印尼热泉菌产热稳定褐藻胶裂解酶的酶学性质研究  

BIOCHEMICAL CHARACTERISTICS OF THERMOSTABLE ALGINATE LYASE OF A STRAIN FROM AN INDONESIAN HOT SPRING

作　　者：高杨 李江[2] 宋益民[1] GAO Yang;LI Jiang;SONG Yi-Min(Qingdao University of Science and Technology, Qingdao 266042, China;The First Institute of Oceanography, SOA, Qingdao 266061, China)

机构地区：[1]青岛科技大学,青岛266042 [2]国家海洋局第一海洋研究所,青岛266061

出　　处：《海洋与湖沼》2018年第3期577-585,共9页Oceanologia Et Limnologia Sinica

基　　金：海洋公益性行业科研专项经费;201505026-4号;国家海洋局海洋生物活性物质与现代分析技术重点实验室开放基金;MBSMAT-2015-06号

摘　　要：以印尼热泉菌为材料,采用以褐藻酸钠为唯一碳源的培养基筛选产褐藻胶裂解酶的菌株,通过16S rDNA序列对产酶菌株进行种属鉴定,并通过硫酸铵沉淀、离子交换层析和凝胶过滤层析从高产酶菌株Aly-B5发酵液上清中纯化获得褐藻胶裂解酶,并对该酶的酶学性质进行了研究。结果表明:高产酶菌株经鉴定为Bacillus属;聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)显示纯化的褐藻胶裂解酶Aly-B5分子量为45kDa,该酶最适作用温度为65°C,75°C时的半衰期为110min,最适pH为7.0;Zn^(2+)、Mg^(2+)、Fe^(2+)对酶活具有明显的抑制作用,但该酶对乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)并不敏感;同时,底物特异性实验表明Aly-B5是一种偏好降解聚古罗糖醛酸(poly G)的双功能裂解酶,降解产物主要是二糖。该酶优良的耐热性使其在海藻高值化利用领域具有潜在的应用前景。A hot spring strain producing alginate lyase, Aly-B5, was screened on a plate using alginate as the sole carbon source and was identified by 16S rDNA sequencing. The alginate lyase Aly-B5 was purified by ammonium sulfate precipitation, and DEAE（diethylaminoethyl）-sepharose Fast Flow and Sephacryl G-100 column chromatography. The biochemical characteristics of Aly-B5 was determined by dinitrosalicylic acid method, including the optimal temperature, pH, thermal and pH stability, metal ions, substrate specificity, and enzymolysis product. The results show that the high-yield alginate lyase strain from Indonesian hot spring was Bacillus sp. The molecular weight of purified alginate lyase Aly-B5 was about 45 kDa by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）. The optimal catalytic activity of Aly-B5 occurred at 65°C and pH 7.0 and it can maintain half of initial activity at 75°C for more than 110 min. The alginate lyase was inhibited by Zn^2＋, Mg^2＋, Fe^2＋, and it was insensitive to EDTA（ethylenediaminetetraacetic acid）. Aly-B5 showed activities toward both α-L-guluronic acid and β-D-mannuronic acid, indicating that it is a bifunctional alginate lyase that prefers α-L-guluronic acid, and the product is mainly disaccharide. The high tolerance toward high temperature implied that this enzyme might be an interesting candidate for seaweeds industrial processes.

关 键 词：印尼热泉菌 褐藻胶裂解酶 酶学性质 热稳定性 

分 类 号：TS201.3[轻工技术与工程—食品科学]