Lipase-catalyzed Reaction Research Articles

The Action of Adenosine Deaminase (E.C. 3.5.4.4.) on Adenosine and Deoxyadenosine Acetates: The Crucial Role of the 5′-Hydroxy Group for the Enzyme Activity

From adenosine 1, 2′-deoxyadenosine 3 and 3′-deoxyadenosine 5 all the acetates were prepared by lipase-catalyzed reactions. Only the acetates with free 5′-hydroxy group were deaminated by adenosine deaminase (ADA), confirming the crucial role of 5′-OH for the enzyme activity.

Lipase-Catalyzed Reaction of Molecularly Pure Linear and Cyclic Poly(3-hydroxybutanoate)s: Evidence of Cyclic Polymer Formation

Abstract Molecularly pure linear and cyclic poly(3-hydroxybutanoate) [P(3HB)] polymer species were subjected to hydrolysis, transesterification and intramolecular esterification by lipase to produce two series of linear and cyclic polymer structures having higher and lower molecular weights. It was found that a cyclic polymer was produced by the lipase-catalyzed reaction of the polymer chain.

Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review

Regiospecificity is one of the major advantages of using lipase technology for the modification of oils and fats to produce high-value added products, such as cocoa butter equivalents, human milk fat substitutes, and other specific-structured lipids. Due to the high cost of biocatalysts, the mainstream applications of lipases for normal oils and fats are still limited. Therefore, positional specificity of lipases has the priority and will be the target property to be exploited for commercial and industrial developments, because no chemical method has such a specificity and is promising or possible for this task. In this paper, encouraging products resulting from this regiospecificity are reviewed together with the critical evaluation of their reaction schemes, side reactions and by-products, sources of substrate oils and acyl donors, and production processes.

Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review

Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a review

Lipase catalyzed reactions of aliphatic and arylaliphatic carbonic acid esters

Symmetrical dialkyl carbonates and dibenzyl carbonates reacted with various nucleophiles in the presence of Candida antarctica lipase B in organic solvents. For example, reaction of dibutyl and dibenzyl carbonate with an alcohol gave a mixture of the mono- and disubstituted products. Aminolysis, however, afforded only the carbamates, without subsequent reaction to the ureum derivatives. The reaction rates were rather low compared with carboxylic esters; the reactivity increased in the order dimethyl<diethyl<dibutyl≈dibenzyl carbonate. Aminolysis of dibenzyl carbonate by chiral benzylamines gave the corresponding enantiomerically pure benzyloxycarbonyl amines, thus, providing a direct, phosgene-free route to the Z-protected amine enantiomer from the racemate.

Formation of hemiacetal esters in lipase-catalysed reactions of vinyl esters with hindered secondary alcohols

Normally many lipases are efficient catalysts for the acetylation of alcohols with vinyl acetate. Unexpectedly, we found that some sterically hindered secondary alcohols react slowly to yield hemiacetal esters as mixtures of diastereomers. Their formation can be explained by the reaction of the alcohol with acetaldehyde that is produced by the lipase-catalysed splitting of vinyl acetate and subsequent acetylation of the resulting hemiacetal by the lipase.

NMR on-line monitoring of esterification catalyzed by cutinase

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.

Ｃｈｅｍｏ‐Ｅｎｚｙｍａｔｉｃ合成法によるジフルオロメチレン基を有するキラル化合物の合成

Asymmetric synthesis of two types of partly difluorinated compounds has been accomplished based on the chemo-enzymatic reaction strategy. Optically active 1, 1-difluoro-2, 3- (bishydroxymethyl) cyclopropane has been synthesized through lipase-catalyzed reaction; prochiral diacetate of cis-1, 1-difluoro-2, 3-bis (hydroxymethyl) cyclopropane was converted to the corresponding monoacetate through Alcaligenes sp. lipase-catalyzed hydrolysis with >99% enantiomeric excess. Racemic acetate of trans-1, 1-difluoro-2, 3-bis (hydroxymethyl) cyclopropane was resolved by Pseudomonas sp. lipase-catalyzed hydrolysis successfully. Lipase-catalyzed reaction was employed to resolve bis-difluorocyclopropane derivatives; the first synthesis of optically pure (trans, trans) -2, 2, 5, 5-tetrafluoro-1, 6-bis (hydroxymethyl) bicyclopropane has thus been accomplished. Carbon radicals from allyl O-trimethylsilyl-α-bromo-α, α-difluoroacetal can cyclize onto the olefinic part regiospecifically to give γ-lactols in good yield. The lactols are then converted to the corresponding α, α-difluoro-γ-lactones. Systematic synthesis of multifluorinated-α, α-difluoro-γ-lactones has been accomplished through intramolecular radical cyclization as a key reaction. The synthesis of difluorinated analogue of an insect sex pheromone has been accomplished through intramolecular radical cyclization and lipase-catalyzed reaction.

