title: Purification and characterization of an esterase involved in poly(vinyl alcohol) degradation by Pseudomonas vesicularis PD.
abstract: An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45 degrees C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K(m) and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 microM, and 6.52 and 12.6 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.
authors.affiliation: Department of Biochemistry, Osaka Municipal Technical Research Institute, Japan. sakai@omtri.city.osaka.jp
authors.forename: K
authors.initials: K
authors.lastname: Sakai
authors.forename: M
authors.initials: M
authors.lastname: Fukuba
authors.forename: Y
authors.initials: Y
authors.lastname: Hasui
authors.forename: K
authors.initials: K
authors.lastname: Moriyoshi
authors.forename: T
authors.initials: T
authors.lastname: Ohmoto
authors.forename: T
authors.initials: T
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Ohe
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D011142:Polyvinyl Alcohol / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D011142:Polyvinyl Alcohol; D002265:Carboxylic Ester Hydrolases; C116227:poly(vinyl alcohol-co-vinyl acetate) esterase