Purification and some properties of extracellular chitinases from Streptomyces sp. S-84.

Abstract

The extracellular chitinases produced by Streptomyces sp. S-84 were purified and characterized. Two chitinases, A and B, were separated from the culture filtrate by anion-exchange chromatography and gel filtration. Both of the enzymes catalyzed the degradation of the 4-methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine disaccharide and trisaccharide. Chitinase A hydrolyzed 4-MU-disaccharide more rapidly than 4-MU-trisaccharide. Chitinase B had the reverse effects. Neither enzyme cleaved 4-MU-monosaccharide. Chitinase B, which accounted for more than 99% of the total activity in the culture, was characterized in greater detail. The molecular weight estimated by SDS-PAGE was 44, 000 and the Pi was 4.8. The optimum activity occurred between pH 6.3 and 6.8. The respective Km and Vmax values of chitinase B for 4-MU-disaccharide were 14μM and 42μmol/min/mg protein, and for 4-MU-trisaccharide they were 2.7μM and 66μmol/min/mg protein. Pb2+ and p-chloromercuribenzoic acid inhibited the activity. The major product of hydrolysis of colloidal chitin by chitinase B was disaccharide with trace amounts of mono- and trisaccharide. Chitinase A had somewhat different properties. Its molecular weight, estimated by SDS-PAGE, and Pi were 41, 000 and 8.3, respectively. The optimum activity was between pH 3.4 and 4.2. The respective Km and Vmax values of chitinase A for 4-MU-disaccharide were 49μM and 0.33μmol/min/mg protein, and for 4-MU-trisaccharide they were 14μM and 0.16μmol/min/mg protein. Chitinase A produced a large amount of disaccharide and a small amount of mono-, tri- and tetrasaccharide from colloidal chitin, but the tetrasaccharide disappeared after longer incubation. The patterns of hydrolysis of p-nitrophenylchitooligosaccharide (monosaccharide to tetrasaccharide) were different in the two chitinases. Monospecific antiserum raised against chitinase B inhibited the activity of chitinase B exclusively. These results showed that the strain S-84 produced 2 distinct chitinases.