Kinetic study of the lipase-catalyzed synthesis of triolein

The kinetics of the synthesis of triolein catalyzed by immobilized Mucor miehei lipase were studied. Equilibrium constants for the synthesis of mono-, di-, and triolein were calculated from the equilibrium compositions for different initial ratios of glycerol and oleic acid by means of multiresponse regression. The 1,3-specific lipase can catalyze the synthesis of triolein because the ester enzymatically formed with the primary alcohol isomerizes, through acyl migration, to an ester on the secondary hydroxyl. The freed primary hydroxyl may then undergo further enzymatic conversion. The rates of isomerization depend on the concentration of oleic acid. (c) 1993 Wiley & Sons, Inc.     

Kinetics of lipase-catalyzed esterification in supercritical CO(2)

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO(2)) and organic solvent (n-hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase from Mucor miehei (Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping-Pong Bi-Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.     

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.     

Production of glycerides from glycerol and fatty acid by immobilized lipases in non-aqueous media

Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid. No organic solvent is necessary to solubilize the substrates, which allows for the use of a reaction medium solely composed of the necessary substrates. Water produced in the reaction evaporates due to the high temperature used for the process. A conversion of 86% of oleic acid into triolein is obtained when using the substrates in stoichiometric amounts. Varying the ratio of glycerol over oleic acid allows for the preferential synthesis of one of the glycerides. Some batch reactors have been set up using different means of removing the water: spontaneous evaporation, molecular sieves, vacuum, and dry air bubbling.     

On the issue of interfacial activation of lipase in nonaqueous media

The question of whether lipases can be activated by adsorption onto an interface in organic solvents was addressed using Rhizomucor miehei lipase as a model. In aqueous solution, this enzyme was shown to undergo a marked interfacial activation. However, lipase (either lyophilized or precipitated from water with acetone) suspended in ethanol or 2-(2-ethoxyethoxy)ethanol containing triolein exhibited no jump in catalytic activity when the concentration of triolein exceeded its solubility in these solvents, thereby resulting in formation of an interface. To test whether the lack of interfacial activation was due to the insolubility of the enzyme in organic media, lipase was covalently modified with poly(ethylene glycol). The modified lipase, although soluble in nonaqueous media, was still unable to undergo interfacial activation, regardless of the hydrophobicity of the interface. This inability was found to be caused by the absence of adsorption of lipase onto interfaces in organic solvents, presumably because of the absence of the hydrophobic effect (the driving force of lipase adsorption onto hydrophobic interfaces in water) in such media. The uncovered lack of interfacial adsorption and activation suggests that the short alpha-helical "lid" covering the active center of the lipase remains predominantly closed in nonaqueous media, thus contributing to diminished enzymatic activity. (c) 1996 John Wiley & Sons, Inc.     

Lipase reaction in AOT-isooctane reversed micelles: effect of water on equilibria

The effect of water on equilibria for hydrolytic reaction in reversed micelles has been investigated using lipase as a model enzyme. The effect of water on equilibria has been ignored for hydrolase reactions in an aqueous phase. In a reversed micellar system, however, the equilibrium of the lipase reaction was changed when water was added during the hydrolytic reaction. Furthermore, equilibrium fractional conversion is affected by the initial water concentration, being shifted to higher values with higher water concentrations, with other reaction conditions being held constant, indicating that the reaction should be regarded as a two-substrate process. Equations corresponding to a two-substrate, second-order reversible model are derived and used for further analysis. The progress curves predicted from the rate equations agree very well with the experimental results under various reaction conditions. The values of the molar ratio of water to surfactant (R) which maximize the initial reaction rate and maximum fractional conversion is predictable from the derived rate equations and the resulting relationship between R and the kinetic constants.     

Thermodynamics of the reactions of carbamoyl phosphate

Two measurements of equilibrium constants by Marshall and Cohen make it possible to calculate standard Gibbs energies of formation of the species of carbamate and carbamoyl phosphate. Carbamate formation from carbon dioxide and ammonia does not require an enzyme, and the equilibrium concentrations of carbamate in ammonium bicarbonate are calculated. Knowing the values of standard Gibbs energies of formation of species of carbamate and carbamoyl phosphate make it possible to calculate the dependencies of the standard transformed Gibbs energies of formation of these reactants on pH and ionic strength and to calculate apparent equilibrium constants for several enzyme-catalyzed reactions and several chemical reactions. These calculations are sufficiently complicated that computer programs in Mathematica are used to make tables and plots. The dependences of apparent equilibrium constants on pH are consequences of the production or consumption of hydrogen ions, which are shown in plots. As usual the increase in the number of enzyme-catalyzed reactions for which apparent equilibrium constants can be calculated is larger than the number of reactions required to obtain the thermodynamic properties of the species involved.     

