Ernst Wehtje

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.

How do additives affect enzyme activity and stability in nonaqueous media

The catalytic activities of lyophilized powders of alpha-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties. Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100-fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions. Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation.The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7 degrees C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein. The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.

Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile leases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohol, or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes. The temperature effects on enantioselectivity, were studied in detail. From measurements of the enantiomeric ratio (C) at different temperatures the activation parameters DeltaDeltaH(#) and DeltaDeltaS(#) were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, DeltaDeltaH(#) and DeltaDeltaS(#) had opposite sign, and therefore the effects cooperated to give high E values (-RTInE = DeltaDeltaG(#) = DeltaDeltaH(#) - TDeltaDeltaS(#)). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations wars discussed. In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions. while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict

Enantioselectivity of lipases : effects of water activity

The enantioselectivity (E) of lipases in esterifications of secondary alcohols with decanoic acid was studied in organic media. The enantioselectivity of 2-octanol differed greatly among the lipases used. Candida antarctica lipase was extremely selective (E = 9 000) while Candida rugosa lipase was much less selective (E = 1.7). Other enzymes (Lipozyme and lipases from Pseudomonas and Rhizopus arrhizus) had intermediate selectivities. In all cases the enantioselectivity for an enzyme was unaffected by changes in water activity. Different methods of determining the enantioselectivity was used: reactions using single enantiomers as well as racemic mixtures. The effect of water activity on enantioselectivity and the enanatioselectivity values themselves were similar irrespective of the method used. The enantioselectivity of other alcohols were also found to be unaffected by the water activity. The enantioselectivity of Pseudomonas lipase was influenced by the organic solvent. The E decreased with increasing hydrophobicity, from 62 in acetonitrile to 40 in toluene and 33 in hexane. In none of these cases was the enantioselectivity affected by the water activity. However, for Lipozyme and Candida rugosa lipase in toluene a trend of increased E with increasing water activity was observed. In summary it can be stated that the water activity does not generally affect the enantioselectivity of the five lipases tested.

Mass transfer studies on immobilized α-chymotrypsin biocatalysts prepared by deposition for use in organic medium

Mass transfer limitations were studied in enzyme preparations of alpha-chymotrypsin made by deposition on different porous support materials such as controlled pore glasses, Celite, and polyamides of different particle sizes. It is the onset of mass transfer limitations that determines the position of the activity optimum with respect to enzyme loading on each support. The evidence of various experiments indicates that internal diffusional limitations are the important mechanism for the observed mass transfer limitations. External diffusion was not found to play an important role under the conditions used, and it was also found that when immobilizing multilayers of enzyme the buried enzyme molecules are active to a large extent. An extreme situation is observed on Celite at very high loadings. Under these conditions, this support is expected to have its pores completely filled with packed enzyme molecules, and then it is the diffusion within the enzyme layer that determines the observed rate. As the enzyme loading increases, the area of contact between the deposited enzyme layers and the liquid solution inside the pores diminishes, causing a decrease on the observed rate of an intrinsically fast reaction which apparently is incongruous with the presence of more enzyme in the system. This work shows that mass transfer limitations can be an important factor when working with immobilized enzymes in organic media, and its study should be carried out in order to avoid undesired reduced enzyme activities and specificities.

Continuous control of water activity during biocatalysis in organic media

This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.

Influence of water activity on the competition between β-glycosidase-catalysed transglycosylation and hydrolysis in aqueous hexanol

Five different β-glycosidases (Pyrococcus furiosus β-glucosidase, Sulfolobus solfataricus β-galactosidase, Caldocellum saccharolyticum β-glucosidase, almond β-glucosidase and Escherichia coli β-galactosidase) were evaluated as transglycosylation catalysts in hexanol containing various amounts of water. All enzymes catalysed both hydrolysis and transglycosylation of the glycosidic substrates (pentyl- and p-nitrophenyl-β-glucoside and p-nitrophenyl-β-galactoside). From the concentration ratio (alcohol/water) it was expected that the transglycosylation/hydrolysis ratio would decrease with increasing water activity in the hexanol. However, for all enzymes tested the selectivity for the alcohol increased with increasing water activity. This counteracted the effect of higher water concentration and in most cases the transglycosylation/hydrolysis ratio increased with increasing water activity. On the other hand, in hexanol/water two-phase systems, hydrolysis was by far the dominating reaction even though the total activity increased for all enzymes. The selectivity values were used to predict the maximal reaction yields in the kinetically controlled reactions. However, deviations were found in cases when the reactions became thermodynamically controlled: at high water contents secondary hydrolysis reduced the transglycosylation yields while higher transglycosylation yields than predicted were obtained at low water activity in some cases using enzymes poorly selective for the alcohol.

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze‐dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.

Characterization and optimization of phospholipase A2 catalyzed synthesis of phosphatidylcholine

The phospholipase A2 (PLA2) catalyzed synthesis and hydrolysis of phosphatidylcholine (PC) was studied in a water activity controlled organic medium. The aim of the study was to find the conditions most favorable for the synthetic reaction. To do this, the impact of various parameters such as water activity, substrate concentration and temperature on enzyme activity and equilibrium yield was determined. The PC to lysophosphatidylcholine (LPC) ratio at equilibrium increases with decreasing water activity and increasing fatty acid concentration, as can be expected from the law of mass action of an esterification reaction. The enzyme activity on the other hand decreases under conditions that favor the esterification. The best yield in the synthetic reaction is 60% at a water activity of 0.11 and an oleic acid concentration of 1.8 M. That is to our knowledge the highest yield ever reported in this reaction. Both the hydrolysis and synthesis reaction follow Michaelis-Menten kinetics, the apparent Km values are the same for PC and LPC, namely 4.9 mM. Vmax is 82.5 and 10.4 nmol h(-1) mg(-1) for the hydrolysis and synthesis reaction, respectively. Studies on PLA2 at water activity controlled conditions resulted in a more complete understanding of the enzymatic reaction and allowed to find the conditions most favorable for the synthetic reaction.

Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones.

The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.