Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli.
An aminotransferase capable of transaminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid to L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG), was purified to apparent homogeneity from Escherichia coli K-12. The enzyme catalyzes the transamination of L-phosphinothricin and various analogs with 2-ketoglutarate as the amino group acceptor. The transaminase has a molecular mass of 43 kilodaltons by sodium dodecyl sulfate-gel analysis and an isoelectric point of 4.35. The enzyme was most active in the high-pH region, with a maximum at pH 8.0 to 9.5, and had a temperature optimum of 55 degrees C. Heat stability was observed up to 70 degrees C. Substrate specificity studies suggested that the enzyme is identical with the 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19). The first 30 amino acids of the N terminus of the protein were determined by gas phase sequencing. The transaminase was immobilized by coupling to the epoxy-activated carrier VA-Biosynth (Riedel de Haen) and used in a column reactor for the continuous production of L-phosphinothricin. The enzyme reactor was operated for 7 weeks with only a slight loss of catalytic capacity. Production rates of more than 50 g of L-phosphinothricin per liter of column per h were obtained.
Schulz
A
A
Hoechst AG, Frankfurt, Federal Republic of Germany.
Taggeselle
P
P
Tripier
D
D
Bartsch
K
K
D000613:Aminobutyrates / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007656:Ketoglutaric Acids / Q000378:metabolism; D015394:Molecular Structure; D013379:Substrate Specificity; D013696:Temperature; D000637:Transaminases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000613:Aminobutyrates; D007656:Ketoglutaric Acids; C003121:phosphinothricin; D005680:gamma-Aminobutyric Acid; D000637:Transaminases