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.     

Thermodynamic properties of nucleotide reductase reactions

Recent thermodynamic measurements have made it possible to calculate the apparent equilibrium constants of the ribonucleoside diphosphate reductase reaction and the ribonucleoside triphosphate reductase reaction with various reducing agents. Third law heat capacity measurements on crystals of d-ribose and other calorimetric measurements make it possible to calculate Delta(f)G degrees for D-ribose and two species of D-ribose 5-phosphate. The experimental value of the apparent equilibrium constant K' for the deoxyribose-phosphate aldolase reaction makes it possible to calculate the standard Gibbs energies of formation Delta(f)G degrees for two protonation states of 2'-deoxy-D-ribose 5-phosphate. This shows that Delta(f)G degrees (2'-deoxy-D-ribose 5-phosphate(2)(-)) - Delta(f)G degrees (D-ribose 5-phosphate(2)(-)) = 147.86 kJ mol(-1) at 298.15 K and zero ionic strength in dilute aqueous solutions. This difference between reduced and oxidized forms is expected to apply to D-ribose, D-ribose 1-phosphate, ribonucleosides, and ribonucleotides in general. This expectation is supported by two other enzyme-catalyzed reactions for which apparent equilibrium constants have been determined. The availability of Delta(f)G degrees values for the species of 2'-deoxy-D-ribose and its derivatives makes it possible to calculate standard transformed Gibbs energies of formation of these reactants, apparent equilibrium constants for their reactions, changes in the binding of hydrogen ions in these reactions, and standard apparent reduction potentials of the half reactions involved as a function of pH and ionic strength at 298.15 K. The apparent equilibrium constant for ADP + thioredoxin(red) = 2'-deoxyADP + H(2)O + thioredoxin(ox) is 1.4 x 10(11) at 298.15 K, pH 7, and 0.25 M ionic strength.     

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.     

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

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.     

Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. (c) 1993 John Wiley & Sons, Inc.     

Enzymatic synthesis of alpha-butylglucoside linoleate in a packed bed reactor for future pilot scale-up

The enzymatic synthesis of a mixture of unsaturated fatty acid alpha-butylglucoside esters, containing more than 60% alpha-butylglucoside linoleate, was achieved through lipase-catalyzed esterification. The continuous evaporation under reduced pressure of the water produced enabled substrate conversions greater than 95% to be reached. Two immobilized lipases from Candida antarctica (Chirazyme L2, c.-f., C2) and Rhizomucor miehei (Chirazyme L9, c.-f.) were compared in stirred batch and packed bed configurations. When the synthesis was carried out in stirred batch mode, C. antarctica lipase appeared to be of greater interest than the R. miehei enzyme in terms of stability and regioselectivity. Surprisingly, a change in the process design to a packed bed configuration enabled the stability of R. miehei lipase to be significantly improved, while the C. antarctica lipase efficiency to synthesize unsaturated fatty acid alpha-butylglucoside esters was slightly decreased. Water content in the microenvironment of the biocatalyst was assumed to be responsible for such changes. When the process is run in stirred batch mode, the conditions used promote the evaporation of the essential water surrounding the enzyme, which probably leads to R. miehei lipase dehydration. In contrast, the packed bed design enabled such water evaporation in the microenvironment of the biocatalyt to be avoided, which resulted in a tremendous improvement of R. miehei lipase stability. However, C. antarctica lipase led to the formation of 3% diesters, whereas the final percentage of diesters reached 21% when R. miehei enzyme was used as biocatalyst. A low content of diesters is of greater interest in terms of alpha-butylglucoside linoleate application as linoleic acid carrier, and therefore the enzyme choice will have to be made depending on the properties expected for the final product.     

Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.     

Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.     

Enantioselective esterification of 2-arylpropionic acids and trans-2-phenyl-1-cyclohexanol: Comparison between immobilised lipases from Candida rugosa and Rhizomucor miehei

2-Phenyl propionic acid, ibuprofen and trans-2-phenyl-1-cyclohexanol were resolved using commercial Rhizomucor miehei lipase (Lipozyme IM20) and Candida rugosa lipase produced in our laboratory immobilised on EP100 polypropylene powder. Important differences were found on the enantioselectivity of both lipases in esterification reactions. Candida rugosa lipase was more enantioselective in the resolution of the tested substrates, especially with trans-2-phenyl-1-cyclohexanol, whereas the lipase from Rhizomucor miehei did not show catalytic activity with this substrate. © Rapid Science Ltd. 1998     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.