Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

Effect of mass-transfer limitations on the selectivity of immobilized alpha-chymotrypsin biocatalysts prepared for use in organic medium.

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.

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.

Mass transfer studies on immobilized alpha-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.

The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can be improved by optimisation of the temperature and by using a high co-substrate concentration.

The stereoselective reduction of ketones by alcohol dehydrogenase from Thermoanaerobium brockii was studied in organic reaction media. 2-Propanol was used as co-substrate to regenerate the coenzyme NADPH. The enantiomeric excess of the alcohol formed from the ketone decreased during the course of the reaction (from 53 to 0% e.e. in the formation of (R)-2-butanol). This was interpreted as being due to the reversibility of all the reactions involved. By using a large excess of 2-propanol this effect was suppressed. In the reduction of 2-butanone to (R)-2-butanol, the enantiomeric excess increased with increasing temperature, but in the reduction of 2-pentanone to (S)-2-pentanol the enantiomeric excess decreased with increasing temperature. The data were evaluated in terms of free energy of activation of the reaction pathways leading to the different possible products.

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.

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.

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.

Calorimetric studies on solid alpha-chymotrypsin preparations in air and in organic solvents.

Differential scanning calorimetry was the method to investigate the thermostability of chymotrypsin. The transition temperature decreased by approx. 30 degrees C when the dry enzyme became highly hydrated. High degree of hydration corresponded to extensive conformational changes during protein denaturation, reflected by large enthalpy values. Sorbitol, lyophilized together with the enzyme, caused the destabilization of the complex within the whole range of water activities. When the enzyme was equilibrated through the apolar solvent, isooctane, stabilization of chymotrypsin was observed at high water activities, compared to equilibration in air. The presence of isooctane resulted in a remarkable stabilization of the chymotrypsin-sorbitol complex. A sorbitol concentration of 5 mmol/g of protein was prerequisite to induce stabilization when equilibrated through isooctane at high water activities. The transition enthalpy increased with increasing amounts of sorbitol. Different hydration isotherms were obtained for the air-equilibrated and solvent-equilibrated enzyme preparations. Increasing amounts of buffer salts within the chymotrypsin preparation caused the enhancement of both the temperature and the enthalpy of the transition at a water activity 0.97. Variations on the hydration of the preparations both offered the explanation to the thermal stability results and supported the evidence obtained from enzyme activity studies. Generally, the catalyst whose hydration was suppressed due to its environment exhibited low enzymatic activity.

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media.

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C).

Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications.

A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent.

Improved activity retention of enzymes deposited on solid supports.

Enzymes deposited on solid support usually show good stability when operated in organic solvents. Decreased stability of the enzyme preparations was noticed when low enzyme loadings were used (e.g., with Celite as support; less than 1 mg enzyme/g). It was possible to avoid the activity loss by the addition of an additive which protects the enzyme during the immobilization. Proteins (such as albumin, gelatin, and casein) and poly(ethylene glycol) were effective additives whereas amino acids, monomeric carbohydrates, and polysaccharides had no effect. The amount of additive needed for stabilization was shown to depend on the structure of the support, more additive being required for a support with high porosity. The stabilizing effect was investigated in a series of glyceryl-controlled-pore glass (CPG) with varying specific surface areas (9.5-180 m(2)/g). The minimum addition of albumin, giving full stabilization, on the different supports correlated to a monolayer coverage of the surface, approximately 2-3 mg protein/m(2). The effect of the additive was less pronounced when increasing amounts of enzyme were immobilized (5-40 mg enzyme/g Celite). The effect of the additives was studied using mandelonitrile lyase, but alpha-chymotrypsin and lipase P were also shown to be stabilized.

Formation of C-C bonds by mandelonitrile lyase in organic solvents.

Mandelonitrile lyase (EC 4.1.2.10) catalyzes the formation of D-mandelonitrile from HCN and benzaldehyde. Mandelonitrile lyase was immobilized by adsorption to support materials, for example, Celite. The enzyme preparations were used in diisopropyl ether for production of D-mandelonitrile. In order to obtain optically pure D-mandelonitrile it was necessary to use reaction conditions which favor the enzymatic reaction and suppress the competing spontaneous reaction, which yields a racemic mixture of D, L-mandelonitrile. The effects of substrate concentrations, water content, and support materials on both the spontaneous and enzymatic reactions were studied. The enzymatic reaction was carried out under conditions where the importance of the spontaneous reaction was negligible and high enantiomeric purity of D-mandelonitrile was achieved (at least 98% enantiomeric excess). The operational stability of the enzyme preparations was studied in batch as well as in continuous systems. It was vital to control the water content in the system to maintain an active preparation. In a packed bed reactor the enzyme preparations were shown to be active and stable. The reactors were run for 50 h with only a small decrease in product yield.