Enzymatic Production of Alkyl Esters Through Lipase-Catalyzed Transesterification Reactions in Organic Solvents: Solvent Effects and Prediction Capabilities of Equilibrium Conversions

The solvent effect on the equilibrium position of the transesterification reaction of hexanol with ethyl acetate catalyzed by a lipase has been investigated in a variety of non-polar and polar solvents - and binary mixtures. The results obtained indicate that the solvent effect on the equilibrium conversion is very small as compared to that for the direct esterification reactions.Equilibrium conversions were then predicted using the equilibrium constant for the reaction obtained from Gibbs free energy of formation information for reactants and products in combination with the UNIFAC activity coefficient model. A solvent independent equilibrium conversion was obtained, which was in good agreement with the observed average value for all solvents. This indicates that UNIFAC provides satisfactory estimates of the activity coefficients but its group contribution structure does not allow the prediction of the small differences in conversion among the solvents examined.Finally plots of these conversions versus the solvent octanol/water partition coefficient or the solubility of water in the solvent, that provide the correct trend in direct esterification reactions, did not achieve the same for transesterification.

Facile synthesis of optically active gamma-lactones via lipase-catalyzed reaction of 4-substituted 4-hydroxybutyramides.

Lipase-catalyzed transesterification of racemic 4-substituted 4-hydroxybutyramides with succinic anhydride proceeded enantioselectively to afford (S)-succinic acid monoester and unreacted (R)-4-hydroxybutyramide derivative, which were separated easily by treatment with an alkaline solution. Both enantiomers were converted easily to optically active gamma-substituted gamma-butyrolactones.

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: II. Multiresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow-fiber reactor.

The hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow-fiber reactor was studied at 30 degrees C. To assess the selectivity of this immobilized enzyme, the effluent concentrations of five different free fatty acids were measured using high-performance liquid chromatography (HPLC). Several rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for this lipase-catalyzed reaction. A multiresponse nonlinear regression method was employed to determine the kinetic parameters associated with these rate expressions. Quasi-optimum operating conditions corresponded to 30 degrees C and a buffer pH value of 7.0. Under these conditions, the concentration of free linoleic acid (C18:2) (the fatty acid of primary interest) in the effluent oil stream for a fluid residence time of 6 h was approximately 0.5 M.

Study of enzyme-catalyzed reactions in organic solvents using multiple linear regression

We have used multiple linear regression to predict either initial rate, log initial rate or specificity for enzyme-catalyzed reactions performed in non-aqueous solvents. The Subtilisin Carlsberg catalyzed transesterification of N-acetyl- l-phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol was assayed in 30 non-aqueous solvents, and the lipase catalyzed transesterification of methyl methacrylate in 23 non-aqueous solvents. Both sets of reactions were performed at fixed thermodynamic water activity. The lipase catalyzed reactions were also performed in water saturated solvents and in dry solvents. The report illustrates that regression analysis may provide insight into how solvents can alter the activity and specificity of enzymes suspended therein. A regression model for the subtilisin catalyzed reaction suggests that solvents which have a flat hydrophobic region inhibit by competing with the substrate for an enzyme cleft. In the lipase catalyzed reaction, tetrachloroethylene is an outlier (i.e., behaves differently to other solvents) for all the regression models. This deviation, together with an element of structural similarity to the substrate, suggests that tetrachloroethylene acts as a competitive inhibitor. Log P is an important descriptor and it, or an expression containing log P, appears in all the regression equations. Log initial rate is predicted by a two-descriptor model for either enzyme system in solvents of high log P at fixed thermodynamic water activity. Regression models with the same two descriptors predict initial rate for the lipase system over the entire log P range for solvents maintained at fixed thermodynamic water activity and for dry solvents, but not for water saturated solvents.

Lipase-catalyzed conversions of trimethylsilyl ethers: deprotection, acetylation, epoxidation and one-pot-multi-step reactions

Unsaturated trimethylsilyl ethers are converted by lipase-catalyzed hydrolysis of ethyl acetate directly to alkenol acetates; the hydrolysis of diethyl carbonate yields unstable carbonic acid monoethylester, which deprotects trimethylsilyl ethers under mild conditions and without remaining acid. By the analogous lipase-catalyzed perhydrolysis of these esters with hydrogen peroxide, epoxyalkanol acetates and epoxyalkanols are obtained in one-pot reactions with selectivities of 90–97%. Using longer chain peroxy fatty acids, generated in-situ by lipase-catalyzed reaction of fatty acid and hydrogen peroxide, trimethylsilyl ethers are selectively (83–95%) epoxidized without removal of the protecting trimethylsilyl group.

