Characterization and Kinetics of 45 kDa Chitosanase from Bacillus sp. P16(Biochemistry & Molecular Biology)

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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℃, and was stable between pH 4.5-10.0 and under 50℃. The K_m and V_<max> were measured with a chitosan of a D. A. of 20.2% as 0.52mg/ml and 7.71×lO^<-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, k_1 values, of 4.98 × lO^<-4>, 2.3× lO^<-4>, and 9.3 × lO^<-6> sec^<-1>, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.
社団法人日本農芸化学会の論文
2003-09-23