Short chain fatty acid esters synthesis by commercial lipases in low-water systems and by resting microbial cells in aqueous medium

Thirteen commercial lipases in hexane and seventeen bacterial cell suspensions in aqueous media were screened for the production of ethyl valerate and ethyl butyrate. The highest esterifying activity was obtained with commercial Pancrealipase (Biozymes Inc.) and Candida rugosa lipase (Amano Enzyme Ltd) and with bacterial cell suspension from Pseudomonas fragi CRDA 446. Commercial enzymes gave molar conversion yield of 68% over 24 h as compared to 17% with whole cells in aqueous medium. However, a comparison of both sources of biocatalyst i.e. whole microbial cells and commercial lipases, based on the amount of ester produced per g of protein for a complete reaction, indicated similar activities. © Rapid Science Ltd. 1998     

Synthesis of various kinds of esters by four microbial lipases

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.     

Enzymatic synthesis of geraniol esters in a solvent-free system by lipases

Geraniol esters were synthesised by direct esterification catalysed by esterases and lipases (five enzymes were tested) in a solvent-free system at 37°C. The best conversions yields, about 85%, on geranyl butyrate and valerate obtained with esterase 30 000 from Mucor miehei. The effect of substrate molar ratio alcohol/acid variation was studied. A study of the water production was made in parallel during the esterification reaction.     

Sensitivity of microbial lipases to acetaldehyde formed by acyl-transfer reactions from vinyl esters

Summary The sensitivity of twenty six microbial lipases towards acetaldehyde (an unavoidable by-product in lipase-catalysed acyl transfer reactions with vinyl esters) was investigated. The sensitivity of an individual enzyme strongly depends on its properties such as microbial source, molecular weight and relative lysine content. Whereas the majority of enzymes (from Pseudomonas, Rhizopus, Chromobacterium, Mucor and Candida antarctica sp.) proved to be remarkably stable, lipases from Candida rugosa and Geotrichum candidum lost most of their activity when exposed to acetaldehyde.     

Continuous synthesis of esters by cell-bound fungal lipases in an organic solvent

Summary Strains of Penicillium cyclopium and Rhizopus arrhizus secrete two extracellular lipases and contain an intracellular lipase. As these intracellular enzymes exhibited good synthetic activities in organic solvent, we designed a loop fixed-bed reactor for the continuous synthesis of esters. In a preliminary study we optimized the yield of ester synthesis using response surface methodology. With dodecanoic dodecyl ester as a model compound, the yields of ester synthesis were higher than 90%. It has been demonstrated that the reactor designed for this study is more efficient than a stirred batch reactor and more efficient than a fixed-bed reactor without a loop current or without a second catalytic column. In application, we have shown that lipases from R. arrhizus and P. cyclopium do not esterify tertiary alcohols like many lipases. Correspondence to: L. C. Comeau     

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l^–1 in 264 h and 142 g l^–1 in 48 h respectively).     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Inhibition of pancreatic and microbial lipases by proteins

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.     

Wax esters synthesis from heavy fraction of sheep milk fat and cetyl alcohol by immobilised lipases

Wax esters were obtained from lipase-catalysed alcoholysis of triglycerides with cetyl alcohol, using n-hexane as solvent. The heavy triglyceride fraction (HTF), obtained by fractionation of sheep milk fat, was used as raw material. In the natural fat mixture GC analysis showed that palmitic, myristic, stearic and oleic acids are the most abundant fatty acids which are useful to produce wax esters. Reactions were tested for different amounts of Lipozyme RMIM catalyst, and the optimum concentration of 10 mg catalyst/ml solution has been determined. The formation of the four main products, i.e. cetyl myristate, cetyl palmitate, cetyl oleate and cetyl stearate, was determined by HPLC/ELSD quantitative analysis. The optimum water activity in the reaction medium a_w=0.35 in the case of Lipozyme RMIM, and a_w=0.53 for Novozym 435 was found. Lipozyme RMIM (immobilised sn-1,3-specific lipase from Rhizomucor miehei) was more active than Novozym 435 (immobilised nonspecific lipase-B from Candida antarctica) towards wax esters production. The acyl migration of 2-monoglycerides was suggested as a crucial step to explain the higher yields produced by the 1,3-specific lipase.     

Purification strategies for microbial lipases

Microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze a wide variety of reactions in aqueous and non-aqueous media. The chemo-, regio- and enantio-specific behaviour of these enzymes has caused tremendous interest among scientists and industrialists. Lipases from a large number of bacterial, fungal and a few plant and animal sources have been purified to homogeneity. This has enabled their successful sequence determination and their three-dimensional structure leading to a better understanding of their unique structure-function relationships during various hydrolytic and synthetic reactions. This article presents a critical review of different strategies which have been employed for the purification of bacterial, yeast and fungal lipases. Since protein purification is normally done in a series of sequential steps involving a combination of different techniques, the effect of sequence of steps and the number of times each step is used is analyzed. This will prove to be of immense help while planning lipase purification. Novel purification technologies now available in this field are also reviewed.     

