Lipase-catalyzed Reaction Research Articles

Production of medium chain glycerides from coconut and palm kernel fatty acid distillates by lipase-catalyzed reactions

Medium chain glycerides (MCGs) containing C 8:0 and C 10:0 fatty acids is very much important for medicinal and nutritional applications. Coconut and palm kernel fatty acid distillates (FADs) can be utilized to produce MCGs by a combination of lipase-catalyzed hydrolysis and esterification reactions. The neutral glycerides present in coconut and palm kernel FADs are hydrolyzed by Candida rugosa lipase. The hydrolysates were then subjected to steam distillation under vacuum (at 120–140 °C) to get fractions rich in medium chain fatty acids (MCFAs). The fractions, from coconut and palm kernel FADs (75.2 and 76.2% MCFAs, respectively), were esterified with Rhizomucor miehei (Lipozyme RM IM) lipase to produce MCGs. Products from coconut FAD contained 64.7–67.5% diacylglycerol (DG), followed by 18.8–22.9% monoacylglycerol (MG) and 9.8–9.3% triacylglycerol (TG). Similarly, products from palm kernel FAD contained 63.5–66.7% DG, 19.1–23.6% MG and 9.5–10.1% TG.

Production of Diglycerol Conjugated Linoleic Acid Monoester by Esterification with Penicillium camembertii Lipase

Polyglycerol fatty acid monoesters are used widely as food emulsifiers, and conjugated linoleic acid (CLA) has various physiological activities. Hence, production of polyglycerol CLA monoester was attempted through a lipase-catalyzed reaction. A commercially available free fatty acid mixture (referred to as FFA-CLA) containing 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA was used as a source of CLA, and its esterification with diglycerol was studied. Screening of several lipases showed that Penicillium camembertii lipase was suitable for production of diglycerol fatty acid monoesters (DGFAMEs). When a mixture of FFA-CLA/diglycerol (1:2, mol/mol), 100 U/g-mixture of the lipase, and 2% water was agitated at 30 °C for 24 h with dehydration at 5 mm Hg using a vacuum pump, the degree of esterification reached 93%, and the ratio of DGFAMEs/diglycerol fatty acid diesters was 1.9:1 (wt/wt). The synthesized DGFAMEs were purified to 97% in a 69% yield by short-path distillation. This process comprising lipase-catalyzed esterification and short-path distillation may be effective for industrial production of an emulsifier for use in nutraceutical applications.

NMR monitoring of lipase-catalyzed reactions of prostaglandins: preliminary estimation of reaction velocities

The synthesis of methyl esters as well as acetylation of prostaglandins (PG) F 2α and E 2 and the corresponding methyl esters catalyzed by Candida antarctica lipase B (CALB) has been monitored by using 1H NMR spectroscopy in order to get a quantitative estimation of the velocities of the reactions occurring in the system. The apparent second-order rate constants of acetylation of the 11-OH group of PGs differed by up to two orders of magnitude while the difference between velocities of the CALB-catalyzed methylation of PGF 2α and PGE 2 was 2.5 times. The curves describing acetaldehyde formation were determined. The initial rapid consumption of water present on Novozym 435 was followed by an acetaldehyde formation matching the acetylation of prostaglandins.

The synthesis of enantiomerically pure novel liquid crystal compounds containing the bis(trifluoromethyl)alkanediol moiety

The synthesis of two types of novel liquid crystals, which possess the optically active bis(trifluoromethyl)alkanediol moiety has been accomplished using the lipase-catalyzed reaction and the Wittig reaction as key reactions.

Hemiacetals and their esters as side-products in lipase-catalysed transesterifications of vinyl esters with sterically hindered alcohols

Lipase-catalysed transesterifications of vinyl esters with various sterically hindered secondary alcohols sometimes give hemiacetals and hemiacetal esters as major side-products along with the expected esters, especially in the presence of aldehydes. The substrates, reaction conditions, and the lipases required for the formation of such hemiacetal products have been studied. Hemiacetals and their esters are very easily hydrolysed. Therefore, when conventional work-up procedures are used, the formation of such products in lipase-catalysed transesterification reactions may easily escape notice, leading to, e.g. an unexpectedly low enantiomeric purity of the isolated remaining substrate in a resolution reaction.

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1:2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40 degrees C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7-36.5 degrees C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5 degrees C; JAOCS 80, 341-346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.

