Purification and some properties of extracellular chitinases from Streptomyces sp. S-84.
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The extracellular chitinases produced by <i>Streptomyces</i> 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 <i>N</i>-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 <i>Pi</i> was 4.8. The optimum activity occurred between pH 6.3 and 6.8. The respective <i>K</i><sub>m</sub> and <i>V</i><sub>max</sub> 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. Pb<sup>2+</sup> and <i>p</i>-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 <i>Pi</i> were 41, 000 and 8.3, respectively. The optimum activity was between pH 3.4 and 4.2. The respective <i>K</i><sub>m</sub> and <i>V</i><sub>max</sub> 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 <i>p</i>-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.
- The Journal of General and Applied Microbiology
The Journal of General and Applied Microbiology 36(6), 377-392, 1990
Applied Microbiology, Molecular and Cellular Biosciences Research Foundation