Effect of reaction parameters on synthesis of citronellyl methacrylate by lipase-catalyzed transesterification

The methacrylate ester of citronellol was synthesized using various lipases as catalyst. The effect of different reaction parameters such as amount of lipase, solvent, temperature, and acylating agent on the conversion of citronellol to citronellyl methacrylate was studied. Methyl methacrylate, vinyl methacrylate, and 2,3-butanedione mono-oxime methacrylate were used as acylating agents. Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Pseudomonas cepacia lipase (Amano-PS) were used as biocatalysts. Diisopropyl ether (DIPE) was found to be the most suitable solvent. The stereoselectivity of CRL in transesterification of (+/-)-citronellol was tested for the optimized reaction parameters.     

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type II). Two immobilization methods were also used, namely: 1). adsorption, using as support the following silica derivatives (150-300 microm e 450micro): phenyl, epoxy, amino and without derivation, and 2). covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/g(support) for CRL and 0.97 U/g(support) for PPL, and of transesterification, 2,8 U/g(support) for CRL and 1,9 U/g(support) for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40 degrees C and 50 degrees C and for PPL at 32 degrees C and 40 degrees C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continuous reactor. Lipases immobilized by covalent binding were used in the assays of operational stability in continuous reactors. For PPL in aqueous medium, at 32 degrees C, and CRL in organic medium at 40 degrees C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40 degrees C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40 degrees C the loss was 33% after 20 days. Comparing both sources with each other, very different results were obtained. Higher activity was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.     

Production of Ricinoleic Acid from Castor Oil by Immobilised Lipases

Abstract

Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Castor bean lipase (CBL) were immobilized on celite by deposition from aqueous solution by the addition of hexane. Lipolytic performance of free and immobilized lipases were compared and optimizations of lipolytic enzymatic reactions conditions were performed by free and immobilized derivatives using olive oil as substrate. Afterwards, the influence on lipolysis of castor oil of free lipases and immobilized lipase derivatives have been studied in the case of production of ricinoleic acid. All of the lipases performances were compared and enzyme derivative was selected to be very effective on the production of ricinoleic acid by lipolysis reaction. Various reaction parameters affecting the production of ricinoleic acid were investigated with selected the enzyme derivative.

The maximum ricinoleic acid yield was observed at pH 7–8, 50°C, for 3 hours of reaction period with immobilized 1,3-specific PPL on celite. The kinetic constants K_m and V_max were calculated as 1.6 × 10^?4 mM and 22.2 mM from a Lineweaver–Burk plot with the same enzyme derivative. To investigate the operational stability of the lipase, the three step lipolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentange of conversion after usage decreased markedly.     

Study of microwave effects on the lipase-catalyzed hydrolysis

The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris–HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

Lipase-catalysed kinetic resolution of secondary alcohols with improved enantioselectivity in propylene carbonate

The lipase-catalysed kinetic resolution of secondary alcohols was studied using vinyl acetate as acyl donor in propylene carbonate. Propylene carbonate offers an environmentally friendly alternative in contrast to conventional solvents. Several different lipases were investigated, and Candida antarctica lipase B (CALB) exhibited better results for all the substrates. It was shown that the addition of non-reactive base triethylamine and silver oxide to the reaction mixture enhanced the reaction rate and enantioselectivity. With propylene carbonate as solvent, CALB could be recycled without significant activity or enantioselectivity losses.     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.     

Cross-linked protein-coated microcrystals as biocatalysts in non-aqueous solvents

Cross-linked protein-coated microcrystals (CLPCMCs) of subtilisin Carlsberg (SC), and lipases from Candida rugosa (CRL) and Burkholderia cepacia (BCL) were prepared by cross-linking protein-coated microcrystals (PCMCs). These showed enhanced reaction rates for transesterification in organic solvents of different polarity compared with PCMCs. CLPCMCs of SC were catalytically active up to 80°C in octane and gave 98% conversion in 6 h, while PCMCs gave only 58% conversion in 8 h and conversions did not further increase with time. The Fourier transform infrared spectra showed that the alpha-helical content of CLPCMCs (unlike that of PCMCs) of SC were similar to what is reported for free SC in water.     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Lipase-catalysed deacetylation of androstane and pregnane derivatives: influence of ring D substitution

A series of acetoxy derivatives of androstane and pregnane was deacetylated in organic solvents by microbial lipases. The best results were obtained with lipase from Candida antarctica (CAL B), Candida rugosa (CRL) and Pseudomonas sp. (PSL). In some derivatives, CAL B and CRL showed a regioselective behaviour towards the removal of the 3β- or 16/16β-acetyl group. The results of the enzymatic deacetylation of pregnanes and androstanes substituted by various groups containing an sp²-hybridised C-atom in ring D could suggest that CAL B activity seems to be conditioned by the occurrence of a polar carbon double bond in this part of the steroid skeleton. Ten new steroid derivatives were obtained through this approach.     

