Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Esterification reactions catalyzed by Chromobacterium viscosum lipase in CTAB-based microemulsion systems

Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been used for multigram-scale ester synthesis, including the kinetic resolution of a secondary alcohol. The stability of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of ethylhexadecanoate as a model reaction. Under pseudo-first-order conditions, the synthesis rates were linearlydependent on the enzyme and fatty acid concentrations and the R dependence shows the characteristic bell-shaped curve (where R = [H(2)O]/[surfactant]). The dependence of enzyme activity toward octyldecanoate synthesis on the pH of the dispersed buffer phase is in marked contrast to that observed for the pH dependence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the former case, the pH-activity profile is approximately sigmoidal, which may reflect the ionization state of the fatty acid substrate. In the latter case, the pH dependence is minimal at both R = 10 and R = 50, suggesting the enzyme does not experience a changed pH environment. Inclusion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected buffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the esterification reactions, thereby providing an additional means of determining initial rates. The method may be of general value for lipase assays in microemulsions since it may provide, at the same time, information regarding enzyme regioselectivity. (c) 1995 John Wiley & Sons, Inc.     

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. (c) 1993 John Wiley & Sons, Inc.     

Pseudomonas cepacia lipase: Esterification reactions in AOT microemulsion systems

Summary The activity of purifiedPseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis(2ethylhexyl)sulfosuccinate sodium salt (AOT). The optima pH, T and water content (w_o) for the enzyme activity in this type of microemulsions have been determined. Studies on the effect of various fatty acids and alcohols on the enzyme specificity have shown a preference of this lipase for palmitic and caprylic acid as well as for propanol, while reactions involving cyclic alcohols can not be catalyzed at all. The differences on the behavior of this lipase as compared to other lipases studied in microemulsion systems as well as in other systems are discussed.     

Lipases have similar water activity profiles in different reactions

The shape of the profiles of enzyme activity versus water activity for four different lipases were independent of the reaction used to determine the activity. The profile for each lipase (Rhizopus arrhizus, Pseudomonas sp., Candida rugosa and Lipozyme) in esterification, hydrolysis and transesterifications profiles were the same. In transesterification the yield was unaffected by the water activity but the hydrolysis yield increased with increasing aW .     

脂肪酶在微乳液和微乳液凝胶中催化辛酸辛醇的酯化反应

脂肪酶在合成反应中具有很高的区域选择性和立体选择性 ,已广泛用于食品工业和药物工业[1,2 ] ,在有机介质中的脂肪酶催化反应已有较多研究[3 ,4 ] 。微乳液一般由表面活性剂、助表面活性剂、油和水等组份组成 ,它是一种热力学稳定、光学透明、宏观均匀而微观不均匀的体系 ,能提供酶催化所需要的巨大油 /水界面[5] 。而将脂肪酶增溶于油包水(Ｗ /Ｏ)微乳液中的纳米级“水池”中 ,可使酶以分子水平分散[6] ,图 1(ａ) ,从而可用来模拟细胞微环境中的反应。油包水微乳液中的酶可通过加入明胶而制成固定化酶 ,含明胶的微乳液凝胶 (ＭＢＧｓ)最早…     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

Esterification reactions catalyzed by lipases in microemulsions: the role of enzyme localization in relation to its selectivity

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. (c) 1993 John Wiley & Sons, Inc.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

华根霉脂肪酶有机相合成酶活的研究

通过比较7种微生物脂肪酶的有机相合成酶活、水相水解酶活及在正庚烷中催化己酸乙酯合成的能力,证明了合成酶活与水解酶活相关性不高,合成酶活比水解酶活更能反映脂肪酶的合成能力。通过比较两株华根霉(Rhizopus chinensis)脂肪酶酶活,发现合成酶活相差较大,表明相同种属微生物的脂肪酶合成酶活存在不同。对．Rhizopus chinensis-2液态发酵产脂肪酶进程研究发现,水解酶活高峰先于合成酶活高峰大约12h。将不同培养时间的Rhizopus chinensis-2全细胞脂肪酶用于催化己酸乙酯合成,具有高合成酶活的全细胞脂肪酶催化己酸乙酯合成反应较快。因此,全细胞脂肪酶用于催化有机相酯合成反应时,具有高脂肪酶合成酶活的菌体具有较好的催化酯合成能力。     

