基于宏基因组学壳聚糖酶挖掘研究  

Studies of chitosanase mining based on metagenomic technology

作　　者：费忠[1] 李恒[1] 龚劲松[1] 杨涛[1] 许正宏[1] 史劲松[1] 

机构地区：[1]江南大学药学院,无锡214122

出　　处：《应用与环境生物学报》2014年第4期597-601,共5页Chinese Journal of Applied and Environmental Biology

基　　金："十二五"国家科技支撑计划课题(2012BA D33B06);国家高技术研究发展计划项目(2012AA022204C);国家自然科学基金项目(21206055);江苏省自然科学基金项目(BK2012127)资助~~

摘　　要：壳聚糖酶(EC3.2.1.132)可有效地将壳聚糖降解为活性良好和应用前景广泛的低分子壳聚糖或壳寡糖,但目前存在专一性强的壳聚糖酶发酵水平低、种类单一、应用性能较差等问题.本研究从虾蟹堆积场淤泥采样,采用宏基因组文库方法,从其Fosmid文库中筛选出壳聚糖酶基因ChiE.测序表明,ChiE由1 362 bp组成,编码453个氨基酸,预测分子量(Mr)为42×103左右.将ChiE与pET-28a(+)载体连接,转化大肠杆菌Escherichia coli Rosetta-gami(DE3)构建了壳聚糖酶重组菌.采用离子交换柱(DEAE Sepharose FF)和凝胶色谱柱(SuperdexTM 75)纯化目的蛋白.酶学性质结果显示,该酶最适作用温度70℃,最适pH 6.0,Li+、Sr2+、K+等离子对其具有一定激活作用,Fe3+、Pb2+、Fe2+等离子则具有不同程度的抑制.在最适作用条件下,该酶比酶活为2 158 U/mg.本研究表明,重组菌E.coli Rosetta-gami(DE3)/ChiE具有培养简便、酶发酵产量大等优点,结果可为利用基因工程菌生产壳聚糖酶奠定基础.Chitosanase（EC3.2.1.132） can effectively degrade chitosan into low molecular chitosan or oligosaccharide, which plays an important role in a wide range area due to its biological activity. However, the application performance of chitosanase is limited by several problems including low fermentation level, lack of variety, and poor catalytic properties. Using chitosanase segregation agar containing chitosan as the medium components, we obtained chitosanase gene ChiE from shrimp activated sludge microbial metagenomic Fosmid library. The chitosanase gene ChiE, which was inserted into the expression vector pET-28a（＋） and transformed into Escherichia coli Rosetta-gami（DE3）, was shown by SDS-PAGE to consist of 1 362 bp, encode 453 amino acid residues, and have a molecular mass of 42 × 10^3. The recombinant chitosanase purified by DEAE Sepharose FF and SuperdexTM 75 was most active at 70 ℃ and pH 6.0. The activity of chitosanase was significantly enhanced by Li＋, Sr2＋, K＋ and obviously inhibited by Fe3＋, Pb2＋, and Fe2＋. The highest chitosanase activity was 2 158 U/mg with an optimal pH and temperature of 6.0 and 70 ℃, respectively. This study demonstrated the advantages of simple cultivation and high fermentation throughput of E. coli Rosetta-gami（DE3）/ChiE, which should be helpful in producing chitosanase by genetically engineered strain.

关 键 词：宏基因组文库 FOSMID 壳聚糖酶 壳寡糖 大肠杆菌 

分 类 号：Q939.97[生物学—微生物学] Q78