| 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。 |
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| BIOCHEMICAL CHARACTERISTICS OF THERMOSTABLE ALGINATE LYASE OF A STRAIN FROM AN INDONESIAN HOT SPRING |
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GAO Yang1, LI Jiang2, SONG Yi-Min1
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1.Qingdao University of Science and Technology, Qingdao 266042, China;2.The First Institute of Oceanography, SOA, Qingdao 266061, China
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| 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 |
