Showing papers in "Biocatalysis and Biotransformation in 1992"

Complex Formation Between Chymotrypsin and Ethyl Cellulose as a Means to Solubilize the Enzyme in Active Form in Toluene

Abstract: Chymotryspin and ethyl cellulose were mixed in an aqueous phosphate buffer solution and freeze dried. Due to complex formation between the substances it was possible to dissolve or at least finely disperse these preparations in toluene. The chymotrypsin-ethyl cellulose complexes were characterized by light scattering measurements. Complexes were also formed by mixing enzyme powder in toluene containing ethyl cellulose and buffer but this was a slow process. Experiments with radioactively labelled bovine serum albumin showed that this protein was also solubilized in toluene in the presence of ethyl cellulose and buffer salts. Chymotrypsin complexes were used to catalyze the esterification of N-acetyl-L-phenylalanine with ethanol in toluene. The presence of buffer salts greatly increased the initial reaction rate of the esterification reaction. The complexes were consideraly more active and stable than enzyme powder in toluene.

The production of catechols from benzene and toluene by Pseudomonas putida in glucose fed-batch culture

Abstract: Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1-1).In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibit...

Modification of the microenvironment of enzymes in organic solvents. Substitution of water by polar solvents

Abstract: Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.

Inspection of an Acyl-Enzyme Intermediate in a Lipase Reaction by Gas Chromatography-Mass Spectrometry and Modelling of the Reaction Mechanism

Abstract: An acyl-enzyme intermediate proposed in the reaction mechanism of lipase was inspected by the exchange of oxygen between substrate (oleic acid) and solvent (18O-labelled water). Gas chromatography-...

Reaction Medium Selection for An Enzymatic Peptide Synthesis in An Aqueous-Organic Two-Phase System

Abstract: Efficient development of enzymatic synthesis in two-phase systems is closely related with appropriate selection of the reaction medium (especially the solvent and phase ratio). A selection procedure based on the calculation of the theoretically allowable conversion and product concentration is presented and applied to a peptide synthesis using papain. For the synthesis of the dipeptide Boc-Gly-Phe-OMe, the operating conditions have been determined, and the two-phase system to be used has been successfully selected (with trichloroethylene being the best solvent).