| 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°C, and was stable between pH 4.5-10.0 and under 50°C. The K m and V max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10?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, k 1 values, of 4.98×10?4, 2.3×10?4, and 9.3×10?6 sec?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer. |
