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A bacterial strain producing a beta-lactam antibiotic acylase, able to hydrolyze ampicillin to 6-aminopenicillanic acid more efficiently than penicillin G, was isolated from soil and characterized. The isolate was identified as Achromobacter sp. using the phenotypic characteristics, composition of cellular fatty acids and 16S rRNA gene sequence. The enzyme synthesis was fully induced by phenylacetic acid (PAA) at a concentration of 2 g l(-1). PAA at concentrations up to 12 g l(-1) had no negative effect on the specific activity of acylase and biomass production, but slowed down the specific growth rate. Benzoic or 4-hydroxyphenylacetic acids can also induce synthesis of the enzyme. The inducers were metabolized in all cases. Acylase activity in cell-free extracts was determined with various substrates; ampicillin, cephalexin and amoxicillin were hydrolyzed 1.5- and 2-times faster than penicillin G. A high stability of acylase activity was observed over a wide range of pH (5.0-8.5) and at temperatures above 55 degrees C.

A novel penicillin G acylase from the bacterial strain Achromobacter sp. CCM 4824 was characterized. The specific activity of purified enzyme was 27.6 U mg −1 protein (6-nitro-3-phenylacetylamidobenzoic acid, NIPAB as the substrate). The enzyme consists of two dissimilar subunits α and β with a molecular mass of 27.0 and 62.4 kDa, respectively. The isoelectric point was about pH 8.8. The N-terminal amino acid sequence of β subunit (SNMWIVGRDHAKDARSILLN) and internal amino acid sequence of α subunit (YGYGYAVAQDRLFQMEMAR) exhibited a significant similarity with penicillin G acylases. The K m values for penicillin G and NIPAB were 1.9±0.1 and 4.5±0.2 μM, respectively. The turnover rates k cat for penicillin G and NIPAB were 29±1 and 19±1 s −1 , respectively. The maximal hydrolytic activity of the enzyme was found at pH 7.5 and temperature of 60 °C. In contrast to the published substrate specificities of penicillin G acylases, the enzyme exhibited an almost two-fold hydrolytic activity with ampicillin, amoxicillin and cephalexin compared to penicillin G.

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.

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-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.

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.