PURIFICATION AND CHARACTERIZATION OF AN ESTERASE INVOLVED IN POLY(VINYL ALCOHOL) DEGRADATION BY PSEUDOMONAS-VESICULARIS PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly (vinyl alcohol) was purified 400-fold to electrophoretic homogeneity f rom the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and ene rgy source. The purified enzyme was a homodimeric protein with a molec ular mass of 80 kDa and the isoelectric point was 6.8. The pH and temp erature optima of the enzyme were 8.0 and 45 degrees C. The enzyme cat alyzed the hydrolysis of side chains of poly(vinyl alcohol), short-cha in p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and w as slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acid s, of poly(vinyl alcohol). The K-m and V-max of poly(vinyl alcohol) (d egree of polymerization, 500; saponification degree, 86.5-89.0 mol%) a nd p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 mu M, and 6.52 and 12.6 mu mol/min/mg, respectively . The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride a nd diisopropyl fluorophosphate at a concentration of 5 mM, which indic ated that the enzyme was a serine esterase. The pathway for the metabo lism of poly(vinyl alcohol) is also discussed.