Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study

In this paper, we report a comprehensive kinetic study on esterification of lauric acid with lauryl alcohol catalysed by commercial porcine pancreatic lipase (PPL) in the form of cross-linked enzyme crystals (CLEC) using glutaraldehyde as the cross linker. The stability of the CLEC was better than the immobilized enzyme for practical applications. Comparative studies using six different solvents having hydrophobicity (log p) values ranging from 0.70 to 3.50 revealed that the esterification reaction was favoured in hydrophobic solvents. The kinetics of the esterification reaction conformed with the so-called Ping-Pong–Bi-Bi mechanism with alcohol inhibition.     

Engineering aspects of immobilized lipases on esterification: A special emphasis of crowding,confinement and diffusion effects

Cross‐linked enzyme crystal (CLEC) and sol‐gel entrapped pseudomonas sp. lipase were investigated for the esterification of lauric acid with ethanol by considering the effects of reaction conditions on reaction rate. The activation energy for the reaction was estimated to be 1097.58 J/mol and 181.75 J/mol for sol‐gel and CLEC entrapped lipase respectively. CLEC lipase exhibited a marginal internal diffusion effect on reaction rate over sol‐gel lipases and found to be interesting. The overall reaction mechanism was found to conform to the Ping Pong Bi Bi mechanism. The higher efficiency of sol‐gel lipases over CLEC lipases in esterification reaction is mainly due to the combined effects of crowding, confinement and diffusional limitations.     

Studies on crystallization and cross-linking of lipase for biocatalysis

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2–5 μm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using β cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Polyvinyl alcohol--trypsin as a catalyst for amino acid ester synthesis in organic media

The bovine trypsin-catalyzed synthesis of N-alpha-benzoyl-DL-arginine esters from N-benzoyl-DL-arginine were studied in various organic solvents. Trypsin was immobilized to polyvinyl alcohol (PVA) by adsorption from its aqueous solutions. Immobilized enzyme showed higher catalytic activities than free enzyme for amino acid esterification in ethanol. The yield of ester is strongly dependent upon the PVA/trypsin ratio and water content in the reaction medium. The rate and equilibrium constant of the ester formation reaction are also dependent on water content.     

Enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol in organic solvent media

The enzymatic esterification of dihydrocaffeic acid with linoleyl alcohol, using immobilized lipases (Lipozyme IM 20 and Novozym 435), was investigated in selected organic solvent media. Novozym 435 was found to be more efficient for catalyzing the esterification reaction. The highest enzymatic activity of 0.89 μmol esterified linoleyl alcohol/g solid enzyme/min was obtained in a hexane/2-butanone mixture of 75:25 (v/v), with an esterification yield of 75%; however, an increase in the 2-butanone proportion in the mixture up to 50% (v/v) resulted in a decrease in enzymatic activity and esterification yield to 0.38 μmol esterified linoleyl alcohol/g solid enzyme/min and 40%, respectively. The maximum esterification yield of 99.3% was obtained with a dihydrocaffeic acid to linoleyl alcohol ratio of 1:8. The electrospray ionization-mass spectroscopic structural analysis of the end products confirmed the biosynthesis of dihydrocaffeic acid ester of linoleyl alcohol, which demonstrated an anti-radical activity using 2,2-diphenyl-1-picrylhydrazyl as a radical model.     

Resolution of (RS)-proglumide using lipase from Candida cylindraceae

Proglumide is used in the treatment of neuropathic pain. It acts by inhibiting peptide cholecystokinin (CCK). Neural injury produces an elevation in plasma CCK. Proglumide has been also shown to augment the analgesic effect of sustained release morphine in neuropathic pain. Currently proglumide is administered as a racemic mixture. In the present study, an attempt is made to separate the racemic mixture of the drug using lipase obtained from Candida cylindracea by stereoselective esterification. Enzymatic stereoselective esterification was carried out in organic solvents. The resolution was studied using a chromatographic column with a chiral support and mass spectrometry. The reaction conditions for stereoselective esterification including amount of substrate, amount of enzyme, alcohol, solvent and temperature were optimised during the present investigation. Butanol and hexanol were found to be suitable for formation of S and R esters, respectively. Hexane was the best solvent for esterification and the optimum temperature was found to be 30 degreesC.     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Enzymatic gallic acid esterification

Gallic acid esters of n-propyl and amyl alcohols have been produced by enzymatic synthesis in organic solvents using immobilized tannase. Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol. The enzyme shows broad alcohol specificity. However, the enzyme exhibits absolute specificity for the acid portion of the ester. Studies were carried out on K(m), V(max), pH, and temperature optima.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

Evaluation of regioselectivity of lipases based on synthesis reaction conducted with propyl alcohol, isopropyl alcohol and propylene glycol

Preliminary investigations on the regioselectiviy of various lipases were performed. Ten commercial lipases from different origins, including three immobilized lipases, were tested by esterification reaction between caprylic acid and propyl or isopropyl alcohol in n-hexane. Reaction products were analyzed with a gas chromatograph. Best yields were obtained with immobilized lipase IM60 from Rhizomucor miehei. Therefore, this enzyme was chosen as biocatalyst for a second step of regioselectiviy study with propylene glycol which bears primary and secondary alcohol groups. It was shown, by using several solvents, that polarity could influence the product profile in situations in which multiple products of various polarities can be formed. Furthermore, the major role of silica gel in reaction mixture was established.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Use of lipases in the resolution of racemic ibuprofen

Summary Resolution of (R,S)-ibuprofen enantiomers by esterification in different organic solvents was studied using Candida cylindracea lipase. This enzyme preparation had high enantiospecificity for S(+)-ibuprofen in the esterification reaction of a racemic ibuprofen with primary alcohols. The esterification yields of secondary alcohols were much lower than those of primary alcohols. Esterification with tertiary alcohols was not observed. The synthesis of esters was profoundly affected by the amount of water in the reaction mixture. C. cylindracea lipase was active only in very hydrophobic solvents. The esterification activity of the lipase was reduced significantly by addition of water. The R- and S-enantiomers of ibuprofen were determined without derivatization by HPLC using a chiral column.     

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

Kinetics of Enantioselective Esterification of Naproxen by Lipase in Organic Solvents

The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Erratum to: Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40?°C, geraniol to oleic acid molar ratio of 5:1, 150?rpm and 10?wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5?wt%) and temperature of 50?°C afforded nearly complete reaction conversion after 1?h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.