摘要:在工業(yè)用酶方面，蛋白酶是最重要的工業(yè)水解用酶，它廣泛應(yīng)用于工業(yè)生產(chǎn)的眾多行業(yè)中，如食品加工，洗滌劑，制藥，多肽合成等領(lǐng)域。但是這些蛋白酶的活性穩(wěn)定性是否能夠經(jīng)受住環(huán)境pH，表面活性劑，高溫等的限制還需進(jìn)一步考究，所以篩選出具有耐表面活性劑嗜熱蛋白酶，具有很重要的應(yīng)用前景。

　　本實(shí)驗(yàn)從徐州工程學(xué)院城南校區(qū)附近的土壤中分離到27株產(chǎn)蛋白酶菌株，用檢測蛋白酶產(chǎn)生水解圈和蛋白酶活性測定相結(jié)合的方法，從中挑選出5株產(chǎn)高活性蛋白酶的菌株，通過搖瓶發(fā)酵得到蛋白酶，對(duì)酶液進(jìn)行表面活性劑耐受性實(shí)驗(yàn)和耐高溫實(shí)驗(yàn)，最終確定一株活力最高的菌株，暫標(biāo)記為12-54，保存。

　　菌株12-54 16S rDNA的PCR擴(kuò)增產(chǎn)物送北京三博遠(yuǎn)志測序公司進(jìn)行測序，核苷酸序列長度為1469bp。測序結(jié)果在NCBI上進(jìn)行BLAST比對(duì)，根據(jù)比對(duì)結(jié)果，利用MEGA 4.0軟件進(jìn)行系統(tǒng)發(fā)育分析，采用鄰接法構(gòu)建系統(tǒng)進(jìn)化樹，比對(duì)結(jié)果表明12-54與地衣芽孢桿菌的同源性最高，為99%。

　　提取菌株基因組為模板DNA，通過PCR方法擴(kuò)增，PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳分離，切膠回收后克隆到pGM-T載體上，將PCR產(chǎn)物過夜連接載體，并轉(zhuǎn)化至大腸桿菌E.coli DH5α感受態(tài)細(xì)胞，通過藍(lán)白班篩選得到了5種陽性轉(zhuǎn)化子，提取這5種轉(zhuǎn)化產(chǎn)物的質(zhì)粒后，進(jìn)行重組質(zhì)粒的酶切鑒定。測序后得到克隆的酶基因序列全長1141bp。

關(guān)鍵詞：蛋白酶；篩選；測序鑒定；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