Effects of organic solvents on activity and conformation of recombinant Candida antarctica lipase A produced by Pichia pastoris

The effect of various organic solvents on the hydrolytic and esterification activities of lipase A from Candida antarctica (CALA) was investigated. Compared to the control group, the esterification and hydrolytic activities of CALA both increased with treatment of acetonitrile or acetone. However, the catalytic activity decreased after treatment with other organic solvents, especially with ethanol or ethyl acetate. Moreover, with treatment by the same organic solvents, the residual esterification activity of CALA was much higher than the hydrolytic activity for the olive oil. Results of Fourier transform-infrared spectroscopy analysis implied that the catalytic activity variance was mainly attributed to the secondary structure changes of CALA. The acetonitrile treatment could also increase the thermal and pH stability of CALA.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Biocatalysis using gelatin microemulsion-based organogels containing immobilized Chromobacterium viscosum lipase

Chromobacterium viscosum (CV) lipase was immobilized in gelatin-containing Aerosol-OT (AOT) microemulsion-based organogels (MBGs). The behavior of this novel, predominantly hydrophobic matrix as an esterification catalyst has been examined. The biocatalyst was most effective when the MBG was granulated to yield gel particles of approximately 500 mum diameter, providing a total surface area of ca. 10(6) mm(2) per 10 cm(3) of gel. The gel was generally contacted with a solution of the substrate(s) in a hydrocarbon oil. Under most conditions reaction was not diffusion limited. Apparent lipase activity was influenced by certain compositional changes in the MBG, but most significantly when the R value, the mole ratio of water to surfactant, was altered. Higher activities were observed at lower R values. Although gels of lowest R value expressed the highest condensation activity, such formulations were physically unsuitable as immobilization matrices due to their proximity to the gel-solution phase boundary. MBGs of intermediate R values (between 60 and 80) were considered most suitable because they offer relatively high condensation activity and good physical stability. The gelatin concentration also exerted a small but measurable influence on the observed condensation rates. Apparent lipase activity was also influenced to some extent by the nature of the parent hydrocarbon used to prepare the MBG. Higher activities were obtained using formulations derived from isooctane and cyclohexane rather than the n-alkanes. Condensation activities expressed by CV lipase in the MBGs were broadly comparable to those expressed in the analogous parent water-in-oil (w/o) microemulsions. The MBGs functioned effectively in neat substrate solutions, but the condensation activity expressed by the MBGs in a series of successive batch syntheses was adversely affected by the formation and retention of the water coproduct. Selective removal of the water was achieved using a concentrated solution of dry reverse micelles, which resulted in recovery of lost activity. Pretreatment of lipase-containing MBGs resulted in the formation of MBGs with enhanced catalytic properties and modified composing the conventional procedure. (c) 1997 John Wiley & Sons, Inc.     

Kinetics and mechanism of lipase catalyzed monoacylglycerols synthesis

Monoacylglycerols are increasingly used in several industrial applications as effective and cheap emulsifiers. In the present work monostearin synthesis has been studied, using lipase as a biocatalyst of the esterification reaction of stearic acid with (R,S)-1,2-O-iso-propylidene glycerol (solketal). The lipase from Candida antarctica (CaL B) was immobilized in AOT/isooctane water in oil microemulsions. Optimization of the reaction conditions have shown that the highest production (80% in 30 min) could be achieved at 40 °C, in microemulsions with relatively low water content (w_o = 8). Kinetic studies have shown that the esterification reaction of stearic acid with solketal catalyzed by CaL B occurs via the ordered bi–bi mechanism, in which inhibition by the acid was identified. Moreover, at high fixed solketal concentrations a negative cooperativity is pronounced, which means that binding of the alcohol lowers the affinity of the enzyme for binding of the acid. Values of all kinetic parameters have been determined.     

