°C, free lipase lost its initial activity within 80?min of time, although the encapsulated lipases retained their initial activities of about 65% by APTES and 60% by EPPTMS after 120?min of heat treatment at 60?°C. The catalytic properties of the encapsulated lipases were utilized to hydrolysis of racemic aromatic carboxylic acid methyl esters (Naproxen and 2-phenoxypropionic acid). The results show that the sporopollenin-based encapsulated lipase (Fe-A-Spo-E) has greater enantioselectivity and conversion in comparison with the encapsulated lipase without supports (lipase-enc).     

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.     

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.     

A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.     

Application of advanced materials as support for immobilisation of lipase from Candida rugosa

Ni/Al-layered double hydroxides (Ni-LDHs) and Ni/Al-sodium dodecyl sulfonate layered double hydroxide nanocomposites (Ni-SDS-LDHs) with a molar ratio of Ni:Al (4:1) have been prepared by a co-precipitation (or salt-base) method. Their structures were determined using Powder X-Ray Diffractometer (PXRD) and the spectra showed that basal spacings for Ni-LDHs and Ni-SDS-LDHs synthesised were around 8.1?Å and 34.8?Å, respectively. Lipase from Candida rugosa was immobilised onto these advanced materials, by physical adsorption. The activity of immobilised lipase was investigated through esterification of palmitic acid and isopropyl alcohol in hexane. The effects of reaction temperature, thermostability, stability in organic solvent, operational stability, leaching and storage studies of the immobilised lipase were investigated. These biocatalysts exhibited higher activities than the native lipase with an optimum temperature of 40°C. Immobilised lipases showed higher storage stability than native lipase (up to 60 days) and during operational studies at 30°C for 5?h, more than 50% of its activity was retained. Leaching studies showed that physical adsorption is suitable for the attachment of enzymes onto LDHs.     

Studies on enantioselective hydrolysis of the acetic ester of a secondary alcohol with Arthrobacter lipase

Summary Characteristics of the enantioselective hydrolysis of the acetic ester of 4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopentenone (HMPC) by Arthrobacter lipase were investigated in a water/oil biphasic reaction mixture. Kinetic studies revealed that the strict enantioselectivity was entirely due to a difference in the catalytic constants for the enantiomeric substrates and that (S)-HMPC acetate acted as a competitive inhibitor. The comparison of enantioselectivity for the acetates of HMPC analogues indicated that hydrophobic substituents in the HMPC molecule were essential for the strict enantioselectivity.Biological preparation of an optically active alcohol. Part II     

Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium

Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs.     

A model for lipase production by Candida rugosa

A model adequately describing the lipase production by Candida rugosa has been developed, calibrated and validated using new experimental data. Process modelling has been done using CAMBIO software (Computer Aided Modelling of BIOprocesses), allowing to easy and interactively test various hypothesis and reaction schemes.Olive oil, oleic acid and glycerol has been used as substrates. The model satisfactorily describes the time evolution of biomass growth as well as lipase production in all cases. In particular diauxic behavior is successfully characterized.Model development process has helped in obtaining a 3-fold increase in lipase production when using oleic acid as substrate instead of the original olive oil used.List of Symbols Oil g/l Oil concentration - Fa g/l Fatty acids concentration - Gly g/l Glycerol concentration - Cr g/l Biomass (dry weight) - Lp U/ml Lipase - _p Oil hydrolysis rate - _gly Uptake rate on glycerol - _fa Uptake rate on fatty acids - _lp Increase rate of lipase - Y _ca Biomass/Fatty acids yield - Y _cg Biomass/Glycerol yield - Y _la Lipase/Fatty acids yield - k _l Specific growth rate on fatty acids - K _c Saturation constant - K _I Inhibition constant for lipase - k11 Specific growth rate on glycerol - k ₃ Oil hydrolysis parameter     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

Immobilization of Candida rugosa lipase on poly(allyl glycidyl ether-co-ethylene glycol dimethacrylate) macroporous polymer particles

Macroporous polymer particles containing surface epoxy groups were synthesized for immobilization of Candida rugosa lipase (CRL). The effect of incorporation of two different sets of monomers [allyl glycidyl ether (AGE) and glycidyl methacrylate (GMA)] and the effect of crosslinking density on immobilization of lipase were studied. AGE-co-EGDM polymers gave higher binding and expression of lipase than GMA-co-EGDM polymers. Optimization of immobilization parameters was done with respect to immobilization time and enzyme loading. Amongst AGE-co-EGDM polymer series, AGE-150 polymer found to give maximum lipase activity yield and therefore evaluated for temperature, pH and storage stability. Under optimum conditions, AGE-150 polymer gave 78.40% of activity yield. Immobilized lipase on AGE-150 showed a broader pH, higher temperature and excellent storage stability.     

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

Summary 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.     

Immobilization of Candida 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. (c) 1996 John Wiley & Sons, Inc.     

Efficient enantioselective hydrolysis of D,L-phenylglycine methyl ester catalyzed by immobilized Candida antarctica lipase B in ionic liquid containing systems

Immobilized Candida antarctica lipase B (Novozym 435)-catalyzed enantioselective hydrolysis of D,L-phenylglycine methyl ester to enatiopure D-phenylglycine was successfully conducted in the systems with ionic liquids (ILs). Novozym 435 exhibited excellent activity and enantioselectivity in the system containing the IL BMIMxBF(4) compared to several typical organic solvents tested. It has been found that the cations and, particularly, the anions of ILs have a significant effect on the reaction, and the IL BMIMxBF(4), which shows to be the most suitable for the reaction, gave the highest initial rate and enantioselectivity among various ILs examined. The reaction became much less active and enantioselective in the systems with BMIMxHSO(4). Also, it was noticed that the enzymatic hydrolysis was strongly dependent on BMIMxBF(4) content in the co-solvent systems and the favorable content of the IL was 20% (v/v). Of the assayed four co-solvents and phosphate buffer, the lowest apparent K(m) and activation energy, and the highest V(max) of the reaction were achieved using 20% (v/v) BMIMxBF(4) co-solvent with phosphate buffer. Additionally, various influential variables were investigated. The optimum pH, substrate concentration, reaction temperature and shaking rate were 8.0, 80mM, 25-30 degrees Celsius and 150rpm, respectively, under which the initial rate, the residual substrate e.e. and the enantioselectivity were 2.46mM/min, 93.8% (at substrate conversion of 53.0%) and 38, respectively. When the hydrolysis was performed under reduced pressure, the initial rate (2.64mM/min) and the enantioselectivity (E=43) were boosted.     

Immobilization of enzymes in liquid membranes for enantioselective hydrolysis

Summary The enantioselective hydrolysis of racemic 4-acetoxy-cyclopentenone by immobilized enzymes is described. Liquid membrane emulsions were used to encapsulate the enzyme. This technique combines the specific enzymatic reaction with a selective transport through the organic phase. The product (–)-4-hydroxy-cyclopentenone was produced with an enantiomeric excess of 82%. The immobilization techniques and all reaction steps, as well as a mathematical model for the complete process are discussed in this report.