Enzymatic catalysis in cosolvent modified pressurized organic solvents

An important advantage of carrying out enzymatic catalysis in organic media is the increased solubility of hydrophobic substrates. This study compares a model lipase catalyzed esterification of cholesterol using vinyl acetate (VA) in two such nontraditional media: high-pressure hexane and supercritical (SCF) ethane. The effect of using one of the reactants (VA) as a cosolvent to increase the solubility of the other reactant (cholesterol) in SCF ethane has been investigated. The thermodynamic activity of water (a(w)) in the reaction media was controlled by the direct addition of the salt hydrate pair Na(4)P(2)O(7)/Na(4)P(2)O(7).10H(2)O. The a(w) of the salt hydrate system is shown to be a function of pressure and its variation over the pressure range 104-173 bar has been estimated. The initial reaction rate in pressurized hexane was found to vary linearly with the cholesterol concentration. The reaction rate was also a function of pressure-the effect being more pronounced in ethane than in hexane. This is consistent with the large negative partial molar volumes observed in SCFs, although the sign of the resulting activation volume differs from previous investigations of lipase-catalyzed reactions in SCFs. When corrected for substrate concentration, the initial rate of catalysis in SCF ethane was determined to be greater than in pressurized hexane over the conditions investigated. This study shows that proper solvent choice can be used to regulate reaction rates in pressurized solvents.

Lipase-catalyzed fractionation of conjugated linoleic acid isomers.

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.

Thiocrown Ether Additive Effects on Diastereoselectivity of the Lipase-Catalyzed Reaction: Preparation of Optically Active 3-Hydroxy-2-methylalkanenitriles through a Double Enzymatic Reaction Strategy

Abstract The additive effect on diastereoselectivity towards the lipase-catalyzed hydrolysis of acetates of 3-hydroxy-2-methyl- or 3-hydroxy-2-ethylalkanenitriles has been investigated. Diastereoselectivity was not influenced by thiocrown ether additives, although a significant modification of enantioselectivity was observed. Origin of the diastereoselectivity of the lipase-catalyzed reaction was thus evidently different from that of enantioselectivity. Based on these results, an easy preparation of optically active 3-hydroxy-2-methylpentanenitrile and 3-hydroxy-2-methylbutanenitrile have been demonstrated through lipase-catalyzed reaction by a double enzymatic reaction strategy.

Causes of unreproducibility of C. rugosa lipase-catalyzed reactions in slightly hydrated organic media

Lipase activity, measured as hydrolysis of tributyrin is a valid assay to quantify the lipase activity of a lyophilized crude lipase in hydrolysis reactions but it is not useful to predict the catalytic activity in lipase-catalyzed reactions in organic media. Three factors control the catalytic activity in these media: i) relative proportion of isoenzymes; ii) amount of water in the lyophilized crude enzyme and iii) amount of lipase protein in the commercial powder. Thus we propose two simple reaction tests: i) heptyl oleate synthesis (specific of lipases), ii) enantioselective esterification of (R) or (S) 2-(3-benzoyl)phenyl propionic acid. This methodology is applied to different crude lipases of Candida rugosa, obtained in different fermenter conditions and shows the origin of the unreproducibility of the synthetic data.

Lipase-catalyzed transesterification of 2-phenyl-1-propanol with vinyl esters having aromatic ring in acyl moiety

Abstract The highly enantioselective transesterification of 2-phenyl-1-propanol, which has not been efficiently resolved by lipase-catalyzed reactions, was attained by using either vinyl 3-( p -tolyl)propanoate or vinyl 3-(2-naphthyl)propanoate as the acyl donor.

Synthesis of optically active bisdifluorocyclopropanes through a chemo-enzymatic reaction strategy

The first synthesis of bisdifluorocyclopropane derivatives has been accomplished via a chemo-enzymatic reaction strategy; ( E)- or ( Z)-3-tributylstannyl-2-propenols were prepared and their conversion led to the ( trans,trans)- and ( cis,cis)-bisdifluorocyclopropanes. The subsequent lipase-catalyzed reaction efficiently afforded optically active ( trans,trans)-2,2,5,5-tetrafluoro-1,6-bis(hydroxymethyl)bicyclopropane.

Synthesis of optically active novel 1,1,2-trifluoro-1-alken-3-ols through lipase-catalyzed reaction

The first synthesis of several types of optically active 1,1,2-trifluoro-1-alken-3-ols has been accomplished through the Pseudomonas cepacia lipase-catalyzed trans-esterefication. The PCL catalyzed acylation using vinyl chloroacetate as the acyl donor provided the corresponding fluorinated-allyl alcohols that possess an aromatic functional group with sufficient enantioselectivity.