Kinetic studies of Chromobacterium viscosum lipase in AOT water in oil microemulsions and gelatin microemulsion-based organogels

Kinetic studies have shown that octyl decanoate synthesis by Chromobacterium viscosum (CV) lipase in sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) water in oil (w/o) microemulsions occurs via the nonsequential (ping-pong) bi bi mechanism. There was evidence of single substrate inhibition by decanoic acid at high concentrations. Initial rate data yielded estimates for acid and alcohol Michaelis constants of ca. 10(-1) mol dm(-3) and a maximum rate under saturation conditions of ca. 10(-3) mol dm(-3) s(-1) for a lipase concentration of 0.36 mg cm(-3). CV lipase immobilized in AOT microemulsion-based organogels (MBGs) was also found to catalyze the synthesis of octyl decanoate according to the ping-pong bi bi mechanism. Reaction rates were similar in the free and immobilized systems under comparable conditions. Initial rates at saturating (but noninhibiting) substrate concentrations were first order with respect to CV lipase concentration in both w/o microemulsions and the MBG/oil systems. Gradients yielded an apparent k(cat) = 4.4 x 10(-4) mol g(-1) s(-1) in the case of w/o microemulsions, and 6.1 x 10(-4) mol g(-1) s(-1) for CV lipase immobilized in the MBGs. A third system comprising w/o microemulsions containing substrates and gelatin at concentrations comparable to those employed in the MBG formulations, provided a useful link between the conventional liquid microemulsion medium and the solid organogels. The nongelation of these intermediate systems stems from the early inclusion of substrate during a modified preparative protocol. The presence of substrate appears to prevent the development of a percolated microstructure that is thought to be a prerequisite for MBG formation. FT-NMR was employed as a semicontinuous in situ assay procedure. The apparent activity expressed by CV lipase in compositionally equivalent liquid and solid phase gelatin-containing systems was similar. An apparent activation energy of 24 +/- 2 kJ mol(-1) was determined by (1)H-NMR for esterification in gelatin-containing w/o microemulsions. This value agrees with previous determinations for CV lipase-catalyzed synthesis of octyl decanoate in "conventional" w/o microemulsions and MBG/oil systems. The similarities in lipase behavior are consistent with the claim, based largely on structural measurements, that the physico-chemical properties of the lipase-containing w/o microemulsion are to a large extent preserved on transformation to the daughter organogel. The close agreement of apparrent activation energies suggests that substrate mass transfer is not rate determining in the three studied systems. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:416-427, 1997.     

Novel kinetic analysis of enzymatic dipeptide synthesis: effect of pH and substrates on thermolysin catalysis.

The point of maximum activity is specific to a particular substrate-enzyme system but may vary with different substrates and the same enzyme. The specificity of enzymes has, however, been generally reported only at their "optimal" pH. In this article, we introduce the Michaelis-Menten equation taking pH into account, and apply it to the pH-activity profile of the thermolysin-catalyzed dipeptide synthesis. It has been reported to date that the pH-activity profile of thermolysin follows a bell-shaped curve with a maximal activity at or near pH 7.0. The profiles obtained in this study, however, indicated that the optimal pH varied from 5.8 (for F-AspPheOMe) to 7.3 (for Z-ArgPheOMe), and the order of thermolysin activity was greatly dependent on the pH of reaction media. We have succeeded in evaluating the substrates-induced change of the dissociation states of the active site of thermolysin using the hydrophobicity of substrates. We have obtained apparent kinetic parameters which are independent of the pH of reaction media. The apparent specificity of thermolysin which were independent of pH of the reaction media was in order L-Leu > L-Asp > L-Arg > L-Ala > L-Gly > L-Val and Z > Boc = F at P1 and P2 positions, respectively.     

