Optimization of immobilized Candida rugosa lipase LIP2-catalyzed resolution to produce l-menthyl acetate

Esterification of l-menthol by lipase is a highly selective method for the resolution of dl-menthol. The present work focuses on the reaction parameters that affect lipase-catalyzed synthesis of l-menthyl acetate in n-hexane using triacetin as acyl donor. Genetically engineered LIP2, an isoform of Candida rugosa lipase, was used as a biocatalyst in the present study. The main objectives of the work were to develop an approach that would enable a better understanding of relationships between the variables (reaction time, temperature, enzyme amount, substrate molar ratio) and the response (molar conversion) for l-menthyl acetate synthesis, and to obtain the optimum conditions for synthesis. By using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis, we found that substrate molar ratio and enzyme amount were the most important variables for the reaction. Based on ridge max analysis, the optimum synthesis conditions were found to be: reaction time 2.2 days, temperature 34.3°C, enzyme amount 0.09 g and substrate molar ratio (dl-menthol:triacetin) 1:1.9, and molar conversion of dl-menthol to l-menthyl acetate was calculated to be 50%. An experiment under optimum conditions was carried out and molar conversion of 48.3% was obtained.     

Hydrolysis of triacetin catalyzed by immobilized lipases: effect of the immobilization protocol and experimental conditions on diacetin yield

The effect of the immobilization protocol and some experimental conditions (pH value and presence of acetonitrile) on the regioselective hydrolysis of triacetin to diacetin catalyzed by lipases has been studied. Lipase B from Candida antarctica (CALB) and lipase from Rhizomucor miehei (RML) were immobilized on Sepabeads (commercial available macroporous acrylic supports) activated with glutaraldehyde (covalent immobilization) or octadecyl groups (adsorption via interfacial activation). All the biocatalysts accumulated diacetin. Covalently immobilized RML was more active towards rac-methyl mandelate than the adsorbed RML. However, this covalent RML preparation presented the lowest activity towards triacetin. For this reason, this preparation was discarded as biocatalyst for this reaction. At pH 7, acyl migration occurred giving a mixture of 1,2 and 1,3 diacetin, but at pH 5.5, only 1,2 diacetin was produced. Yields were improved at acidic pH values and in the presence of 20% acetonitrile (to over 95%). RML immobilized on octadecyl Sepabeads was proposed as optimal preparation, mainly due to its higher specific activity. Each enzyme preparation presented very different properties. Moreover, changes in the reaction conditions affected the various immobilized enzymes in a different way.     

Eryngase: a Pleurotus eryngii aminopeptidase exhibiting peptide bond formation activity

An aminopeptidase that has peptide bond formation activity was identified in the cell-free extract of carpophore of Pleurotus eryngii. The enzyme, redesignated as eryngase, was purified for homogeneity and characterized. Eryngase had a molecular mass of approximately 79 kDa. It showed somewhat high stability with respect to temperature and pH; it was inhibited by iodoacetate. Among hydrolytic activities toward aminoacyl-p-nitroanilides (aminoacyl-pNAs), eryngase mainly hydrolyzed hydrophobic l-aminoacyl-pNAs and exhibited little activity toward d-Ala-pNA and d-Leu-pNA. In terms of peptide bond formation activity, eryngase used various aminoacyl derivatives as acyl donors and acceptors. The products were all dipeptidyl derivatives. Investigation of time dependence on peptide synthesis revealed that some peptides that are not recognized as substrates for hydrolytic activity of eryngase could become good targets for synthesis. Furthermore, eryngase has produced opioid dipeptides––l-kyotorphin (l-Tyr-l-Arg) and d-kyotorphin (l-Tyr-d-Arg)––using l-Tyr-NH₂ and d- and l-Arg-methyl ester respectively as an acyl donor and acceptor. Yield evaluation of kyotorphin synthesis indicated that the conversion ratio of substrate to kyotorphin was moderate: the value was estimated as greater than 20%.     

