An entire structural gene coding for ${Delta}^5$-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni was overexpressed in E. coli. The resultant overexpressed enzyme was purified to homogeniety by fractional precipitation using ethanol and a steroid affinity column which specifically binds to KSI. Purified KSI has the same size as KSI isolated from Pseudomonas testosteroni based on migration in SDS-PAGE. Aspartic acid 38 at the putative active-site, which is supposed to abstract a proton during catalysis, was changed to phenylalanine by an oligonucleotide directed mutagenesis. The resulting mutant ksi gene (D38F) was overexpressed in E. coli and the D38F KSI was purifed to homogeniety. D38F KSI was specifically bound to the deoxycholate affinity column and completely lost enzyme activity. This suggests that the aspartic acid plays an essential role as a basic residue abstracting the $4{eta}$-proton of the steroid substrate in the catalytic mechanism of the isomerization reaction.