PURIFICATION AND SOME PROPERTIES OF EXTRACELLULAR CHITINASES FROM STREPTOMYCES SP S-84

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 K(m) and V(max) values of chitinase B for 4-MU-disaccharide were 14-mu-M and 42-mu-mol/min/mg protein, and for 4-MU-trisaccharide they were 2.7-mu-M and 66-mu-mol/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 K(m) and V(max) values of chitinase A for 4-MU-disaccharide were 49-mu-M and 0.33-mu-mol/min/mg protein, and for 4-MU-trisaccharide they were 14-mu-M and 0.16-mu-mol/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.