Lipase-catalyzed transesterification in aqueous medium under thermodynamic and kinetic control using carboxymethyl cellulose acetylation as the model reaction

Enzymatic acetylation of carboxymethyl cellulose (CMC) with vinyl acetate by lipase A12 from Aspergillus niger ( A. niger) in buffer was used as a model reaction for investigating lipase-catalyzed ester synthesis in aqueous solution. An increase in CMC concentration (1–15 wt.%) enhanced the reaction yield via thermodynamically favoring the CMC ester synthetic reaction in aqueous medium. The use of vinyl acetate as activated acetyl donor kinetically drove the forward transesterification over product hydrolysis, but partially inhibited enzyme activity when used at large amounts (>5 mL/g CMC). The increase in enzyme amount initially improved the reaction markedly, but less efficiently at larger enzyme amounts (>1000 lipase activity units/g CMC) due to the substrate diffusion limitation. The difference in reaction course demonstrated that the lipase-catalyzed transesterification was under kinetic control, while the non-enzymatic reaction under thermodynamic control. The acetylation yield of the lipase-catalyzed reaction was 5.6 times higher than that of the non-enzymatic reaction. This increase in reaction yield was attributed partly (by 19%) to the side cellulase activity of the lipase due to its substrate degradation role, but largely (81%) to the lipase activity by virtue of its transesterification catalysis function. The A. niger lipase was identified as a hydrolase capable of catalyzing ester synthesis in aqueous solution.

How Does Lipase Flexibility Affect Its Enantioselectivity in Organic Solvents? A Possible Role of CH×××π Association in Stabilization of Enzyme—Substrate Complex.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Different recognitions of ( E)- and ( Z)-1,1′-binaphthyl ketoximes using lipase-catalyzed reactions

Lipase-catalyzed hydrolysis of ( E)-2-[α-(acetoxyimino)benzyl]-1,1′-binaphthyl [ (±)- 1a ] and ( Z)-2-[α-(acetoxyimino)benzyl]-1,1′-binaphthyl [ (±)- 1b ] yielded optically active ( E)-2-[α-(hydroxyimino)benzyl]-1,1′-binaphthyl [ ( S)- 2a ] and ( Z)-2-[α-(hydroxyimino)benzyl]-1,1′-binaphthyl [ ( R)- 2b ], respectively, with high enantiomeric excess. Selectivity for the opposite enantiomer of the axial binaphthyl skeleton was shown by ( Z)-isomer 1b against ( E)-isomer 1a .

Synthesis of optically active gem-difluorocyclopropanes through a chemo-enzymatic reaction strategy

The synthesis of a chiral difluorocyclopropane building block has been accomplished using lipase-catalyzed reaction; the prochiral diacetate of cis-1,3-bishydroxymethyl-2,2-difluorocyclopropane was converted to the corresponding chiral monoacetate by the Alcaligenes lipase (lipase QL)-catalyzed hydrolysis with >99% enantiomeric excess. Enantiomerically pure trans-1,3-bishydroxymethyl-2,2-difluorocyclopropane was also obtained through the Pseudomonas cepacia SL-25 lipase (lipase SL)-catalyzed reaction. The synthesis of the chiral trans, trans-bis- gem-difluorocyclopropane derivatives has been accomplished using the same lipase technology; trans, trans-1,6-bishydroxymethyl-2,2,5,5-tetrafluorobicyclopropane was obtained in optically active form using the lipase SL-catalyzed hydrolysis of the corresponding diacetate.

How Does Lipase Flexibility Affect Its Enantioselectivity in Organic Solvents? A Possible Role of CH···π Association in Stabilization of Enzyme–Substrate Complex

Abstract For lipase-catalyzed reactions of 2-(4-substitued phenoxy)propionic acids with alcohols in organic solvents containing a small amount of water, the increase of the lipase flexibility brought about by addition of water is found to be favorable for the induced-fit motion of the lipase for the correctly binding enantiomer of the substrate used, thus resulting in the improvement of the lipase enantioselectivity. In particular, for the reaction of the substrate with rich π electron density on its aromatic ring, the enantioselectivity was much more sensitive to the change of the lipase flexibility. Thus, in the induced fit motion, the CH···π association between amino acids side chains around the lipase’s active site and the aromatic ring of the correctly binding enantiomer is assumed to accelerate the accommodation of the substrate into the lipase’s active site and the stabilization of the complex between the enzyme and the substrate. This assumption is also supported by a discussion based on the value of the Michaelis constant obtained. Furthermore, on the basis of a model concerning the acyl-enzyme structure for the incorrectly binding enantiomer, the long alkyl chain alcohols as a nucleophile are found to improve the lipase enantioselectivity markedly.

