n-benzyloxycarbonyl-aspartylphenylalanine-methyl-ester and ethyl-acetate

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency.

Topics: Acetates; Bacillus; Biotechnology; Dipeptides; Enzyme Stability; Enzymes, Immobilized; Hydrolysis; Kinetics; Microwaves; Pentanols; Sodium Chloride; Solvents; Substrate Specificity; Temperature; Thermolysin

In order to clarify the mechanism for the peptide synthesis with an immobilized enzyme in a water-immiscible organic solvent system, we studied the synthesis of Z-AspPheOMe from Z-Asp and PheOMe catalyzed by thermolysin immobilized onto Amberlite XAD-7. As an organic solvent, ethyl acetate was used. The reaction was also done in the aqueous/organic biphasic system and in ethyl acetate containing a small amount of water using free enzyme for comparison. The substrate concentration dependencies of the initial rate for the synthesis with the immobilized enzyme in ethyl acetate were quite different from those in aqueous buffer with the free enzyme, but similar to those measured in the biphasic system or in ethyl acetate containing a small amount of water. Therefore, it was considered that as a first approximation the reaction in the water-immiscible organic solvent with the immobilized enzyme could be treated by the aqueous/organic biphasic reaction. Based on this consideration, the optimum reaction condition for the reaction with the immobilized enzyme in ethyl acetate for the synthesis of Z-AspPheOMe could be estimated.

Topics: Acetates; Dipeptides; Enzymes, Immobilized; Kinetics; Peptides; Solvents; Water

We studied kinetics and the equilibrium relationship for the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-Asp-PheOMe) from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) in an aqueous-organic biphasic system. This is a model reaction giving a condensation product with dissociating groups. The kinetics for the synthesis of Z-Asp-PheOMe in aqueous solution saturated with ethyl acetate was expressed by a rate equation for the rapid-equilibrium random bireactant mechanism, and the reverse hydrolysis reaction was zero-order with respect to Z-Asp-PheOMe concentration. The courses of synthesis of Z-Asp-PheOMe in the biphasic system were well explained, by the rate equations obtained for the aqueous solution and by the partition of substrate and condensation product between the both phases. The rate of synthesis in the biphasic system was much lower than in aqueous solution due to the unfavorable partition of PheOMe in the aqueous phase. The equation for the equilibrium yield of Z-Asp-PheOMe in the biphasic system was derived assuming that only the non-ionized forms of the substrate and condensation product exist in the organic phase. It was found theoretically and experimentally that the yield of Z-Asp-PheOMe is maximum at the aqueous-phase pH of around 5, lower than for synthesis in aqueous solution. The effect of the organic solvent on the rate and equilibrium for the synthesis of Z-Asp-PheOMe could be explained by the variation in the partition coefficient. The effect of the partitioning of substrate on the aqueous-phase pH change was also shown.

Topics: Acetates; Buffers; Catalysis; Dipeptides; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Mathematics; Models, Chemical; Solubility; Solvents; Thermolysin