Continuous Production of N-(Benzyloxycarbonyl)-L-Glycyl-L-Phenylalanine Methyl Ester Utilizing Extractive Reaction in Aqueous/Organic Biphasic Medium
ABSTRACT N-(Benzyloxycarbonyl)-L-glycyl-L-phenylalanine methyl ester was continuously synthesized, enzymatically, utilizing an extractive reaction in an aqueous/organic biphasic system. The extremely high yield, ca.100%, was obtained continuously in a water/butyl acetate biphasic medium.
New method for analysis of enzymatic reactions: Thermolysin-catalyzed synthesis of aspartame precursor
Aspartame is an artificial sweetener which is 200 times sweeter than sucrose. A precursor of aspartame, N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) was synthesized and hydrolyzed with thermolysin in an aqueous monophasic system. Graphical plotting methods and/or nonlinear regression have been used to analyze enzymatic reactions because of the nonlinearity of enzymatic rate equations. There are, however, problems: the former require accurate measurement of the initial reaction rate and the latter gives no analytical information. The authors proposed a method to solve the nonlinear enzymatic rate equations with respect to the concentration of a product with the Taylor expansion, especially for the reversible Theorell-Chance mechanism (Murakami, Y., Hirata, M., and Hirata, A., J. Ferment. Bioeng., 82, 246–252, 1996). This solution, however, is complex for practical use. In this paper, a simpler solution to explain the thermolysin-catalyzed synthesis of Z-APM is obtained. New methods to determine the kinetic parameters, especially for the reversible Theorell-Chance mechanism, based on the approximated solution are proposed. The results calculated using the obtained kinetic parameters can satisfactorily explain the experimental results, which demonstrates the effectiveness of the proposed analysis. We must know the relation between the concentrations of substrates and a product, and accurately evaluate of the result obtained using Taylor expansion when we determine the kinetic parameters using our newly developed methods. The methods to determine the kinetic parameters of the reversible Theorell-Chance mechanism described in this paper is, however, expected to be applied to general enzymatic reactions.
Atypical kinetics of immobilized firefly luciferase
The kinetic properties of collagen‐bound firefly luciferase have been investigated. Under definite hydrodynamic conditions with low agitation in the reaction medium, the observed behavior is modified compared to the enzyme free in solution: reducing the stirring rate decreases the observed enzymatic activity. But diffusional resistances alone cannot account for these atypical kinetics though mass transfer may certainly play an important role during the transient state of the bioluminescent reaction. After immobilization, the time necessary to reach the steady state increased from 300 ms to 3 min and the two substrates, luciferin and ATP, behave differently with respect to the enzyme: The nature of the saturating substrate first in contact with the bound enzyme is not indifferent suggesting that immobilization can reveal behaviors or mechanisms which are not visualized with the free enzyme.