Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.     

Lipase-catalyzed synthesis of ethyl (R)-2-benzyloxy-2-isopropylhydrogenmalonate: a useful combination of chemical synthesis with enzymatic methods

Ethyl (R)-2-benzyloxy-2-isopropylhydrogenmalonate is a key intermediate for the synthesis of the side chain in ergopeptines. In this work, we adopted a method to prepare enantiomerically pure title monoester via immobilized Candida antarctica lipase B (Novozym 435)-catalyzed hydrolysis of the corresponding diester.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.     

Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents

Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed. (c) 1995 John Wiley & Sons, Inc.     

Lipase-catalyzed hydrolysis of 2-naphtyl esters in biphasic system

The authors measured the rate of hydrolysis of the homologs of 2-naphtyl ester by using a Lewis cell with constant interfacial area to elucidate the kinetic mechanism of the lipase-catalyzed hydrolysis in biphasic system. On the basis of the two-film model, it was found from the analysis of experimental results that the hydrolysis of these substrates proceeds at the interface between the aqueous and organic phases. The interfacial reaction rate could be correlated by Michaelis-Menten mechanism. The values of the rate constant and the Michaelis constant were almost independent of the kinds of 2-naphtyl ester. The values of the interfacial kinetic parameters for 2-naphtyl ester were much greater than those for the hydrolysis in the aqueous phase.     

The water-dependence of the catalytic activity of bilirubin oxidase suspensions in low-water systems.

We investigated the enzymic activity of bilirubin oxidase when it is suspended as a lyophilized powder in a low-water system. The enzyme required buffer salts and a source of water to show activity. This study investigated the complete range of water thermodynamic activity (a(w)) by combining the use of salt hydrates and two-phase systems with concentrated solutes in the aqueous phase. When free water was added, activity reached a maximum at a defined water content, but this maximum increased with buffer content, suggesting that there was competition for water with the buffer salts from which the enzyme was lyophilized. Alternatively, a range of salt hydrates was used, each able to fix the water activity (a(w)) at a different value. By providing water to the organic solvent phase in this way, the dependency of enzyme activity upon a(w) was investigated and shown to be independent of buffer concentration. However, the optimum a(w) was uncertain because the available a(w) range for salt hydrates is < or = 0.90. Investigation of the remaining water activity range was made possible by using an a(w) depressor (sorbitol) to lower the a(w) of a two-phase system. The optimum a(w) for the bilirubin oxidase activity in this two-phase system was a(w) = 0.936, independent of buffer concentration. The study therefore confirmed the need to control the water 'available' to low-water systems and the dependence of enzyme activity on water thermodynamic activity (a(w)) not water content.     

Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic (liquid/solid) media

Ester synthesis catalyzed by Candida cylindracea lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was investigated in solid/liquid biphasic media containing the enzyme preparation and reactants without addition of organic solvents not participating in the reaction. Although the effects of water on enzyme kinetics have been abundantly studied in nearly anhydrous media, reactions in which water is produced have not been investigated. The effect of water produced by the reaction itself on the enzymatic activity was studied. The dispersion of water in a shaken, nearly anhydrous medium was shown to be responsible for the lack of activity of the enzyme. In contrast, when slowly shaken, the enzyme was fully activated by the water furnished as a product of the reaction. However, when experiments were performed in a two-phase aqueous/organic system with previously solubilized enzyme in water, the enzyme activity was increased by shaking and was of the same order of magnitude as in nearly anhydrous media. Under low water activity conditions, a powerful agitation can lead to slower reaction rate, because water, a product of esterification, is not retained in the microenvironment of the enzyme to activate it. The activation effect of water produced by the reaction was clearly shown using enzyme preparations shaken in an anhydrous medium and previously equilibrated at low water activities (a_w = 0.13 and 0.69). This activation did not occur for an enzyme preparation equilibrated at high a_w (0.89) or for a preparation gently shaken in a water-saturated medium. The lag time preceding activation of the enzyme increased with the extent of enzyme dehydration. The mass of the enzyme preparation was shown to be a parameter affecting the capacity of the lipase to produce enough water in its immediate environment. The lack of activity observed for a small quantity of enzyme was eliminated by addition of heat-denaturated lipase.     

Enzymatic alcoholysis of SO2-uracil analog diacyclonucleosides. Long-distance effect of the substituents on the regioselectivity

Diacetoxy SO₂-uracil analog diacyclonucleosides have been deprotected via lipase-mediated alcoholysis. The reactivity and regioselectivity of the reaction are dependent on the steric hindrance of distant 5-substituents, even if they are as small as a methyl or a 4,5-trimethylene chain, and, in a lesser extent, the electronic or hydrogen bond interactions of 3-carbonyl and hydroxyl-chain groups.     

Screening of various glycosidases for the synthesis of octyl glucoside

Thirteen glycosidases of microbial origin and almond beta-glycosidase were assayed in octanol/DMF (80:20, v/v), using a combination of hydrolysis, transglycosylation, and condensation reactions, in order to assess their potential for the production of alkyl glucosides. The two mesophile enzymes were highly impaired by the organic media. Three of the 11 thermophile enzymes gave interesting results in the hydrolysis and transglycosylation reactions, but they were highly inhibited by glucose. This made their use in a condensation reaction less interesting than the use of almond beta-glucosidase, which has a lower activity but shows less inhibition by the glucose.     