Lipase catalyzed formation of flavour esters

Summary Thirteen commercial lipase preparations were checked for their ability to catalyse the formation of flavour esters (isoamyl or geranyl acetate, propionate and butyrate) by either direct esterification or ester solvolysis in n-heptane. The formation of isoamyl or geranyl butyrates and propionates by direct esterification was catalyzed by the majority of the tested lipases. Acetic acid esters were more difficult to obtain. Transesterification reactions were found to be a good alternative way for ester synthesis.     

Recombinant microbial lipases for biotechnological applications

Lipases, mainly of microbial origin, represent the most widely used class of enzymes in biotechnological applications and organic chemistry. Modern methods of genetic engineering combined with an increasing knowledge of structure and function will allow further adaptation to industrial needs and exploration of novel applications. Production of such tailored lipases requires their functional overexpression in a suitable host. Hence, this article describes the functional heterologous production of commercially important microbial lipases. Based on the knowledge of different lipases' substrate binding sites, the most suitable lipase for a particular application may be selected.     

Formation of polyol-fatty acid esters by lipases in reverse micellar media

The synthesis of polyol-fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2-monoglyceride, and sugars and their dervatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50-60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl-enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of paritioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of "mixed" glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2-monoglyceride to 1-monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no convesion.     

Overproduction of microbial lipids and lipases

The nomenclature of simple and complex lipids, and their analysis are introduced and the overproduction of lipids and lipases are discussed. Translated by J. Neužil     

Studies on synthesis of short chain alkyl esters catalyzed by goat pregastric lipase

Goat pregastric lipase, in the form of a suspended enzyme powder, was found to be active in catalyzing the synthesis of alkyl esters in anhydrous organic solvents. The rate of catalyzed synthesis of esters was very dependent on the solvent medium, and maximum activity was found when a hydrocarbon was used as the solvent. The optimal temperature for the catalyzed synthesis ranged from 30 to 40°C and the maximal temperature was 35°C for the synthesis of butyl caproate in isooctane. The selectivity for the carbon-chain length of the fatty acid by the lipase was similar to that seen in hydrolysis reactions in aqueous solution, and the optimal rate of synthesis of alkyl esters was found for synthesis of the esters which had 8 or 10 carbons in the alkyl moieties from the two individual substrates. The rate of synthesis was also dependent on the water content in the system, with maximum activity occurring at 1% w/w water in isooctane.     

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.     

Selectivity of lipases and esterases towards phenol esters

The selectivity of 28 lipases and esterases in the hydrolysis of butanoates of o-, m- or p-substituted phenols was investigated in a microtiterplate format. The phenols released during enzyme-catalyzed hydrolysis were converted in situ with Gibbs’ reagent to form a blue indophenol complex, which was quantified spectrophotometrically at 600 nm. Substantial differences in rates were found, which exhibits that the type and position of the substituent at the alkyl group has a strong influence on the selectivity of the enzymes. For various enzymes, the p-nitro derivative was the best substrate, whereas for other enzymes the m-Cl-derivative was preferentially hydrolyzed. Analysis of the data using the Hammett equation showed that sometimes the observed changes followed a predictable trend, but in several cases the result is very unexpected.     

Fluorescent organophosphonates as inhibitors of microbial lipases

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the omega-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.     

Regioselective synthesis of ethoxylated glycoside esters using beta-glucosidase in supersaturated solutions and lipases in organic solvents

Three ethoxylated glycosides, tetraethylene glycol beta-D-glucoside, tetraethylene glycol beta-D-xyloside, and methoxy triethyleneglycol beta-D-glucoside, were prepared via almond beta-glucoside-catalyzed (trans)glycosylation carried out in supersaturated solutions of glucose or p-nitrophenyl beta-D-xyloside and the respective polyethylene glycols. The products were isolated and further modified by enzymatic esterification with Candida antarctica and Mucor miehei lipases. The latter enzyme showed a much greater selectivity for the primary hydroxyl group on the polyethylene glycol chain of the glucoside substrate, thus enabling us to obtain exclusively the corresponding monoester, omega-O-oleoyl tetraethylene glycol beta-D-glucoside. Novozyme was used for the preparative synthesis of two other monoesters, 6-O-oleoyl (methoxy triethyleneglycol) beta-D-glucoside and omega-O-oleoyl tetraethylene glycol beta-D-xyloside. Two diesters, di-oleoyl tetraethylene glycol beta-D-glucoside and tetraethylene-bis(6-0-oleoyl glucoside) were also synthesized in good yields using this lipase. Copyright 1998 John Wiley & Sons, Inc.