Penicillin G acylase from Achromobacter sp. CCM 4824 : an efficient biocatalyst for syntheses of beta-lactam antibiotics under conditions employed in large-scale processes.

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of ß-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme.

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 degrees C in amount corresponding to 8% of the cell soluble protein (0.6 Umg(-1)). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 degrees C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.

Cross-linked cell aggregates of Trigonopsis variabilis: D-amino acid oxidase catalyst for oxidation of cephalosporin C.

Trigonopsis variabilis CBS 4095 was treated with alkali (pH 11, 30 min), heated (65 degrees C, 60 s) and immobilized. Glutaraldehyde, polyethyleneimine and a cross-linking reagent formed by reaction of polyethyleneimine with glutaraldehyde were used for stabilization of D-amino acid oxidase in the cells, as well as for aggregation and binding of the cells. A specific activity of 82-98 U of D-amino acid oxidase per g dry mass was produced with a yield of about 20%. The half-life time of 142 repeated conversion cycles corresponds to a productivity of 130 kg cephalosporin C oxidized per kg catalyst dry mass.