Efficient kinetic resolution of dl-menthol by lipase-catalyzed enantioselective esterification with acid anhydride in fed-batch reactor

Acid anhydrides were used as highly reactive and non-water-producing acyl donors for hydrolase-catalyzed enantioselective esterification. Efficient kinetic resolution of dl-menthol has been achieved via lipase-catalyzed enantioselective esterification in cyclohexane when propionic anhydride as an acyl donor was continuously fed into a reactor containing dl-menthol and Candida cylindracea lipase OF 360, while a high concentration of the acid anhydride in a batch reaction system with a dehydrated organic solvent did not facilitate the reaction, because water necessary for the enzyme function was consumed by the competing hydrolysis of the anhydride catalyzed by the same enzyme. The efficiency of this fed-batch reaction system using acid anhydride was higher and the enzyme stability in repeated use was much better than those of conventional batch and fed-batch reaction systems using propionic acid as an acyl donor. The optical purity (more than 98% e.e.) of the l-menthyl ester produced in the fed-batch system using the anhydride was comparable to that in the system using the corresponding acid. *** DIRECT SUPPORT *** AG903062 00002     

Enzymic synthesis of fructose monooleate in a reduced pressure pilot scale reactor using various acyl donors

Enzymic synthesis of fructose esters was studied under reduced pressure. Different acyl donors were tested, and immobilized Candida antarctica lipase was used as biocatalyst. Influences of pressure, nature of the acyl donor, molar ratio sugar/acyl donor were investigated. Pressure had the greatest influence. At 200 mbar, more than 90% of fructose was acylated compared to 50% under atmospheric pressure. This is explained by the evaporation of reaction by-product (methanol or water) that shifted the equilibrium. C. antarctica lipase catalyzed sugar ester synthesis very efficiently using rapeseed oil as acyl donor. Moreover, synthesis performed with an equimolar mixture of both substrates gave promising results. Although the reaction rate was slower than synthesis performed with an excess of fatty acid, fructose monooleate concentration was still high (44 g l⁻¹ instead of 56 g l⁻¹) and the residual acyl donor concentration was very low. Downstream processes for the recovery of pure fructose monooleate were simplified in this case.     

Glycerol triacetate as solvent and acyl donor in the production of isoamyl acetate with Candida antarctica lipase B

Glycerol triacetate was successfully used as a green solvent and as the acyl donor in the transesterification of isoamyl alcohol to produce isoamyl acetate using free and immobilized Candida antarctica lipase B. Immobilized lipase was more catalytically active than free lipase and could be easily separated from the reaction mixture by filtration. In addition, it was found that increasing either the reaction temperature or the enzyme to substrate ratio increased the conversion of isoamyl alcohol. Using triacetin as the solvent also enabled the separation of product by simple extraction with petroleum ether and catalyst recycling.     

Enzymatic synthesis of l-ascorbyl linoleate in organic media

A novel l-ascorbyl fatty acid ester, l-ascorbyl linoleate was successfully prepared by enzymatic esterification and transesterification in a non-aqueous medium using immobilized lipase as biocatalyst. Changes in enzymatic activity and product yield were studied for the following variable: the nature of the fatty acid, the fatty acid concentration and water content. The yield of synthesis for the C18 unsaturated fatty acids were higher than for the C18 saturated fatty acid. Initial enzyme concentration does not affect the equilibrium of the reaction. And the product yield (33.5%) in the transesterification was higher than that of the esterification (21.8%) at a high-substrate concentration 0.3 M. The medium water content was found to have a distinct influence on the l-ascorbyl linoleate synthesis.These authors contributed equally to the article.     