Production of Medium Chain Glycerides and Monolaurin from Coconut Acid Oil by Lipase-Catalyzed Reactions

Medium chain glycerides (MCGs) containing C8:0 and C10:0 fatty acids have got commercial importance by considering their medicinal and nutritional applications. Monolaurin is also a value added commercial product. Coconut acid oil, which is mainly utilized in soap manufacturing process, can be effectively utilized to produce MCGs and monolaurin by a combination of lipase-catalyzed hydrolysis and esterification. Coconut acid oil containing 68% free fatty acid (as lauric acid), and 31.4% neutral glycerides are hydrolyzed by Candida rugosa lipase. The hydrolyzed acid oil was then subjected to steam distillation under atmospheric pressure to get a fraction (yield, 5%) rich in medium chain fatty acids (C8:0, 46.2%, C10:0, 19.2%), and the residual fraction was fractionally distilled under vacuum to get a fraction rich in lauric acid (68.4%). These two fractions were esterified with glycerol with Rhizomucor miehei (Lipozyme RM IM) lipase to produce MCGs from the first fraction and monolaurin from the second fraction.

有機酸の選択的分離が可能なリパーゼ‐オルガノゲル膜システムの開発

We have developed a lipase-facilitated organogel membrane for selective separation of organic acids. We previously demonstrated that lipase-catalyzed reactions drove transport of organic acids through a supported liquid membrane. In this study, a lipase-facilitated liquid membrane was gelled with 12-hydroxystearic acid to stabilize the liquid membrane phase. The lipase-facilitated organogel membrane exhibited the improved stability of the liquid membrane phase compared with a non-gelled supported liquid membrane, although the permeate flux of an organic acid through the organogel membrane was less than that through the non-gelled liquid membrane. The lipase-facilitated organogel membrane demonstrated the selective separation of 3-phenoxypropionic acid from various organic acids due to the substrate specificity of lipases. Enantioselective separation of (S) -ibuprofen from racemic ibuprofen was also achieved owing to the enantioselectivity of lipase.

Poly(ethylene glycol)-lipase complexes catalytically active in fluorous solvents.

Lipase-catalyzed alcoholysis between vinyl cinnamate and benzyl alcohol in fluorous solvents was investigated. This is the first report of a lipase-catalyzed reaction in a fluorous solvent. Forming the poly(ethylene glycol)(PEG)-lipase PL complex enhanced lipase activity over 16-fold over that of native lipase powder. The PEG-lipase PL complex exhibited markedly higher alcoholysis activities in fluorous solvents than in conventional organic solvents such as isooctane and n-hexane. The optimum reaction temperature for FC-77 (perfluorooctane) was 55 [degree]C and the optimum pH for the preparation of the PEG-lipase complex was 9.0; similar to the conditions for lipase PL-catalyzed reaction in aqueous solution. The alcoholysis reaction in fluorous solvent requires the addition of a FC77-miscible organic solvent (isooctane) in order to dissolve non-fluorinated substrates. Lipase activity in the fluorous solvent was significantly influenced by the volume ratio of isooctane in the reaction medium. Vinyl cinnamate inhibition of the lipase-catalyzed reaction occurred at a much lower concentration in the fluorous solvent than in isooctane. These results can be explained by the localization of substrates around lipase molecules, induced by adsorption of the substrates to the PEG layer of the PEG-lipase complex.

Enzyme‐Catalyzed Regioselective Transesterification of Peracylated Sophorolipids.

Abstract Regioselective transesterifications and hydrolysis of peracylated sophorolipid (SL) derivatives catalyzed by lipases was investigated. This study is the first evaluation of the lipase-catalyzed reactions on the non-lactonic SL derivatives. Four lipases, namely from porcine pancreas (PPL, Type II), Candida rugosa (AYS, TypeVII), Pseudomonas cepacia (PS-30), and Candida antarctica (Novozym 435, carrier fixed lipase fraction B) were used in anhydrous THF or in phosphate buffer (pH=7.4, 0.2 M). It was confirmed from the detailed spectral analysis of the products that transesterification failed to furnish any free hydroxyls on the sophorose ring. Instead, transesterification took place on the methyl ester located at the carboxylic end of the 17-hydroxyoctadecenoic acid chain attached to the C-1′ position of the sophorose ring. It is proposed that in absence of the lactonic structural motif, the binding of the peracylated non-lactonic SLs in the lipase binding pocket takes place such that the carboxyl group of the octadecenoic acid, not the sophorose sugar, is preferentially accessible to the active site.