Immobilization of lipases on different carriers and their use in synthesis of pentyl isovalerates

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.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Lipase-catalyzed synthesis of L-phenylalanyl-D-glucose

Regioselective synthesis of L-phenylalanyl ester of D-glucose with unprotected L-phenylalanine and D-glucose was carried out in organic solvents using lipases from Rhizomucor miehei (RML) and porcine pancreas (PPL). The reaction was investigated in terms of free unprotected L-phenylalanine, D-glucose, RML and PPL concentrations, buffer salts (pH and concentration), enzyme reusability and incubation period. Under the experimental conditions employed, both the enzymes exhibited good esterification potentialities, with RML exhibiting better conversions (maximum yield, 98%) than PPL (maximum yield, 75.6%). Reactions in the presence of buffer salts gave about 10% higher yields than those in their absence. Two-dimensional heteronuclear single quantum coherence transfer (HSQCT) NMR spectral analysis confirmed the formation of five diastereomeric esters: three different L-phenylalanyl-D-glucose monoesters (6-O: 24.1%, 3-O: 23.3% and 2-O: 19.2%) and two different diesters (2,6-di-O: 16.6% and 3,6-di-O: 16.8%) in an esterification yield of 92.4%.     

pH-optima in lipase-catalysed esterification

Though lipases are frequently applied in ester synthesis, fundamental information on optimal pH or substrate concentration, can almost only be found for the reverse reaction – hydrolysis. This study demonstrates that the pH-optima of lipase-catalysed esterifications differ significantly from the optima of the hydrolysis reaction. In the esterification of n-butanol and propionic acid with lipases of Candida rugosa (CRL) and Thermomyces lanuginosa (TLL) pH-optima of 3.5 and 4.25, respectively, were found. This is about 3–4 units (CRL) and 7 units (TLL) in pH lower than optimum for hydrolysis. Enzyme activity increased with increasing concentrations of protonated acid indicating that the protonated acid rather than the deprotonated form is substrate for esterification. The rate of esterification can be drastically increased by ensuring acid concentrations up to 1000 mmol L^?1 for CRL and 600 mmol L^?1 for TLL in the reaction system.     

Lipase catalysed synthesis of optically enriched alpha-haloamides

An efficient lipase catalysed synthesis of optically enriched alpha-halogenated amides with concomitant optical enrichment of the starting alpha-haloesters is described. Candida antarctica lipase (CAL) was found to be a better catalyst over porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL). The effect of different organic solvents was also studied.     

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents

BackgroundUnderstanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood.MethodsDynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPL_mut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed.ResultsPPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPL_mut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents.ConclusionsOnly the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid.General significanceThis study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue–residue interactions during lid opening movement in water and in octanol.     

A Contrtbutlon to Evaluate the Size of the Aciiveslte of the Lipasex from Candida Rugasa Pseudomonas Cepecia

The synthesis of some azido hydroxy butanoaks in enantiomexidly pure fonn is presented. The racemic educts were prepared in few steps. These munates att resolved with the aid of lipases hdseudomm cepia(KL) and Candida rugasa (CRL) in order to evaluate not only the size of the active sites but also to get some hints about the distance between active sites and the anchor groups.     

Lipase-catalyzed chemo- and enantioselective acetylation of 2-alkyl/aryl-3-hydroxypropiophenones

The chemo- and enantioselective capabilities of porcine pancreatic lipase (PPL) in tetrahydrofuran, and Candida rugosa lipase (CRL) in diisopropyl ether have been investigated for the acetylation of racemic 2-alkyl/aryl-3-hydroxypropiophenones, which are important precursors in the synthesis of biologically active chromanones and isoflavanones. A highly chemoselective acetylation of primary hydroxy group in preference to phenolic hydroxy group leading to the formation of enantiomerically enriched monoacetates has been observed.     

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.     

Effect of ionic liquids,organic solvents and supercritical CO2 pretreatment on the conformation and catalytic properties of Candida rugosa lipase

The objective of this work is to assess the structure and activity of Candida rugosa lipase (CRL) pretreated with seventeen ionic liquids (ILs), five organic solvents and super-critical carbon dioxide (SC-CO₂). The results revealed that anion selection of ILs showed generally much greater effects on CRL esterification activity than cation choice, and CRL pretreated by ILs with strong water miscible properties showed very low esterification activity. The highest CRL activity treated with ILs [Hmim][PF6] was obtained with the value of 45078.0 U/g-protein. Furthermore, the CRL activities pretreated with five conventional organic solvents were also examined and the values increased with the log P decrease of organic solvents when log P was lower than 2.0. Finally, the CRL activities were respectively 1.2- and 1.3-fold higher over the untreated ones after pretreatment with sub- and super-critical CO₂ under the pressures of 6 MPa and 15 MPa at 40 °C for 20 min. Further analyses via FT-IR demonstrated that the high activity of CRL pretreated with ILs, organic solvents and SC-CO₂ was probably caused by the changes of CRL secondary structure. In conclusion, the results in this work will be helpful for us to choose the suitable reaction medium in CRL biocatalysis and biotransformation reactions.