Lipase-catalyzed esterification of conjugated linoleic acid with <Emphasis Type="SmallCaps">l</Emphasis>-carnitine in solvent-free system and acetonitrile

Lipase-catalyzed esterification of conjugated linoleic acid (CLA) with l-carnitine in solvent-free system and acetonitrile was studied. Three lipases (Novzym 435, Amamo AY30 and Amano AYS) have been assayed as suitable biocatalysts in the reaction. It was found that Amano AY30 was the most effective biocatalyst in both solvent-free system and acetonitrile. The conversion rate varied from 8.05 to 60.9% in terms of reaction conditions such as the amount of lipase, the presence of water, the amount of molecular sieves and reaction time. The conversions of substrate in solvent-free system were higher than that in acetonitrile. When the substrates were 1 mmol CLA and 1 mmol l-carnitine, the maximum conversion (60.9%) was obtained in solvent-free system with 150 mg lipase AY30, 50% water content and 150 mg molecular sieves at the reaction time of 24 h. A novel CLA ester product was successfully isolated and characterized by ESI-MS and ¹H NMR.     

Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.     

Efficient synthesis of enantiomeric ethyl lactate by <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (CALB)-displaying yeasts

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid.     

A mutant leucine aminopeptidase from <Emphasis Type="Italic">Streptomyces cinnamoneus</Emphasis> with enhanced <Emphasis Type="SmallCaps">l</Emphasis>-aspartyl <Emphasis Type="SmallCaps">l</Emphasis>-amino acid methyl ester synthetic activity

l-Aspartyl l-amino acid methyl ester was synthesized using a mutant of a thermostable leucine aminopeptidase from Streptomyces cinnamoneus, D198 K SSAP, obtained in previously. A peptide of high-intensity sweetener, l-aspartyl-l-phenylalanine methyl ester, was selected as a model for demonstrating the synthesis of l-aspartyl l-amino acid methyl ester. The hydrolytic activities of D198 K SSAP toward l-aspartyl-l-phenylalanine and its methyl ester were, respectively, 74-fold and fourfold higher than those of wild type. Similarly, the initial rate of the enzyme for l-aspartyl-l-phenylalanine methyl ester synthesis was over fivefold higher than that of wild-type SSAP in 90% methanol (v/v) in a one-pot reaction. Furthermore, other l-aspartyl l-amino acid methyl esters were synthesized efficiently using D198 K SSAP. Results show that the substitution of Asp198 of SSAP with Lys is effective for synthesizing l-aspartyl l-amino acid methyl ester.     

α-Chymotrypsin-Catalyzed Peptide Synthesis Using <Emphasis Type="Italic">N</Emphasis>-Protected <Emphasis Type="SmallCaps">D</Emphasis>-Amino Acid Carbamoylmethyl Esters as Acyl Donors

α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml⁻¹; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h.     

Stereoselective esterification of dl-menthol by polyurethane-entrapped lipase in organic solvent

Stereospecific esterification of dl-menthol was studied by the use of immobilized lipase in an adequate water-saturated organic solvent system. Lipase from Candida cylindracea immobilized by entrapment with urethane prepolymers and 5-phenylvaleric acid as the acyl donor were chosen based on the stereoselectivity and the yield of l-menthyl ester. Water-saturated cyclohexane or isooctane was found to be the most suitable solvent system. Entrapment significantly enhanced the operational stability of lipase.     

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid

Summary. Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from N ^α -(tert-butyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained d-series rather than l-series (in the present case; N ^α -Boc-d-Ala and N ^α -Boc-l-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible. Received August 10, 2000 Accepted December 2, 2000     

Lipase catalyse glycerolysis for kinetic resolution of racemates

Candida antarctica lipase B catalyzed kinetic resolution of representative secondary alcohols, esters, and amine was successfully performed using triacetin or glycerol as solvents and acyl donor/acceptor. High conversions and enantioselectivities were achieved and the product was easily separated by simple extraction with diethyl ether.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Synthesis of 6-O-palmitoyl L-ascorbic acid catalyzed by Candida antartica lipase

The synthesis of ascorbyl palmitate was achieved in non aqueous medium with an immobilized lipase from Candida antartica as biocatalyst. This enzymatic synthesis is perfectly regioselective. When palmitic acid methyl ester is used as acyl donor, 68 % of ascorbic acid is converted against 56 % in the presence of palmitic acid. These optimal values were obtained when the initial molar ratio of ascorbic acid to acyl donor is 1:5.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.