Highly enantioselective lipase-catalyzed reactions at high temperatures up to 120°C in organic solvent

Lipase-catalyzed kinetic resolutions of 1,1-diphenyl-2-propanol were performed at high temperatures up to 120°C. Burkholderia cepacia lipase immobilized on porous ceramic particles, lipase PS-C II (Amano Enzyme Inc.), gave an enantiopure product at 40–120°C, with the highest conversion (39%) at 80–90°C. The mechanism of high enantioselectivity retained even at 120°C is also described briefly.

Enzyme-catalyzed regioselective transesterification of peracylated sophorolipids

Regioselective transesterifications and hydrolysis of peracylated sophorolipid (SL) derivatives catalyzed by lipases was investigated. This study is the first evaluation of the lipase-catalyzed reactions on the non-lactonic SL derivatives. Four lipases, namely from porcine pancreas (PPL, Type II), Candida rugosa (AYS, TypeVII), Pseudomonas cepacia (PS-30), and Candida antarctica (Novozym 435, carrier fixed lipase fraction B) were used in anhydrous THF or in phosphate buffer (pH=7.4, 0.2 M). It was confirmed from the detailed spectral analysis of the products that transesterification failed to furnish any free hydroxyls on the sophorose ring. Instead, transesterification took place on the methyl ester located at the carboxylic end of the 17-hydroxyoctadecenoic acid chain attached to the C-1′ position of the sophorose ring. It is proposed that in absence of the lactonic structural motif, the binding of the peracylated non-lactonic SLs in the lipase binding pocket takes place such that the carboxyl group of the octadecenoic acid, not the sophorose sugar, is preferentially accessible to the active site.

Modeling the effect of free water on enzyme activity in immobilized lipase-catalyzed reactions in organic solvents

The influence of water content on the lipase-catalyzed acidolysis of triolein (glycerol-trioleate, TO) and caprylic acid (CA) in hexane, using an immobilized enzyme was studied. An adequate water content ( R W) ranged from near zero to 0.1 g of water/g of dry enzyme. Over these values there was a decrease in the rate of incorporation of CA into triglyceride. This decrease was attributed to the progressive flooding of the carrier’s pores, in which the enzyme was immobilized. The flooding reduced the number of the enzyme molecules at the water–hexane interface and therefore, hindered the accessibility of the hydrophobic substrates (TO and CA) to the enzyme. A simple physical model based on a characterization of the immobilized enzyme particle by mercury porosimetry was developed. The model agreed well with both the experimental data and the prior published data. The model may partly explain the observed inhibition when using low molecular weight alcohols and carboxylic acids in immobilized lipase-catalyzed processes.

Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil

Biodiesel as alkyl esters derived from vegetable oils has considerable advantages in terms of environmental protection. In the present work, methyl esters were produced from sunflower oil by lipase-catalyzed reactions. The effect of organic solvent on alcoholysis rate was investigated and highest conversion (80%) was found in n-hexane and petroleum ether. Among several microbial lipases tested for alcoholysis activity in a solvent-free system, the best conversion (>90%) was found with lipase from Pseudomonas fluorescens (Amano AK) at the highest molar equivalent of oil:methanol (1:4.5). To reduce inactivation of commercial immobilized lipases by methanol, a three-step protocol consisting of the stepwise addition of 1 M equivalent of methanol at 5 h intervals, was developed. Also with immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) high conversion (>80%) was possible. Moreover, the transesterification could be conducted for at least 120 h during five batch runs without significant loss of activity.

Chemoenzymatic polymer-supported liquid phase synthesis of glucose γ-aminobutyric ester

A concise, polymer-supported solution synthesis of 6- O-(γ-aminobutyryl)- d-glucose has been achieved. Glucose was attached to polyethylene glycol monomethyl ether (MPEG) through an α,α′-dioxyxylyl diether linker, and, subsequently, the HO-6 hydroxyl of the glucose was regioselectively esterified with γ-aminobutyric acid by a lipase-catalyzed reaction.