深海沉积物宏基因组文库中产蛋白酶克隆的筛选及性质分析

Screening and Characterization of Protease from Metagenomic Library of Deep Sea Sediment

提取东太平洋深海沉积物宏基因组DNA,构建了未培养微生物宏基因组文库.该文库平均插入片段30kb以上,含有7000个克隆,含外源DNA总长度约为210Mb.从文库中筛选到18个产胞外蛋白酶的克隆,16SrRNA分析表明,它们分别来源于假单胞菌(Pseudomonas)、寡养单胞菌(Stenotrophomonas)和弧菌(Vibrio)3个菌属.根据活性大小及来源菌株的16SrRNA序列比较,选择10个蛋白酶进行酶学性质分析,结果表明,它们的最适作用pH值在7～10之间,最适作用温度在10～40℃之间.其中Pro20蛋白酶最适作用温度最低,为10℃;Pro1蛋白酶具有最适作用温度低(20℃)、pH耐受性好、热稳定性较好等特性,具有良好的应用开发潜力.

Metagenomic cosmid library, containing 7 000 clones with more than 30 kb inserts and covering about 210 Mb metagenomic DNA of uncultured microorganisms from the deep sea sediment of east Pacific, was constructed, and 18 independent clones expressing protease activity were isolated from the library. The analysis of 16S rRNA showed that these proteases originated from Pseudomonas, Stenotrophomonas and Vibrio, respectively. Ten proteases were selected for further study based on the comparison of their activities and 16S rRNA sequence. The results indicated that the optimal pH and temperature for their activities ranged between 7-10 and 10-40 ℃, respectively. The Pro20 protease had the lowest optimal temperature at 10 ℃. The Prol protease had better potential for exploitation owing to some virtues including high activity at low temperature, better resistant for high pH and temperature. Fig 6, Ref 16