Glycosidase activities of the 293 and NS0 cell lines, and of an antibody-producing hybridoma cell line

We have previously demonstrated that Chinese hamster ovary (CHO) cell lysates harbor sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities that can accumulate extracellularly in CHO cell culture, thereby potentially leading to extracellular modification of glycoprotein oligosaccharides. The sialidase activity in CHO cell lysates was surprisingly active and stable at pH 7.5, with a half-life of 57 h at 37 degrees C.We have extended this work to determine whether 293, NS0, or hybridoma cell lysates contain similar glycosidase activities. The pH-activity profiles of beta-galactosidase and beta-hexosaminidase in lysates of these three cell lines resemble the pH-activity profiles for these enzymes in CHO cell lysate, whereas the pH-activity profiles of sialidase and fucosidase appear to be cell-type dependent. Sialidase activities were relatively stable at pH 4.5 in 293, NS0, and hybridoma cell lysates. However, the activities in 293 and NS0 cell lysates were unstable at pH 7.5, with no activity remaining after a 2-h incubation at 37 degrees C. The sialidase activity in hybridoma cell lysate was moderately stable at pH 7.5 with 30% of the activity remaining after a 2-h incubation at 37 degrees C. We conclude that the sialidase activites from 293, NS0, and hybridoma cells have characteristics similar to the vast majority of reported mammalian sialidase activities, and that these activities are markedly differant from the CHO cell sialidase activity.Finally, sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities were measured at pH 7 in cell-free bioreactor supernatants of the hybridoma cell line. As previously observed in CHO cell culture, all four glycosidase activities were present in the hybridoma supernatants. However, the sialidase activity in hybridoma supernatant was an order of magnitude lower than in CHO cell culture supernatant despite the fact that the hybridoma cell lysis rate was an order of magnitude higher. (c) 1994 John Wiley & Sons, Inc.     

Activity and Stability Studies Of Mucor miehei Lipase Immobilized in Novel Microemulsion-based Organogels

Lipase from Mucor miehei was immobilized in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT) as well as lecithin water-in-oil (w/o) microemulsion-based organogels (MBGs) formulated with biopolymers such as agar and hydroxypropylmethyl cellulose (HPMC), respectively. These lipase-containing MBGs prove to be novel solid-phase catalysts for use in organic media. Using these organogels at 25°C, various esterification reactions in non-polar solvents as well as in solvent free systems were possible. Apparent lipase activity was influenced to some extent by the nature and the concentration of biopolymers used. Lipase stability in such MBGs is much higher than that observed in w/o microemulsions. MBGs containing lipase functioned effectively in repeated batch syntheses of fatty esters. Kinetic studies have shown that ester synthesis catalyzed by immobilized lipase occurs via the Ping-Pong bi-bi mechanism in which only inhibition by excess of alcohol has been identified. Values of all kinetic parameters were determined.     

Activity and Stability Studies Of Mucor miehei Lipase Immobilized in Novel Microemulsion-based Organogels

Lipase from Mucor miehei was immobilized in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT) as well as lecithin water-in-oil (w/o) microemulsion-based organogels (MBGs) formulated with biopolymers such as agar and hydroxypropylmethyl cellulose (HPMC), respectively. These lipase-containing MBGs prove to be novel solid-phase catalysts for use in organic media. Using these organogels at 25°C, various esterification reactions in non-polar solvents as well as in solvent free systems were possible. Apparent lipase activity was influenced to some extent by the nature and the concentration of biopolymers used. Lipase stability in such MBGs is much higher than that observed in w/o microemulsions. MBGs containing lipase functioned effectively in repeated batch syntheses of fatty esters. Kinetic studies have shown that ester synthesis catalyzed by immobilized lipase occurs via the Ping-Pong bi-bi mechanism in which only inhibition by excess of alcohol has been identified. Values of all kinetic parameters were determined.