Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

Conversion of a pentane-1,3,5-triol derivative using lipases as chiral catalysts and possible function of the lid for the regulation of substrate selectivity and enantioselectivity

Abstract

The enantioselective desymmetrization of a prochiral 3-O-silyl protected pentane-1,3,5-triol derivative was achieved by lipase-catalysed hydrolysis. The lipase from B. cepacia led to 95.4% enantiomeric excess (ee)of the (R)-configured compound (R)-4 at a theoretical yield of 79% and was isolated with 98.2% ee and 27% yield. Furthermore, it was found that the ee switched from an excess of (R)-4 to an excess of (S)-4 during the course of the reaction using crude lipase from C. rugosa under the same conditions. This finding was investigated in detail and compared to the change of substrate selectivity known for the lipase from M. miehei. It is supposed that both phenomena may result from a movement of the lid.     

Selective synthesis of panthenyl esters by a kinetically controlled enzymatic process

Abstract

Panthenyl esters (panthenyl monoacetate and panthenyl diacetate) were synthesized in high yields (≈100%) by a kinetic reaction control using a commercial immobilized Candida Antarctica lipase B (Novozyme 435) in acetonitrile. The enzyme showed excellent synthetic activity, regioselectivity, and operational stability under the conditions used.     

Enrichment of pinolenic acid from pine nut oil via lipase-catalyzed ethanolysis with an immobilized Candida antarctica lipase

Abstract

Pinolenic acid (PLA) enrichment as an ethyl ester from pine nut oil was successfully accomplished in a batch reactor by lipase-catalyzed ethanolysis using Novozym 435 lipase from Candida antarctica as a biocatalyst. PLA is predominantly an sn-3 substituent of the pine nut oil triacylglycerol (TAG), where it accounts for about 39 mol% of the fatty acids esterified at that position. In the presence of ethanol, Novozym 435 exhibited sn-3 regiospecificity with respect to the TAG of pine nut oil. The effect of the molar ratio of reactants on PLA enrichment by ethanolysis was investigated. The molar ratios of pine nut oil to ethanol were varied from 1:20 to 1:100. A fatty acid ethyl ester (FAEE) fraction with higher PLA content was obtained in the early stage of the reaction, although the yield of PLA was small. However, the PLA content of the FAEEs decreased with increasing reaction time, while the yield of PLA increased. The molar ratio of pine nut oil to ethanol that produced the optimum content and yield of PLA in FAEEs was 1:80.     

Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media

Isoamyl butyrate, an important fruity flavor ester, was synthesized using Rhizomucor miehei lipase immobilized on a weak anion exchange resin (Lipozyme IM-20) by the esterification of isoamyl alcohol and butyric acid. The effects of various reaction parameters such as substrate and enzyme concentrations, substrate molar ratio, temperature and incubation time have been investigated. Yields above 90% were obtained with substrate concentrations as high as 2.0 M. No evidence of enzyme inhibition by butyric acid was present up to 1.0 M concentration. Acid inhibition and, to a small extent, alcohol inhibition were evident above 1.0 M substrate concentration. Conversions reached a saturation value by the end of 24–48 h of incubation due to the accumulation of the water of reaction. The equilibrium was successfully pushed forward towards esterification by removing the accumulated water using a molecular sieve.Journal of Industrial Microbiology & Biotechnology (2000) 25, 147–154. Received 09 February 2000/ Accepted in revised form 24 June 2000     

Isolation of a novel lipase producing fungal isolate Aspergillus fumigatus and production optimization of enzyme

Abstract

Fungal lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. In the present study, Aspergillus fumigatus isolated from oil-contaminated soil produced good amount of lipase activity with galactose (1%) as carbon source and peptone (0.1%) as nitrogen source after 72?h of incubation in the production medium at 45?°C and pH 10.0. The isolated enzyme was found to give its optimum reaction temperature at 40?°C and pH 9.0 with the substrate used as p-nitrophenyl benzoate. The activity of lipase was inhibited by the presence of metal ions. A 6.68-fold increase for lipase production was obtained by one variable at a time. Based on the findings of present study, lipase of A. fumigatus is a potential lipase and a candidate for industrial applications such as bioremediation, detergent, leather and pharmaceutical industries.     

Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

Improving performance of Yarrowia lipolytica lipase lip2-catalyzed kinetic resolution of (R,S)-1-phenylethanol by solvent engineering

Extracellular Yarrowia lipolytica lipase Lip2 (YLIP2) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-1-phenylethanol by solvent engineering with different kinds of binary solvent. The enantioselectivity was significantly improved by the addition of 1, 4-dioxane. The reaction parameters including co-solvent concentration, reaction temperature, and the reaction time were optimized. When the reaction was carried out with n-hexane in the presence of 0.8% 1,4-dioxane at 50°C for 72 h, the enantiomeric excess of product markedly increased to 99.1% from 66% in pure n-hexane; the enantiomeric ratio was higher than 200, which was 500-fold compared with that in pure n-hexane. The results indicated that it is very important to design the proper co-solvents, especially to create appropriate micro-environment for YLIP2 for catalyzing the resolution of (R,S)-1-phenylethanol.     

Highly efficient production of monoglycerides by the continuous removal of fatty acids from lipase-catalyzed oil hydrolysis

Highly efficient production of monoglycerides was achieved from lipase-catalyzed oil hydrolysis by the continuous addition of CaCl₂ to remove the fatty acids produced. A fusion protein produced by connecting a cellulose-binding domain of Trichoderma hazianum cellulase to Bacillus stearothermophilus L1 lipase was used as a model 1,3-regiospecific lipase. The reaction was performed at pH 10 and 50°C, and the relationship between continuous removal of fatty acids and the production of monoglyceride was investigated by microscopic and HPLC analysis of oil emulsions and the reaction products. Without the addition of Ca^{2 +} the reaction was inhibited by fatty acids, with the decrease in reaction rate being proportional to the concentration of fatty acids. When CaCl₂ was continuously added in a 1:2 molar ratio with the released fatty acids, the reaction progressed unimpeded due to the formation of Ca-soaps. Both the yield and the fraction of monoglyceride in the reaction product increased due to the continuous removal of fatty acids.     

The inhibitory kinetics and mechanism of glycolic acid on lipase

Abstract

Obesity is prone to cause a variety of chronic metabolic diseases, and it has aroused people’s attention that the rapid increase in the global population of obese people in the past years. As a kind of weight-loss drug acting in the intestine, lipase inhibitor does not enter the bloodstream without producing central nervous side effects. Because they do not affect the metabolism system, lipase inhibitors and obesity have become one of the hot spots in recent years. Glycolic acid is a new substrate analog inhibitor with the value of the semi-inhibitory concentration of lipase is estimated to be 17.29?±?0.14?mM. Using the plots of Lineweaver-Burk, the inhibition mechanism of lipase by glycolic acid was reversible and the inhibition type belongs to competitive inhibition with a K_I value of 19.61?±?0.26?mM. The inhibitory kinetics assay showed that the microscopic velocity constant k₊₀ of inhibition kinetics is 1.79?×?10^?3?mM^?1s^?1, and k_?0 is 0.73?×?10^?3 s^?1. The results of UV full-wavelength scanning on product cumulative, fluorescence quenching and molecular simulation also indicated that glycolic acid and substrate competitive with lipase by binding to Lys137. Thereby glycolic acid inhibiting the oxidation-catalyzed reaction and reducing the product of the enzyme and substrate. This adds a new direction for the search for lipase inhibitors and provides new ideas about the development of anti-obesity drugs.

Communicated by Ramaswamy H. Sarma     

Microwave-assisted synthesis of new benzimidazole derivatives with lipase inhibition activity

Abstract

A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester hydrochlorides of phenylacetic with 4,5-dichloro-1,2-phenylenediamine under microwave irradiation leads to the benzimidazole derivatives with good yields and in short reaction times. After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.     

Immobilized lipases from Candida antarctica for producing tyrosyl oleate in solvent-free medium

Abstract

Two immobilized lipases from Candida antarctica have been compared for the direct esterification of tyrosol with oleic acid in equimolar conditions and in the absence of organic solvent. Candida antarctica lipase B was immobilized on octyl-silica agglomerates and compared with commercial Novozym 435. Reduction of tyrosol particle size to 0.1 mm significantly increased the reaction rate with both immobilized lipases, and reduced pressure improved the final tyrosyl oleate yield up to 95% (w/w) in both cases. Immobilized lipases were recovered and reutilized in three consecutive trials with negligible inactivation. Under optimum conditions, a product mixture comprising more than 95% of tyrosyl oleate (w/w) was attained in less than 2 hours. Finally, the index of antioxidant activity obtained, according to the Rancimat method, indicated that tyrosyl oleate was slightly more effective than tyrosol as an antioxidant in a low polar matrix.     

