摘要:在工业用酶方面，蛋白酶是最重要的工业水解用酶，它广泛应用于工业生产的众多行业中，如食品加工，洗涤剂，制药，多肽合成等领域。但是这些蛋白酶的活性稳定性是否能够经受住环境pH，表面活性剂，高温等的限制还需进一步考究，所以筛选出具有耐表面活性剂嗜热蛋白酶，具有很重要的应用前景。
本实验从徐州工程学院城南校区附近的土壤中分离到27株产蛋白酶菌株，用检测蛋白酶产生水解圈和蛋白酶活性测定相结合的方法，从中挑选出5株产高活性蛋白酶的菌株，通过摇瓶发酵得到蛋白酶，对酶液进行表面活性剂耐受性实验和耐高温实验，最终确定一株活力最高的菌株，暂标记为12-54，保存。
菌株12-54 16S rDNA的PCR扩增产物送北京三博远志测序公司进行测序，核苷酸序列长度为1469bp。测序结果在NCBI上进行BLAST比对，根据比对结果，利用MEGA 4.0软件进行系统发育分析，采用邻接法构建系统进化树，比对结果表明12-54与地衣芽孢杆菌的同源性最高，为99%。
提取菌株基因组为模板DNA，通过PCR方法扩增，PCR产物经琼脂糖凝胶电泳分离，切胶回收后克隆到pGM-T载体上，将PCR产物过夜连接载体，并转化至大肠杆菌E.coli DH5α感受态细胞，通过蓝白班筛选得到了5种阳性转化子，提取这5种转化产物的质粒后，进行重组质粒的酶切鉴定。测序后得到克隆的酶基因序列全长1141bp。
关键词：蛋白酶；筛选；测序鉴定；克隆
Abstract:The protease is the most important industrial hydrolysis enzymes, which are widely used in industrial production such as food processing, detergents, pharmaceuticals, peptide synthesis, and other fields. However, the environment such as pH, surface active agents, high temperature are effecting the activity and stability of these proteases, so screening of thermophilic Surfactant-stable protease production has a very important application prospect.
We got 27 strains that can produce protease from Xuzhou Institute of Technology, detection protease hydrolysis ring and protease activity was a method of screening. We got five strains that produced highly active protease, and then we obtained protease by the shake-flask fermentation. I made an experiment to make sure if the enzyme solution can remain highly activity in the effect of heat and Surfactant. Finally, I defined a good strain named as 12-54 and saved.
The strain’s 16S rDNA PCR amplification products were sent to Beijing Sunbiotech to sequence, nucleotide sequence length is 1469bp. The sequencing results made a BLAST alignment on NCBI. According to the comparison results, using MEGA 4.0 software to phylogenetic analysis and using the neighbor-joining method to construct phylogenetic tree, the results showed that 12-54 is 99% similar to Bacillus licheniformis.
As the strain genomic to cDNA, we use the special primer for PCR amplification to specific fragment, use PGM-T to build a carrier, the PCR product was connected carrier overnight, and transformed into E. coli competent cells, by blue and white screening, I got 5 kinds of transformation strain extract five kinds of transformation products plasmid, restriction enzyme digestion of the recombinant plasmid. The gene length is 1141bp after sequence.
Keywords :protease screening DNA sequencing cloning
