Stereospecific production of the herbicide phosphinothricin (Glufosinate): Purification of aspartate transaminase from Bacillus stearothermophilus, cloning of the corresponding gene, aspC, and application in a coupled transaminase process

Abstract

We have isolated and characterized an aspartate transaminase (glutamate:oxalacetate transaminase, EC 2.6.1.1) from the thermophilic microorganism Bacillus stearothermophilus. The purified enzyme has a molecular mass of 40.5 kDa by sodium dodecyl sulfate gel analysis, a temperature optimum of 95°C, and a pH optimum of 8.0. The corresponding gene, aspC, was cloned and overexpressed in Escherichia coli. The recombinant glutamate:oxalacetate transaminase protein was used in immobilized form together with 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19) from E. coli for the production of L-phosphinothricin [L-homoalanin-4-yl- (methyl)phosphinic acid], the active ingredient of the herbicide Basra (AgrEvo GmbH), from its nonchiral 2-keto acid precursor 2-oxo-4- [(hydroxy)(methyl)phosphinoyl]butyric acid {PPO). In this new coupled process conversion rates of ca. 85% were obtained with substrate solutions containing 10% PPO by using only slight excesses of the amino donors glutamate and aspartate. The contamination of the reaction broth with amino acid by-products was <3%.