摘要
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180r·min^-1，30℃的摇床中培养60h．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6．8×10^3；以筛选的S．maltophilia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6．8×10^3（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．
A strain of Stenotrophomonas maltophilia which produced extacellular chitinase was isolated from chitin-rich soils of shrimp drying fields. The fermentation experiments showed that the suitable chitinase producing media were containing 1.5% colloid chitin and 1.0% yeast extract, and cultured for 60 hours （180 r· min^-1 ） in shake flask. The optimal pH and temperature were 7.0 and 30 ℃, respectively. The crude enzyme was obtained from the fermentation broth by ammonium sulfate precipitation process, the enzyme optimal temperature was 50 ℃ and pH was 7.2, respectively. The stability of chitinase decreased rapidly when the temperature get above 55℃. The chitinase was 6.8× 10^3 by SDSPAGE analysis. Some properties of this enzyme were deduced by bioinformatics method： this is a secretary protease; its molecular mass is 6.8 ×10^3 （consistent with the result by extraction chitinase electrophoresis）. The predicted pI is 5.42. The enzyme amino acid sequence has five domains： signal peptide （residues 1-41）, type Ⅲ chitin-binding domain （Cht-BD3）（residues 47-92）, polycystic kidney disease （PKD） domain （residues 107-194）, fibronectin type Ⅲ （Fn3） domain （residues 201-278）,and family 18 glycosyl hydrolases （Glyco-18） catalytic domain. Spanning each of the five domains are short sequences full of Gly,Ala,Pro,Ser,and Thr. There is a helix structure between 400th-500th residues.
出处
《华侨大学学报（自然科学版）》
CAS
北大核心
2008年第2期245-249,共5页
Journal of Huaqiao University(Natural Science)
基金
福建省自然科学基金资助项目(C04010011)
关键词
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
chitinase
Stenotrophomonasmaltophilia
fermentation culture
molecular structure
bioinformatics
