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.
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 degrees 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 Basta (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%.
Bartsch
K
K
Hoechst Schering AgrEvo GmbH, Frankfurt, Germany.
Schneider
R
R
Schulz
A
A
D000612:4-Aminobutyrate Transaminase; D000586:Amination; D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006540:Herbicides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000613:Aminobutyrates; D002087:Butyrates; D006540:Herbicides; D011993:Recombinant Fusion Proteins; C003121:phosphinothricin; D001219:Aspartate Aminotransferases; D000612:4-Aminobutyrate Transaminase