Patrick Adlercreutz

ADSORPTION OF LIPASE ON POLYPROPYLENE POWDER

Adsorption of different lipases by EP-100 polypropylene powder from crude and pure lipase preparations was studied. Langmuir isotherms described the adsorption equilibria well both for protein and lipase activity adsorption. Adsorption isotherms for five different proteins all gave a similar saturation level of 220 mg protein per g carrier. Twelve commercial lipase preparations were tested for selectivity in the adsorption of lipase. For all preparations the selectivity factor was larger than one. In a crude lipase preparation from Pseudomonas fluorescence, the specific activity in solution decreased by two orders of magnitude after adsorption. The adsorption was not significantly influenced by pH changes in the adsorption buffer, indicating that hydrophobic and not electrostatic interactions are the dominating adsorption forces. Adsorption of a crude lipase from Candida rugosa (Sigma) was fast and equilibrium was reached in 30 and 100 min for protein and lipase activity adsorption respectively. Desorption in aqueous solution was negligible. Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent.

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.

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.

Preparation of diglycerides by lipase-catalyzed alcoholysis of triglycerides

Lipase from Penicillium roquefortii immobilized on porous polypropylene particles was used for enzymatic preparation of 1,2-diglycerides by alcoholysis in organic media. A screening of commercially available lipases showed that lipases from Penicillium species produced high amounts of 1,2-diglycerides from triglycelirles. Reaction parameters such as solvent, alcohol, water activity, and fatty acid chain length wet-e investigated. The positional selectivity of the lipase as well as the selectivity for type of glyceride specie were studied using pure isomers of the partial glycerides. The enzyme showed high selectivity for triglycerides and 1-monoglycerides and very low activity towards diglycerides. The lipase had a clear preference for the 1- and 3-positions. The highest lipase activity was observed at low water activity, but the yield increased with increasing water activity. Above all, the regio-isomeric purity of the diglycerides increased with increasing water activity. The yield of dilaurin was 75% and furthermore, 95% of the the total dilaurin was 1,2-dilaurin. Alcohol concentration and chain length of the alcohol had insignificant effect on yield and enzyme activity, but product stability increased when alcohol was present in the reaction medium. The best solvents were ethers; higher enzyme activities were obtained in aliphatic hydrocarbons bur yields and regio-isomeric purities were low presumably due to acyl migration. (Less)

Lipase catalyzed esterification of lactic acid

Reactions between lactic acid and alcohols or carboxylic acids catalyzed by lipase from Candida antarctica were evaluated with hexane as solvent. Lactic acid was a good acyl donor and esters of both primary and secondary alcohols were effectively synthesized. No interfering dimer formation due to lactic acid acting as both nucleophile and acyl donor was observed. In agreement with this, no esterification occurred between lactic acid and carboxylic acids.

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 .

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.

Water transfer kinetics in a water activity control system designed for biocatalysis in organic media

Abstract The performance of a water activity-controlling system in organic solvents using saturated salt solutions circulating in silicone tubing submerged in the solvent was studied. The mass transfer could be regarded as a flux through a cylindrical geometry. Integration over the barrier gave diffusion coefficients of water ( D aw ) which were similar for the different tubing sizes used. The driving force for the transfer was shown to be the difference in water activity and not the water concentration across the membrane. Hydrophilic solvents (ethyl acetate) gave higher transfer rates than more hydrophobic ones (diisopropyl ether). The D aw obtained in different solvents was influenced by the swelling behavior of the tubing and the solubility of water. The water transfer was studied in a water “producing” system consisting of 2.5 m silicone tubing submerged into a 250-ml tank reactor with a constant influx of water-saturated solvent. Different steady-state levels were obtained at different flow rates and the corresponding D aw values were calculated. The data obtained can be used to predict the required amount of tubing necessary to achieve a desired water transfer in a new application.

Lipase-catalyzed esterification of ethylene glycol to mono- and diesters. The effect of process parameters on reaction rate and product distribution

Diol fatty acid esters find a variety of industrial applications. The use of lipases in microaqueous environment provides an attractive route for synthesis of fatty acyl esters of ethylene glycol. The effect of various process parameters that play key roles in delineating reaction kinetics and distribution of mono- and diesters in the product have been studied.