Solvent-free lipase-catalysed synthesis of saccharide-fatty acid esters: closed-loop bioreactor system with in situ formation of metastable suspensions

Abstract

Saccharide-fatty acid esters – biodegradable, biocompatible and non-ionic bio-based surfactants derived from inexpensive renewable agricultural sources, utilized in foods, cosmetics and pharmaceuticals, have been synthesized under solvent-free conditions using a closed-loop system operated under continuous recirculation consisting of a reservoir, a peristaltic pump and a packed-bed bioreactor containing immobilized Rhizomucor miehei lipase (RML). Metastable suspensions of saccharide crystals were formed through continuous stirring in the reservoir with an in-line filter of 180 µm normal size preventing larger crystals from clogging the tubing. The initial reaction medium consisted of oleic acid/fructose-oleic acid esters 95/5 w/w, with saccharide added periodically to the reservoir to replace consumed acyl acceptor substrate. The liquid-phase water concentration was retained at a previously determined optimal value of ～0.4 wt % through free evaporation of the co-product, water, in the reservoir during the initial phase of the reaction, and N₂(g) bubbling and vacuum pressure after 40 h of reaction. After 200 h, the reaction mixture contained 84 wt % ester, of which 90% of the ester consisted of monoester. Equivalently, the productivity was 0.195 mmol_Ester h^??1 g_RML^??1. The resultant technical grade product can potentially be used directly, without further purification. A mathematical model based on mass balances and a zeroth-order kinetic model was successfully developed to predict the concentration of substrates (oleic acid and saccharide) in the reservoir during the time course of reaction.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

Temperature-dependence of enantioselectivity and desymmetrization in the acetylation of 2-mono- and 2,2-di-substituted 1,3-propanediols by a novel lipase isolated from the yeast Cryptococcus spp. S-2

The effect of temperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d) by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the temperature range of ?20°C to 40°C, while diacetylation of 1b occurred considerably even at 0°C. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).     

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.     

Chemical modification of lipases with various hydrophobic groups improves their enantioselectivity in hydrolytic reactions

Semi-purified lipases from Candida rugosa, Pseudomonas cepacia and Alcaligenes sp. were chemically modified with a wide range of hydrophobic groups such as benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, t-butoxycarbonyl, lauroyl and acetyl moieties. The Candida rugosa lipase MY modified with the benzyloxycarbonyl group (modification ratio = 84%) brought about a 15-fold increase in enantioselectivity (E value) towards the hydrolysis of racemic butyl 2-(4-ethylphenoxy)propionate in an aqueous buffer solution, although the enzymatic activity was decreased. The origin of the enantioselectivity enhancement by chemical modification of the lipase is attributed to a significant deceleration in the initial reaction rate for the incorrectly binding enantiomer.     

Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library

The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60°C and at alkaline pH of 10.5.     

Biocatalytic synthesis of chiral N-(2-hydroxyalkyl)-acrylamides

Abstract

The preparation of a series of novel chiral N-(2-hydroxylalkyl)-acrylamides through a lipase-catalyzed resolution of racemic alkanolamines is described. The absolute stereochemistry and enantiomeric excess of the products were determined by a modified Mosher's method. The method was validated for this particular case by the synthesis of an enantiomerically pure product. Moreover, the stereoselective behavior of the lipase in this reaction is discussed.     

Preparation of a Fludarabine Intermediate via Selective Alkylation of 2-Fluoroadenine

The reaction between 2-fluoroadenine (3) and 1,3,5-tri-O-benzyl-1-α-d-chloroarabinofuranose (4) with potassium t-amylate was evaluated in various solvents to afford 9-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (5) and the corresponding α-anomer (6). In addition, 7-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (7) and an unusual “bis-fluoroadenine” nucleoside (8) were isolated as by-products. The highest anomeric ratio (β/α > 10) and conversion (>80%) were observed with the highly polar solvent sulfolane. This reaction was demonstrated on gram scale as a practical laboratory synthesis of 5, a known intermediate in the synthesis of fludarabine.