Jo You-Young, Jo Kyu-Jong, Jin Yu-Lan, Kim Kil-Yong, Shim Jae-Han, Kim Yong-Woong, Park Ro-Dong
Department of Agricultural Chemistry, Chonnam National University, Gwangju, Korea.
Biosci Biotechnol Biochem. 2003 Sep;67(9):1875-82. doi: 10.1271/bbb.67.1875.
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.
从芽孢杆菌属P16的培养上清液中纯化并鉴定了一种细胞外45 kDa的内切壳聚糖酶。纯化后的酶最适pH为5.5，最适温度为60℃，在pH 4.5 - 10.0之间以及50℃以下稳定。以脱乙酰度为20.2%的壳聚糖为底物时，测得的Km和Vmax分别为0.52 mg/ml和7.71×10⁻⁶ mol/sec/mg蛋白质。该酶不降解几丁质、纤维素或淀粉。壳聚糖酶能部分消化N - 乙酰化壳聚糖，对15 - 30%乙酰化壳聚糖活性最高，对0 - 15%乙酰化壳聚糖活性较低。壳聚糖酶在反应的早期阶段就能迅速降低壳聚糖溶液的粘度，表明其对聚合壳聚糖链的切割方式为内切型。壳聚糖酶以内切方式水解(GlcN)7，产生(GlcN)(2 - 5)的混合物。时间进程研究表明，底物降解速率从(GlcN)7到(GlcN)6再到(GlcN)5逐渐降低，表观一级速率常数k1值分别为4.98×10⁻⁴、2.3×10⁻⁴和9.3×10⁻⁶ sec⁻¹。该酶几乎不催化小于五聚体的壳寡糖的降解。
