A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304.
KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-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 beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-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 alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-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 alpha-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-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.
Yano
Shigekazu
S
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Japan.
Rattanakit
Nopakarn
N
Wakayama
Mamoru
M
Tachiki
Takashi
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000502:physiology; D001487:Basidiomycota / Q000166:cytology; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000302:isolation & purification / Q000502:physiology*; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000502:physiology; D011523:Protoplasts / Q000166:cytology*; D012567:Schizophyllum / Q000166:cytology*
D001426:Bacterial Proteins; D002686:Chitin; D006026:Glycoside Hydrolases; D002688:Chitinases