节杆菌β-1,3葡聚糖内切酶的分离纯化与特性研究

Characterization of an endo-1,3-β-glucanase from Arthrobacter sp.

通过硫酸铵沉淀、阴离子交换层析和柱层析从节杆菌（Arthrobacter sp．）粗酶制剂分离纯化得到β-1，3葡聚糖内切酶。经SDS凝胶电泳和柱层析测得纯化酶为单体酶，分子量为32．5kDa；纯化酶N末端的10个氨基酸序列为APGDLLWSDE，在pH5～8之间稳定，最适pH6．5，最适温度55℃，但50℃以上失活很快。纯化酶对昆布4，5，6，7糖及昆布多糖底物的米氏常数分别为0．12，0．11，0．067，0．066mM和0．16mg／mL，也可水解热凝胶和地衣多糖，葡糖苷内切酶H（Streptomyces griseus）不能分解它，证明该酶不含糖基。该酶属于糖基水解酶16族系。

An endo-1,3-β-glucanase fromArthrobacter sp. was purified by ammonium sulfate precipitation, anion-exchange and gel-filtration chromatographies. SDS/PAGE and gel-filtration chromatography suggested a monomer enzyme with molecular mass of 32, 5 kDa, N-terminal sequence for 10 residues was Apgdllwsde. Stable and optimum pHs were 5 to 8 and 6.5, respectively, Optimum temperature was 55 ℃ ; however, above 50 ℃ its activity was lost rapidly. The Michaelis constant Km for laminaritetraose, laminaripentaose, laminarihexaose, laminariheptaose, and laminarin were 0. 12, 0. 11,0.067, 0. 066 mM, and 0, 16 mg/mL, respectively. Curdlan and lichenan were also hydrolyzed, The purified enzyme did not give product by digestion with endoglycosidase H （ Streptomyces griseus）, indicating that the carbohydrate moiety was little consisted in the enzyme protein, It was suggested that the enzyme belong to the family 16 of glucosyl hydrolase from the properties as above mentioned.