Immobilization of glutaryl-7-aminocephalosporanic acid acylase on silica gel and enhancement of its stability

Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase isan enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20°C, and immobilization time of 120 min. Unreacted aldehydegroups were quenched by reaction with a low-molecular-weight material such as l-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.     

Purification and stability of glutaryl-7-ACA acylase from pseudomonas sp.

The enzyme glutaryl-7-ACA acylase fromPseudomonas sp. NCIMB 40474, produced by a recombinantEscherichia coli host, was purified to homogeneity. The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HC1 concentration. The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.     

Monitoring of cephalosporin C during bioconversion

A simple method based on Schiff’s base formation betweenp-dimethylaminobenzaldehyde and cephalosporin C is developed for estimation of cephalosporin C. The calibration curve was linear up to 500 μg of cephalosporin C. The application of the method in monitoring bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid is described.     

Single-step conversion of cephalosporin-C to 7-Aminocephalosporanic acid by free and immobilized cells of <Emphasis Type="Italic">Pseudomonas diminuta</Emphasis>

7-Aminocephalosporanic acid (7-ACA), the starting material for the production of a number of clinically used semisynthetic cephalosporins, is produced by deacylation of cephalosporin-C. The production of 7-ACA was studied in various modes, at the optimal conditions using free and immobilized whole cells of Pseudomonas diminuta.     

Improvement in thermal stability and substrate binding of pig kidney d-amino acid oxidase by chemical modification

Chemical modification was evaluated to stabilize pig kidney d-amino acid oxidase (pkDAAO), which is required for analytical determination of d-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical modification at 30°C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of activity. pkDAAO was stable at less than 55°C at pH 6.0, while the conjugated enzyme was stable even at 70°C. In addition, the conjugated enzyme showed decreased K _m values for d-amino acids. Because of these outstanding charcteristics, this new material is expected to be available for use as a liquid assay reagent.     

Regioselective and stereospecific opening of an oxirane system mediated by trifluoroacetic acid and halide anions. A new direct approach to C3-vicinal halohydrins

Glycidol derivatives bearing ester, ether or silyl functionality upon treatment with trifluoroacetic acid (TFA) in the presence of a halide anion (e.g., Bu₄NX; X = Cl, Br or I) at room temperature undergo regioselective and stereospecific opening of the oxirane ring to produce the corresponding C3-vicinal haloalkanols in practically quantitative yields.