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.     

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.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

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.     

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.     

Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems

The hydrolysis of olive oil catalyzed by Chromobacterium viscosum lipase (EC 3.1.1.3) in a water/isooctane two-phase system was carried out both under ultrasound and conventional stirring. The maximum activity of lipase in the ultrasonicated system was 1.75 times higher than that in the stirred system. The lipase activity was dependent on ultrasonic power and volume ratio of isooctane to water. The optimum reaction temperature in both systems was around 25°C. The stability of lipase at 25°C in the ultrasonicated system decreased more rapidly than that in the stirred system. In the presence of exogenous oleic acid, however the half-life of lipase in the ultrasonicated system was improved to a value, which was respectively half and twice of that in stirred systems with and without oleic acid. The maximum reaction rate (V_max) was increased by ultrasonication whereas the Michaelis constant (K_m) remained unaltered.     

Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.     

Organic solvents strip water off enzymes

Exchange of enzyme-bound H(2)O with T(2)O in aqueous solution followed by freeze drying provided tritiated water bound to chymotrypsin, subtilisin Carlsberg, and horseradish peroxidase. The desorption of T(2)O from these enzymes suspended in various organic solvents showed that all three enzymes lost enzyme-bound water with peroxidase losing the most T(2)O of the three in solvents of moderate to high polarity. Polar solvent resulted in the highest degree of T(2)O desorption (e.g., methanol desorbed from 56%-62% of the bound T(2)O), while nonpolar solvents resulted in the lowest degree of desorption (e.g., hexane desorbed from 0.4%-2% of the bound T(2)O). Desorption is nearly immediate with most of the desorbable T(2)O being released from the enzymes within the first 5 min. Both solvent dielectric and a measure of the saturated molar solubility of water in a given solvent provide accurate correlations between the properties of the organic solvents and the extent of T(2)O desorption. This investigation shows that water stripping from an enzyme into a nonaqueous medium does occur and can be significant in polar solvents.     

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.     

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.     

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.     

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...     

Facile biocatalytic synthesis of a macrocyclic lactone in sub- and supercritical solvents

Abstract

High yield and selectivity in the intramolecular synthesis of the macrocyclic lactone oxacyclohexadecan-2-one were achieved via a straightforward biocatalytic synthesis utilising non-conventional solvents: supercritical carbon dioxide (scCO₂), ethane, fluoroform and 1,1,1,2-tetrafluoroethane. Batch and continuous syntheses were demonstrated with a higher yield attained in a continuous synthesis using in-situ product extraction by scCO₂. A remarkably high yield was obtained in 1,1,1,2-tetrafluoroethane at batch conditions.     

Enzyme thermoinactivation in anhydrous organic solvents

Three unrelated enzymes (ribonuclease, chymotrypsin, and lysozyme) display markedly enhanced thermostability in anhydrous organic solvents compared to that in aqueous solution. At 110-145 degrees C in nonaqueous media all three enzymes inactivate due to heat-induced protein aggregation, as determined by gel filtration chromatography. Using bovine pancreatic ribonuclease A as a model, it has been established that enzymes are much more thermostable in hydrophobic solvents (shown to be essentially inert with respect to their interaction with the protein) than in hydrophilic ones (shown to strip water from the enzyme). The heat-induced aggregates of ribonuclease were characterized as both physically associated and chemically crosslinked protein agglomerates, with the latter being in part due to transamidation and intermolecular disulfide interchange reactions. The thermal denaturation of ribonuclease in neat organic solvents has been examined by means of differential scanning calorimetry. In hydrophobic solvents, the enzyme exhibits greatly enhanced thermal denaturation temperatures (T(m) values as high as 124 degrees C) compared to aqueous solution. The thermostability of ribonuclease towards heat-induced denaturation and aggregation decreases as the water content of the protein powder increases. The experimental data obtained suggest that enzymes are extremely thermostable in anhydrous organic solvents due to their conformational rigidity in the dehydrated state and their resistance to nearly all the covalent reactions causing irreversible thermoinactivation of enzymes in aqueous solution.     

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.