Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity.
Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60 degrees C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the beta-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity.
Tanabe
Toshiaki
T
Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan.
Morinaga
Kazuko
K
Fukamizo
Tamo
T
Mitsutomi
Masaru
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000235:genetics; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D048271:Chitosan; D015698:Genomic Library; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013305:Streptomyces griseus / Q000201:enzymology*; D013379:Substrate Specificity
D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium