Enhancement of Trigonopsis variabilis D-Amino Acid Oxidase Overproduction in Fed-batch Cultivation of Escherichia coli

D-Amino acid oxidase is a key enzyme in the production of semisynthetic cephalosporins. The Trigonopsis variabilis D-amino acid oxidase with 12amino-acid peptide at N-terminus was expressed in Escherichia coli BL21(DE3), in a series of short fed-batch cultivations. The strategy of gradual induction and gradual feeding with D-methionine during expression resulted in active enzyme production twice as high as that in which the inducer and methionine were added all at once at the expression start. In optimal conditions during 14 h of aerobic cultivation, 350 mg L of active enzyme was produced.