The influence of cosolvent concentration on enzymatic kinetic resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives

A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives in water–acetone solution is presented. Two different approaches were compared: enzyme-catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a substantial decrease in enantioselectivity from almost E = 150 to less than 5.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Arylsulfotransferase from Clostridium innocuum-A new enzyme catalyst for sulfation of phenol-containing compounds

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions.     

Chemoenzymatic synthesis of four diastereomers of (6-fluoro-2-chromanyl) oxirane: An intermediate of a potent β-blocker

The synthesis of optically active 5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, for the synthesis of the potent β-blocker R-67555, bis[2-(2-chromanyl-6-fluoro)-2-hydroxyethyl]amine 1, was investigated. The acetylation of 3-(p-fluorophenoxy)-1,5-pentanediol 5a using lipozyme and the hydrolysis of 1,5-diacetoxy-3-(p-fluorophenoxy)pentane 5b using lipase Amano P yielded (3S)- and (3R)-5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, respectively, with high enantiomeric excess. Four diastereomers of (6-fluoro-2-chromanyl)oxirane 2, important intermediates for the synthesis of R-67555, were synthesized by chemical methods using (S)-4 and (R)-4.     

Salt hydrates for in situ water activity control have acid-base effects on enzymes in nonaqueous media

Salt hydrates very frequently are utilized as in situ water activity buffers in reaction mixtures of enzymes in nonaqueous media. In addition to buffering water activity, there is evidence that salt hydrates also often affect initial rates in other ways. This has been generally overlooked or thought to be related to water transfer effects. Here we show that salt hydrates can have important acid-base effects on enzymes in nonaqueous media. We performed transesterification reactions in n-hexane and in supercritical ethane catalyzed by cross-linked crystals of subtilisin, differing in the method used to set a(W), and confirmed that the presence of salt hydrate pairs significantly affected the catalytic performance of the enzyme. However, in the presence of a solid-state acid-base buffer, salt hydrates had no effect on enzymatic activity. Direct evidence for the acid-base effects of salt hydrates was obtained by testing their effect on the protonation state of an organo-soluble H(+)/Na(+) indicator. The four salt hydrate pairs tested affected the indicator to very different extents. By promoting the exchange of H(+) for Na(+), salt hydrates will tend to affect the ionization state of acidic residues in the protein and, hence, enzymatic activity. In fact, salt hydrates were able to affect the pH memory of subtilisin lyophilized from different aqueous pHs, bringing about up to 20-fold enhancements and up to 5-fold decreases in catalytic activity. The possibility of such acid-base effects need to be considered in all experiments using salt hydrates to control water activity.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础.方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的...     

Ionic liquids as alternative co-solvents for laccase: study of enzyme activity and stability

The activity and stability of commercial laccase (DeniLite base) in three different water soluble ionic liquids (ILs) (1-ethyl-3-methylimidazolium 2-(2-methoxyethoxy) ethylsulfate, [emim][[MDEGSO4], 1-ethyl-3-methylimidazolium ethylsulfate, [emim][EtSO4], and 1-ethyl-3-methylimidazolium methanesulfonate, [emim][MeSO3]) have been studied and compared to that in two organic solvents (acetonitrile and dimethyl sulfoxide). Initial enzyme activities were similar among the ILs if the same conditions were used. A high reduction on initial enzyme activity was found with acidic pH (5.0). The effect of pH and solvent concentration on enzyme stability were investigated in more detail for 1 week. The enzyme maintained a high stability at pH 9.0 for all ILs tested. [emim][MDEGSO4] was the most promising IL for laccase with an activity loss of about 10% after 7 days of incubation. The kinetic studies in the presence of ABTS as substrate allowed to calculate the Michaelis- Menten parameters. Good agreement was found between experimental data and calculated values using the Michaelis-Menten mechanism, with a total average relative deviation of 2.1%.     

Study on the kinetics of enzymatic interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor

A new route for biodiesel production using methyl acetate instead of methanol as the acyl acceptor was proposed in our previous research, and it has been found that this novel route could enhance the stability of the immobilized lipase greatly. In this paper, the kinetics of lipase-catalyzed interesterification of triglycerides for biodiesel production with methyl acetate as the acyl acceptor was further studied. First, a simplified model based on Ping Pong Bi Bi with substrate competitive inhibition mechanism was proposed to describe the reaction kinetics of the interesterification. During our further study, it was observed that three consecutive and reversible reactions occurred in the interesterification of triglycerides and methyl acetate. So, a kinetic model based on mass balance of three second-order reversible reactions was developed and the reaction rate constant, k, was determined by solving the differential rate equations of the reaction system. The results showed that k_DG–MG (0.1124) and k_MG–TA (0.1129) were much higher than k_TG–DG (0.0311), which indicated that the first step reaction was the limit step for the overall interesterification.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

The Use of Salt Hydrates as Water buffers To Control Enzyme Activity in Organic Solvents

The conversion of o-diphenols to o-quinones was carried out in chloroform using a dry powder prepared from mushrooms as the catalyst. Several salt hydrates proved effective in supplying the small amount of water necessary for catalysis. The efficacy of the hydrates was related to their water activities and their use provided a convenient method for controlling water activity in nearly non-aqueous conditions.