Bis-phenacetyl and phenoxyacetyl groups as substrates for penG and penV amidases

o-, m-, p-Bis-phenylacetic and -bis-phenoxyacetic acid esters with solketal are prepared and submitted to enzymatic hydrolysis with penicillin V (PVA) and G (PGA) amidases. While the para-isomers are recovered unchanged, ortho- and meta-bis-esters are completely hydrolysed. PVA shows a reversed substrate specificity, hydrolysing phenylacetates faster than its natural substrate. The use of the bis-acids as alcohol-protecting groups is proposed.     

Controlling the hydration of covalently immobilised penicillin G amidase in low-water medium: properties and use of Celite R-640

The study describes how Celite R-640 adsorbs liquid water in toluene and vapour water from a gas phase. In toluene, Celite R-640 is able to maintain the water activity (a_w) constant within broad ranges of water concentrations. The a_w values are strongly related to the original hydration of the Celite batches, but prolonged drying confers comparable and reproducible properties to the different batches. The use of Celite R-640 in controlling the hydration and activity of covalently immobilised PGA in toluene is reported.     

Canavanine derivatives useful in peptide synthesis

Summary The objective of this work is to investigate the possibilities for introducing the currently used N-, N_G- and C-protective groups into the canavanine molecule and the preparation of canavanines selectively blocked at the guanidino function. These novel compounds will find application in the synthesis of canavanine derivatives expected to be amino acid antimetabolites and of canavanine modified biologically active peptides.     

Experimental design for optimal parameter estimation of an enzyme kinetic process based on the analysis of the Fisher information matrix

The investigation of enzyme kinetics is increasingly important, especially for finding active substances and understanding intracellular behaviors. Therefore, the determination of an enzyme's kinetic parameters is crucial. For this a systematic experimental design procedure is necessary to avoid wasting time and resources. The parameter estimation error of a Michaelis-Menten enzyme kinetic process is analysed analytically to reduce the search area as well as numerically to specify the optimum for parameter estimation. From analytical analysis of the Fisher information matrix the fact is obtained, that an enzyme feed will not improve the estimation process, but substrate feeding is favorable with small volume flow. Unconstrained and constrained process conditions are considered. If substrate fed-batch process design is used instead of pure batch experiments the improvements of the Cramer-Rao lower bound of the variance of parameter estimation error reduces to 82% for mu(max) and to 60% for K(m) of the batch values in average.     

Kinetically controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase

Advantages of performing penicillin G amidase catalysed synthesis of ampicillin and cephalexin by enzymatic acyl transfer to the β-lactam antibiotic nuclei in a highly condensed system using mainly undissolved substrates, with no apparent aqueous liquid phase, were demonstrated. It was shown that synthesis can be performed in the absence of a liquid phase formed by water or an organic co-solvent. This highly condensed system is formed by a liquid phase given by one of the reactant, the phenylglycine methyl ester (PGM), that remains liquid in these operative conditions and the partially dissolved β-lactam nucleus. Operating in such highly condensed system, the water that causes the hydrolysis of PGM is limited to the water hydrating the support on which the enzyme is covalently immobilised. In this way the reaction system is maintained at a controlled degree of hydration.

In the present work the reaction system was modulated by eliminating the solvent (aqueous or aqueous/organic), reducing the amount of water to the minimum for the biocatalytic activity and using PGM as solvent and reagent at the same time. The synthesis was conducted with equimolar amounts of PGM and the β-lactam nucleus, with a reduced hydrolysis of the activated acyl donor. We have also studied a simple and efficient method for the workup of the reaction where the unreacted reagents can be recovered after selective filtration and precipitation.     

Identification of enzyme inhibitory mechanisms from steady-state kinetics

Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists.Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.     

Estimation of kinetic parameters for substrate and inhibitor in a reaction with an enzyme sample containing different types of inhibitor

The method of kinetic analysis is developed to obtain the maximum velocity (V_m), the Michaelis constant (K_m) and the parameters characterizing the inhibitors in an impure enzyme reaction, contaminated with one of four types of inhibitor (competitive, noncompetitive, uncompetitive and mixed-type). Although the reaction rate decreases with the increasing concentration of the enzyme sample containing an inhibitor, the double-reciprocal plot of the rate against the sample concentration becomes linear. The slopes of these linear plots at several different concentrations of substrate provide K_m and the specific enzyme activity, which is proportional to V_m, in the sample. These linear straight lines intersect in a point, of which the coordinates give the unique parameters for the inhibitor. To prove the validity of this kinetic method, the model experiments were carried out with acetylcholinesterase and its inhibitors, phenyltrimethylammonium and trimethylammonium. The present method was applied to the measurement of the specific activity of galactosylceramide galactosidase in the mouse cerebral homogenate. In addition, a kinetic method is indicated for the inhibition of an enzymatic reaction by a contaminant which binds the substrate to reduce the fraction available to the enzyme.     

Parameter estimation in a special reaction-diffusion system modelling man-environment diseases

An approximation scheme for a reaction-diffusion system with distributed feedback through the boundary is developed. It is used to estimate the strength of the feedback mechanisms from measurements of the states. The results are illustrated by numerical examples.