Purification of 7α-hydroxysteroid dehydrogenase from Escherichia coli strain 080

Abstract

Purification studies of 7α-hydroxysteroid dehydrogenase (7α-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 °C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54 000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera. Key words: 7α-hydroxysteroid dehydrogenase, Escherichia coli.