海洋Pseudomonas aeruginosa sp. DES-3的鉴定以及基因ap02对其产蛋白酶的影响研究

Identification of a Pseudomonas aeruginosa sp. DES-3 From Marine and the Effect of Protease-producing by a Gene of ap02

海洋是地球上最大的生态系统,其多样独特的生态环境造就了其有别于其他环境的微生物资源。本研究利用蛋白酶的活性筛选策略,从辽宁省营口市某海洋淤泥中分离得到一株产蛋白酶的优势菌株DES-3。通过微生物形态学特征、生理生化性质和16SrDNA基因鉴定以及系统发育进化关系的分析,将该菌株鉴定为铜绿假单胞菌属,命名为Pseudomonas aeruginosa sp.DES-3。其胞外蛋白酶的最适反应温度和最适pH分别为55℃和9.0。将来自于碱性污染土壤宏基因组文库的编码碱性蛋白酶基因ap02与广宿主表达载体pBBR1MCS-5连接,转化至野生型菌株P.aeruginosasp.DES-3细胞中,成功构建了基因工程菌株P.aeruginosasp.DES-3/pBBR1MCS-5-ap02。基因工程菌株的最适反应温度为60℃,较野生型菌株提高了5℃。本研究构建基因工程菌株的策略可为菌株的改造和相应工业应用提供一定的技术参考。

Ocean is the largest ecosystem on the earth,and the microbial resources in the marine environment are significantly different from the others natural eco-systems.In this study,aprotease-producing strain DES-3was obtained by using functional screening strategy from the marine sludge in Yingkou city,Liaoning Province of China.The DES-3strain was identified as the genus of Pseudomonas aeruginosa and named as Pseudomonas aeruginosasp.DES-3based on the results of microbial morphological characterization,physiological and biochemical properties,and 16 SrDNA gene identification and phylogenetic tree analysis.The optimal temperature and pH of the purified protease were 55 ℃ and 9.0,respectively.Furthermore,an ap02 gene encoding alkaline protease identified from an alkaline polluted soil metagenomics library,was ligated into a widely host expression vector of pBBR1MCS-5and transferred into the competition cells of Pseudomonas aeruginosasp.DES-3.Finally,agenetically engineered strain of the P.aeruginosasp.DES-3/pBBR1MCS-5-ap02 was constructed.The enzymatic data indicated that the optimal temperature and pH of the P.aeruginosasp.DES-3/pBBR1MCS-5-ap02 were 60℃and 9.0,respectively,the final optimal temperature increased 5 ℃ than that of the original P.aeruginosa sp.DES-3.This study showed a technical reference for the genetic modification of the wild type strain and also provided the potential industrial application.