农业生物技术学报2018，Vol.26Issue(2)：302-310,9.DOI:10.3969/j.issn.1674-7968.2018.02.013
嗜热地衣芽胞杆菌Y6角蛋白酶的异源表达与表征
Cloning and Heterologous Expression of the Keratinase from Thermophilic Bacillus licheniformis Y6
摘要
Abstract
Bacillus licheniformis could secrete a kind of keratinase which can hydrolyze keratin such as feather,hair,nail and so on.In previous work,the results indicated that a thermophilic Bacillus licheniformis Y6 was able to live at elevated temperature,it also could degrade feather rapidly,and lived on feather as the only source of carbon and nitrogen.Based on these properties,this paper finally extracted a kind of thermostable keratinase with excellent characteristics was extracted.Hence,it possess the potential to manage poultry waste and can be exploited in some specific industries,aiming at cloning a keratinase gene from Bacillus licheniformis Y6,this paper designed a pair of primers,and finally obtained the gene (GenBank No.MF327392) which included a complete Open Reading Frame with the size of 1 140 bp.Aligned in NCBI,this keratianse remained to be conserved during evolution,and shared the same catalytic triad Ser-His-Asp with other serine proteases.Analyzed by some online software,this entire gene encoded 379 amino acids with pre-,pro-and mature protein regions,and its mature protein,with the molecular weight 27.27 kD and the theory isoelectric point 6.57,was classified as stable protein.After cloning the gene into vector pET-32a,The recombinant vector pET-32a kerY6 was transformed into Escherichia coli BL21(DE3) induced with 0.5 mmol/L isopropyl β-D-thiogalactoside(IPTG),then the precipitation and culture supernatant were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis which indicated that the recombinant keratinase was successfully induced with a band at 58 kD corresponding to expectation,but could not be detected in the broth.In order to detect whether it developed into inclusion body,precipitation and supernatant of bacteria lysate were subjected to SDS-PAGE,results showed no band in the supematant lane.Subsequently,the recombinant keratinase was purified by Ni-chelating affinity chromatography.Followed by characterization,the recombinant keratinase displayed a wide range of reaction temperature 60～80 ℃,and retained 85％ maximum activity for a long time which indicated an excellent thermost ability.As the time accumulation,deactivation of enzyme was observed by incubating the recombinant keratinase over 80 ℃.Incubated with a variety of pH values,the maximum activity was obtained at pH value 8.5.The results of pH values assay demonstrated that the recombinant keratinase was a typical alkaline protease.Ethylene diaminete traacetic acid (EDTA),sodium dodecyl sulfate (SDS),phenylmethanesulfonyl fluoride (PMSF),Mn2+ and Fe3+ could remarkly inhibited its activity by 76％,49％,59％,34％,75％,respectively;Ca2+ and Mg2+ prompted its activity to 118％,125％,respectively.Organic solvent,for instance,isopropylalcohol,glycerin and dimethyl sulfoxide (DMSO) almost had no influence on its activity.In conclusion,the keratinase from thermophilic bacillus licheniformis Y6 has great potential to apply to industry.关键词
地衣芽胞杆菌/角蛋白酶/克隆/表达Key words
Bacillus licheniformis/Keratinase/Clone/Expression分类
农业科技引用本文复制引用
刘建新,姚大伟,刘艳海,杨德吉..嗜热地衣芽胞杆菌Y6角蛋白酶的异源表达与表征[J].农业生物技术学报,2018,26(2):302-310,9.基金项目
农业部“948”研究项目(No.2015-Z40) （No.2015-Z40）
