Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16.
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.
Jo
You-Young
YY
Department of Agricultural Chemistry, Chonnam National University, Gwangju, Korea.
Jo
Kyu-Jong
KJ
Jin
Yu-Lan
YL
Kim
Kil-Yong
KY
Shim
Jae-Han
JH
Kim
Yong-Woong
YW
Park
Ro-Dong
RD
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
D009844:Oligosaccharides; D011134:Polysaccharides; D012492:Salts; D012997:Solvents; D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase