Microbial catalyzed esterification of primary and secondary alcohols in organic solvent

Summary Microbial esterification of primary and secondary short chain alcohols with butyric acid in organic solvent has been studied. A screening for 2-octylbutyrate hydrolysis between microorganisms belonging to different genera allowed the selection of 12 microbial strains able to hydrolyze this substrate. The potential of these microorganisms in catalyzing ester formation was checked for various 1- and 2-alkylbutyrate derivatives:Rhizopus delemar,Rhizopus oryzae andSarcina lutea promoted both 1- and 2-alkylbutyrate synthesis with almost complete molar conversion of the primary alcohols, whileAspergillus niger andYarrowia lipolytica only catalyzed 1-alkanol esterification.     

Efficient and selective microbial esterification with dry mycelium of Rhizopus oryzae

The use of dry mycelium of Rhizopus oryzae as biocatalyst for ester production in organic solvent has been studied. Mycelia with notable carboxylesterase activity were produced when different Tweens (20, 40, 60 and 80) were employed as main carbon source for the growth. Dry mycelium of four strains of Rhizopus oryzae proved effective for efficiently catalysing the synthesis of different flavour esters (hexylacetate and butyrate, geranylacetate and butyrate) starting from the corresponding alcohol and free acid, including acetic acid. The esterification of the racemic mixture of 2-octanol and butyric acid proceeded with high enantioselectivity (R-ester produced with enantiomeric excess > or =97%) when Rhizopus oryzae CBS 112.07 and Rhizopus oryzae CBS 260.28 were employed.     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Rice bran lipase catalyzed esterification of palm oil fatty acid distillate and glycerol in organic solvent

Rice bran lipase (RBL) was delipidated to enhance its stability in organic solvent and its esterification activity at elevated temperature. The esterification activity of delipidated RBL increased as temperature was increased from 45 to 65°C. The esterification activity of delipidated RBL at 65°C was about 14 times greater than that of the non-delipidated RBL. As temperature was further increased to 75°C, the non-delipidated RBL lost all esterification activity, whereas the delipidated RBL retained approximately 48% of its esterilication activity. The delipidated RBL maintained a relative esterification activity greater than 80% after 16 h of incubation in hexane, whereas the non-delipidated RBL maintained a relative esterification activity of only 50%. A method for production of acylglycerol using delipidated RBL to esterify palm oil fatty acid distillate (PFAD) with glycerol in hexane was successfully developed. The effects of reaction temperatures and type of water removal agents (silica gel and molecular sieve) on the degree of esterification were also examined. A 4 h reaction at 65°C, catalyzed by delipidated RBL and using silica gel as the water removal agent resulted in 53.8% esterification. Thin layer chromatography analysis suggested that the esterified product was primarily comprised of mono-and di-acylglycerols.     

Specificity and glyceride synthesis by mycelial lipases of Rhizopus delemar

Mycelial lipase activity of the mould Rhizopus delemar was purified by gel filtration chromatography to three distinct proteins of notable lipase activity. The three enzymes were designated A′, B′ and C′, according to elution volumes from a Sephadex G150 column. The capacity of the three lipases to catalyse glyceride synthesis from free fatty acids and glycerol indicated a tendency towards short-chain and unsaturated fatty acids in preference to long-chain saturated fatty acids. The postional specificity of all lipases involved in such synthetic reactions indicated the formation of ester bonds at positions 1 and 3 of glycerol.     

