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嗜麦芽窄食单胞菌产生的几丁质酶的特性

DOI: 10.11830/ISSN.1000-5013.2008.02.0245

Keywords: 几丁质酶, 嗜麦芽窄食单胞菌, 发酵培养, 分子结构, 生物信息学

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Abstract:

从土壤中筛选出一株产几丁质酶的革兰氏阴性细菌,通过16S rDNA法对酶活力最高的菌株C进行鉴定,确定为嗜麦芽窄食单胞菌(S.maltophilia)菌株.通过单因素优化法和均匀设计法确定S.maltophilia产几丁质酶的最佳条件:在pH值为7.0的基础培养基中添加质量分数为1.0%的酵母膏、质量分数为0.5%的胶体几丁质,于180 r.min-1,30℃的摇床中培养60 h.对硫酸铵沉淀获得的粗酶进行酶学性质研究,结果表明,该几丁质酶的最适反应温度为50℃,最适反应pH值为7.2,在55℃以上的温度条件下容易失活.通过聚丙烯酰胺凝胶电泳分析,确定S.maltophilia几丁质酶的相对分子质量为6.8×103; 以筛选的S.malto-philia菌株的总DNA为模板,利用聚合酶链式反应(PCR)扩增出几丁质酶DNA测序; 应用生物信息学手段推导S.maltophilia几丁质酶为分泌型蛋白酶,预测其等电点pI值为5.42,相对分子质量为6.8×103(与实验纯化的酶蛋白电泳结果一致).预测该酶氨基酸序列具有信号肽区(氨基酸残基1~41)、Ⅲ型几丁质结合区(残基47~92)、多囊肾病域(107~194)、类纤维连接蛋白Ⅲ型区(Fn3,201~278)和18家族糖基水解域等5个结构功能区,5个区域被富含Ala,Gly,Pro,Ser和Thr的短序列连接起来; 在第400~500个氨基酸残基间有一个螺旋结构.

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