Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB

An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45 degrees C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The K-m and V-max for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 mu M as acetyl content in the polymer) and 36.0 mu M, and 66.8 and 39.1 mu mol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.