title: Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16.
abstract: An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60 degrees C, and was stable between pH 4.5-10.0 and under 50 degrees C. The Km and Vmax were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71 x 10(-6) mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)7 in an endo-splitting manner producing a mixture of (GlcN)(2-5). Time course studies showed a decrease in the rate of substrate degradation from (GlcN)7 to (GlcN)6 to (GlcN)5, as indicated by the apparent first order rate constants, k1 values, of 4.98 x 10(-4), 2.3 x 10(-4), and 9.3 x 10(-6) sec(-1), respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.
authors.affiliation: Department of Agricultural Chemistry, Chonnam National University, Gwangju, Korea.
authors.forename: You-Young
authors.initials: YY
authors.lastname: Jo
authors.forename: Kyu-Jong
authors.initials: KJ
authors.lastname: Jo
authors.forename: Yu-Lan
authors.initials: YL
authors.lastname: Jin
authors.forename: Kil-Yong
authors.initials: KY
authors.lastname: Kim
authors.forename: Jae-Han
authors.initials: JH
authors.lastname: Shim
authors.forename: Yong-Woong
authors.initials: YW
authors.lastname: Kim
authors.forename: Ro-Dong
authors.initials: RD
authors.lastname: Park
mesh_terms: D000107:Acetylation; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D048271:Chitosan; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D012492:Salts / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D014783:Viscosity
chemical_list: D009844:Oligosaccharides; D011134:Polysaccharides; D012492:Salts; D012997:Solvents; D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase