Collimonas sp.ZL261蛋白酶基因的克隆、表达及其酶学特性

Gene Cloning,Expression and the Characterization of the Protease Produced by Collimonas sp. ZL261

分离自西藏色季拉山海拔4530 m高山草甸土壤的山冈单胞菌(Collimonas sp.)ZL261代谢产物具有很高的蛋白酶活性,为了明确其蛋白酶种类及其作用特点,本文采用限制性内切酶法构建了菌株ZL261的基因组文库,利用透明圈法筛选其胞外蛋白酶活性克隆,对其基因及蛋白结构进行了分析,进一步研究了该酶的表达和酶学特性。结果表明:该蛋白酶基因(命名为capro)包含一个长为1092 bp的完整开放阅读框(GenBank:KF992845),其编码蛋白由363个氨基酸组成;序列比对和同源性分析结果显示,该氨基酸序列与食真菌山冈单胞菌(Collimonas fungivorans)Ter331胞外蛋白酶序列(NC015856)相似性最高,为83%;一级、二级和三级结构分析均显示该蛋白含M35金属蛋白酶家族(EC 3.4.24.20)保守结构域。蛋白酶基因在大肠杆菌DH5α中异源表达获得了约38 kDa的目的蛋白。ZL261粗酶液对金属蛋白酶抑制剂EDTA敏感,最适作用pH值为7,最适温度30℃,在10℃时仍保持70%以上酶活力。综合分析可知,该蛋白酶为中性低温金属蛋白酶。具有较好的开发应用潜能。研究结果为微生物蛋白酶的开发提供了新型种质资源,也为探求不同生境微生物蛋白酶的系统进化关系提供了基础信息。

Collimonas sp. ZL261 was isolated from the alpine meadow soil at Sejila Mountain in Tibet of China, of which the altitude is 4530 meters. The strain was able to produce a high active protease which formed large transparent zones on protease testing medium plates. In order to clarify the category and the function characteristics of the protease, the protease gene （named as capro） was cloned by constructing the gene library with restriction endonuclease method, and successfully expressed in Escherichia coli DH5α. An open reading frame of 1092 bp （GenBank：KF992845） encoding a 363 -amino acid （aa） protein with a calculated molecular weight （Mr） of 38.0 kDa and a pI of 6.65. Protein BLAST and phylogenetic tree analysis of the deduced amino acid sequence from capro showed that the encoded protein had 83% homology to a protease of Collimonas fungivorans Ter331 （NC015856）. The analysis of primary, secondary and three- dimensional structures showed that the amino acid sequece of Capro include zinc-binding conservative regions. One recombinant protein with the size of 38 kDa was obtained by expressing the gene capro in E. coli DH5α. The protease Capro was sensitive to EDTA which was an inhibitor of metalloproteases. The optimum pH and optimum temperature of Capro activities were pH 7 and 30℃ , respectively. When exposed to 10℃ , the enzyme relative activity still remained more than 70%. Summarizing the results above, we reached the conclusion that the protease Capro is a cold-active proteases belonging to matrix metalloproteinases M35. Summarizing the experimental data above, The protease Capro is a cold-active proteases belonging to matrix metalloproteinases M35 and has a great development potential. The research result offered a new type of germplasm resource for the development of microbial proteases, and provided the fundamental information for exploring the phylogenetic relationship among proteases produced by microorganisms in different eco-environments.