A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

Abstract

KA-prep, a culture nitrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. α-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and β-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and β-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the α-1,3-glucanase. The β-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of α-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with α-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N′-diacetylchitobiose and N,N′,N″-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-β-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (ALATPTLNV SASSGM) had no sequential identity to those of known chitinases.