A NOVEL ARYLESTERASE ACTIVE TOWARD 7-AMINOCEPHALOSPORANIC ACID FROM AGROBACTERIUM-RADIOBACTER IFO-12607 - PURIFICATION AND CHARACTERIZATION

Through the screening of yeasts and bacteria, some strains of Agrobact erium radiobacter have been found to efficiently concert 7-aminocephal osporanic acid (7-ACA) to deacetyl 7-ACA. The enzyme responsible for t he conversion was constitutively formed by the bacterium. The 7-ACA de acetylating enzyme was purified from a cell-free extract of A. radioba cter IFO 12607 to homogeneity. The purified enzyme was a tetramer, com posed of identical subunits with a molecular mass of 26 kDa, and showe d activity toward some derivatives of 7-ACA, aryl acetates, monoacetin and triacetin, but was not active towards alkyl acetates and cephalos porin C. The K-m and k(cat) values of the enzyme for 7-ACA were 8.24 m M and 81 s(-1), respectively. Serine inhibitors and sulfhydryl reagent s were both found to inhibit the activity. An amino acid sequence, GDS LT, which is the active center motif of an arylesterase, was identifie d in the amino terminal region. These results indicate that the enzyme belongs to the family of arylesterase (EC 3.1.1.2), and is a new 7-AC A-deacetylating enzyme.