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.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

Production of mycelium biomass and ethanol from paper pulp sulfite liquor by Rhizopus oryzae

The cultivation conditions for Rhizopus oryzae grown in synthetic medium and paper pulp spent sulfite liquor (SSL) were investigated to achieve high biomass and ethanol yields using shake flasks and bioreactors. The fungus assimilated the hexoses glucose, mannose and galactose, and the pentoses xylose and arabinose as well as acetic acid which are present in SSL. The assimilation of hexoses was faster than pentoses during cultivation in a synthetic medium. However, all sugars were assimilated concomitantly during growth in SSL supplemented with ammonium, magnesium, calcium, phosphate, sulfate and trace amounts of some other metal ions (SSL-S). The medium composition had an important influence on biomass yield. The highest biomass yields, viz. 0.18 and 0.43 g biomass/g sugar were obtained, when the cells were cultivated in shake flasks with a synthetic medium containing glucose as carbon and energy source and SSL-S, respectively. The corresponding yields in a bioreactor with more efficient aeration were 0.22 and 0.55 g/g. In addition to the biomass, ethanol, lactic acid, and glycerol were important extracellular metabolites of the cultivation with maximum yields of 0.37, 0.30 and 0.09 g/g, respectively. When the source of sugars in the medium was exhausted, the fungus consumed the metabolites produced, such that the liquid medium was depleted of potential oxidizable nutrients. In general, there was a direct competition between lactic acid and ethanol among the metabolites. Poor medium compositions and cultivation conditions resulted in higher yields of lactic acid, whereas the ethanol and biomass yields were higher in rich media. SSL-S supported good growth of mycelium and a high ethanol yield.     

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.     

Removal of water produced from lipase-catalyzed esterification in organic solvent by pervaporation

Esterification of oleic acid with n-butanol in the presence of Lipozyme(R) was carried out at 25 degrees C in isooctane with various initial water activities. Initial reaction rate as well as equilibrium conversion decreased at high initial water activity. Therefore, removal of water present in the reaction mixtures was essential. A pervaporation process was applied to the lipase-catalyzed synthesis of n-butyloleate to remove water. Pervaporation selectively separated water from the reaction mixture using a nonporous polymeric membrane, cellulose acetate. Therefore, pervaporation is potentially applicable to remove the water produced from various enzymatic processes, such as synthesis of various esters, peptides, and glycosides in a solvent system as well as in a solvent-free system. (c) 1995 John Wiley & Sons, Inc.     

Effect of temperature on lipase-catalyzed esterification in organic solvent

Summary For the esterification of 2-(4-ethylphenoxy)propionic acid catalyzed by lipase MY (Candida rugosa) in isopropyl ether containing a suitable amount of water, the enantioselectivity for the reaction has become higher as the reaction temperature increasing. In contrast, the reverse trend of the temperature effect has been observed for lipase AY (Candida rugosa). A model for these temperature dependence has been proposed.     

A novel transesterification catalyzed by phosphotriesterase in an organic solvent

Summary Phosphotriesterase (PTE) from Flavobacterium sp. was utilized for transesterification of the organophosphate insecticide, Paraoxon, using 2-phenylethyl alcohol as a nucleophile and dimethly sulfoxide as an organic solvent forming the converted phosphotriester compound, diethyl 2-phenylethyl phosphate.     

Kinetic cutinase-catalyzed esterification of caproic acid in organic solvent system

Practical application of any chemical reaction requires the knowledge of its kinetics; in particular if one wishes to be able to describe a chemical reactor over an extended range of reaction conditions or if one intends to optimize the reaction conditions, a suitable kinetic model must be obtained. In order to ensure that the model is applicable over a wide range of experimental conditions it should be based on a mechanistic scheme describing the fundamental steps involved in the reaction; the development of these kind of models can also be used to provide insight into the processes that are taking place.A kinetic study, using experiments carried out in a batch stirred reactor, has been made for the enzymatic esterification of caproic acid with ethyl alcohol catalyzed by Fusarium solani pisi cutinase. Different acid and alcohol concentrations (whilst also varying the acid/alcohol molar ratio) were tested and the results were used to identify the best reaction scheme to describe the results obtained over an extended range of conditions. Several different approaches were used to identify the most adequate mechanistic model, namely by resorting to the quasi stationary state and the rate-limiting hypothesis. The main kinetic characteristics observed in esterification reaction were found to follow an ordered Ping-Pong Bi–Bi mechanism but different modifications were used o ensure that the kinetic model was applicable over the entire range of experimental conditions that were covered.     

Lipase-catalyzed ammonolysis of trimethylsilylmethyl acetate in organic solvent

Lipase could catalyze the ammonolysis of trimethylsilylmethyl acetate in organic solvents and Novozym 435 was the best biocatalyst for the reaction. The influences of some factors on the reaction were investigated. Cyclohexane, n-hexane and heptane were found to be suitable reaction media and ammonium carbamate was the best ammonium source. The optimal initial water activity, temperature and pH value were 0.55-0.75, 35°C and 6.5 respectively, under which a substrate conversion of 97.6% could be achieved after reaction for 140 h.     

Efficient kinetic resolution of organosilicon compounds by stereoselective esterification with hydrolases in organic solvent

Stereoselective esterification of three isomers of trimethylsilylpropanol, 1-trimethylsilyl-2-propanol, 1-trimethylsilyl-1-propanol, and 2-trimethylsilyl-1-propanol, was systematically studied with five kinds of hydrolases in an organic solvent system in connection with the structure of the compounds. The hydrolases were found to be able to esterify these organosilicon compounds, even -hydroxyalkylsilanes, which are unstable under the conditions of acid-catalysed esterification, and the highly optically active organosilicon compounds were successfully prepared with the selected hydrolases. Even a primary alcohol, 2-trimethylsilyl-1-propanol, was stereoselectively esterified by lipase. Furthermore, comparative studies were made by using their carbon counterparts. The silicon atom in the substrates was found to enhance the enzyme stereoselectivity in some cases, but its effect on the substrate reactivity was dependent on the structure of the substrates. These results are discussed based on the specific characters of the silicon atom. Correspondence to: A. Tanaka     

Lipase-catalyzed ammonolysis of trimethylsilylmethyl acetate in organic solvent*

Lipase could catalyze the ammonolysis of trimethylsilylmethyl acetate in organic solvents and Novozym 435 was the best biocatalyst for the reaction. The influences of some factors on the reaction were investigated. Cyclohexane, n-hexane and heptane were found to be suitable reaction media and ammonium carbamate was the best ammonium source. The optimal initial water activity, temperature and pH value were 0.55–0.75, 35°C and 6.5 respectively, under which a substrate conversion of 97.6% could be achieved after reaction for 140 h.     

Ester synthesis in organic solvent catalyzed by lipases immobilized on hydrophilic supports

Summary Eight microbial lipases and one animal lipase were immobilized on hydrophilic supports either by adsorption or entrapment. All preparations catalyzed the synthesis of geranyl or menthyl butyrate or laurate using heptane as solvent. This is a simple and easy method for ester synthesis.     

Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity

We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide. (c) 1994 John Wiley & Sons, Inc.     

Transacetylations to carbohydrates catalyzed by acetylxylan esterase in the presence of organic solvent

Various conditions were applied to test the ability of acetylxylan esterase (AcXE) from Schizophyllum commune to catalyze acetyl group transfer to methyl beta-D-xylopyranoside (Me-beta-Xylp) and other carbohydrates. The best performance of the enzyme was observed in an n-hexane-vinyl acetate-sodium dioctylsulfosuccinate (DOSS)-water microemulsion at a molar water-detergent ratio (w(0)) of about 4-5. Although the enzyme was found to have a half-life of about 1 h in the system, more than 60% conversion of Me-beta-Xylp to acetylated derivatives was achieved. Under identical reaction conditions, the enzyme acetylated other carbohydrates such as methyl beta-D-cellobioside (Me-beta-Cel), cellotetraose, methyl beta-D-glucopyranoside (Me-beta-Glcp), 2-deoxy-D-glucose, D-mannose, beta-1,4-mannobiose, -mannopentaose, -mannohexaose, beta-1,4-xylobiose and -xylopentaose. This work is the first example of reverse reactions by an acetylxylan esterase and a carbohydrate esterase belonging to family 1.