title: Purification and characterization of inducible cephalexin synthesizing enzyme in Gluconobacter oxydans.
abstract: Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS-PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS-PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 degrees C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free alpha amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.
authors.affiliation: Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Chia-Yang
authors.initials: CY
authors.lastname: Shiau
authors.forename: Shun-Chung
authors.initials: SC
authors.lastname: Pai
authors.forename: Wen-Po
authors.initials: WP
authors.lastname: Lin
authors.forename: Dar-Der
authors.initials: DD
authors.lastname: Ji
authors.forename: Yu-Tien
authors.initials: YT
authors.lastname: Liu
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis* / Q000737:chemistry; D002506:Cephalexin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020600:Gluconobacter oxydans / Q000201:enzymology*; D007525:Isoelectric Focusing
chemical_list: D000217:Acyltransferases; C019862:cephalosporin synthesizing enzyme; D002506:Cephalexin