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印尼热泉菌产热稳定褐藻胶裂解酶的酶学性质研究
高杨1, 李江2, 宋益民1
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
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℃,75℃时的半衰期为110min,最适pH为7.0;Zn2+、Mg2+、Fe2+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(polyG)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。
关键词:  印尼热泉菌  褐藻胶裂解酶  酶学性质  热稳定性
DOI:10.11693/hyhz20170800213
分类号:TS201.3
基金项目: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。
BIOCHEMICAL CHARACTERISTICS OF THERMOSTABLE ALGINATE LYASE OF A STRAIN FROM AN INDONESIAN HOT SPRING
GAO Yang1, LI Jiang2, SONG Yi-Min1
1.Qingdao University of Science and Technology, Qingdao 266042, China;2.The First Institute of Oceanography, SOA, Qingdao 266061, China
Abstract:
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 45kDa by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal catalytic activity of Aly-B5 occurred at 65℃ and pH 7.0 and it can maintain half of initial activity at 75℃ for more than 110 min. The alginate lyase was inhibited by Zn2+, Mg2+, Fe2+, 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.
Key words:  hot spring bacterium  alginate lyase  biochemical characteristics  thermostability
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