Penicillin G acylase from Achromobacter sp. CCM 4824

Abstract

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.

Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases

“…Enzyme purification and hydrolytic activity assay. aPGA was purified as described by Škrob et al 81,82 and ecPGA according to Kutzbach and Rauenbusch. 83 The activity of 1 unit (U) was defined as the amount of aPGA or ecPGA cleaving 1 μmol of corresponding HSL per min in 0.05 M sodium phosphate buffer at pH 8.0 and 7.0, respectively, containing 2 % (w/v) HSL at 35 °C.…”

Strategies to Improve the Biosynthesis of β-Lactam Antibiotics by Penicillin G Acylase: Progress and Prospects

“…Comparatively, the half-lives of EcPGA and AfPGA under the same conditions are 5 and 15 min, respectively ( Skrob et al, 2003 ). Moreover, in the synthesis of ampicillin and amoxicillin, AxPGA presents significantly higher S/H ratios than EcPGA ( Becka et al, 2014 ). In our previous studies, we evaluated the activities of three different PGAs in terms of ampicillin and amoxicillin synthesis.…”

“…Based on the reaction types, the biosynthesis procedure is further divided into thermodynamic control or kinetic control ( Cobos-Puc et al, 2020 ). In the first case, a non-activated acyl donor (free carboxylic acid) is used, and the synthesis is mainly governed by the reaction equilibrium constant, whereas the enzyme only accelerates the reaction rate by lowering the activation energy, which does not affect the product yield ( Becka et al, 2014 ). Although this approach has been successfully applied in the synthesis of some antibiotics, its efficiency is extremely low.…”

Draft Genome Sequence of Pantoea agglomerans JM1, a Strain Isolated from Soil Polluted by Industrial Production of Beta-Lactam Antibiotics That Exhibits Valacyclovir-Like Hydrolase Activity

“…The strain Pantoea agglomerans JM1 (Czech Collection of Microorganisms, CCM 8766) was isolated by extensive screening of microorganisms from a soil sample polluted by a factory producing beta-lactam antibiotics (Slovenská L’upča). The aim of the study was to identify new enzymes useful for the biotransformation of beta-lactams, with activities analogous to alpha-amino acid ester hydrolases (AEHs) ( 13 ) or penicillin G acylases (PGAs) ( 14 , 15 ). The enzymes were detected by the hydrolysis of a chromogenic substrate, 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB) ( 16 , 17 ).…”