摘要
从地衣芽孢杆菌(Bacillus licheniformis) ATCC16580中克隆得到角蛋白酶基因kerA,将其构建在芽孢杆菌表达载体p LIYU-2上,再转化到解淀粉芽孢杆菌(Bacillus amyloliquefaciens) TCCC111018中,构建出高产角蛋白酶重组菌株TCCC111018/p LIYU-2-kerA。重组菌株在羽毛发酵培养基中12h内即可将羽毛全部降解,在摇瓶发酵培养基中36h酶活力达到1 361. 54U/m L。重组角蛋白酶最适温度为65℃,最适p H值为9,在40～60℃和p H值9～11范围内具有良好的稳定性。蛋白电泳(SDS-PAGE)分析表明,重组角蛋白酶分子质量约为31kD。Na^+,K^+、Ca^(2+)及Mg^(2+)能显著促进角蛋白酶活力,Cu^(2+)和Fe^(3+)对重组角蛋白酶有明显的抑制作用。高效液相色谱(HPLC)分析显示,羽毛酶解液中以缬氨酸、异亮氨酸和苯丙氨酸为主,另外也含有较多的酪氨酸和胱氨酸,游离氨基酸浓度为14. 59μmol/m L。
The kerA gene was cloned from nacillus licheniformis ATCC16580and inserted into expression vector pLIYU-2,then transformed into bacillus amyloliquefaciens TCCC 111018,constructed high yield keratinase recombinant strain TCCC111018/pLIYU-2-kerA.The recombinant strain can degrade all the feather within 12hours and the enzyme activity of recombinant strain reached 1361.54U/mL after 36h in the fermentation medium.For the degradation reaction,the optimum temperature of the recombinant keratinase is 65℃ and the optimum pH is 9.The recombi- nant keratinase has good stability at the temperature of 40-60℃.and pH value of 9-11.SDS-PAGE shows that the molecular weight of recombinant keratinase is about 31kD.The recombinant keratinase activity can be promoted by Na^+,K^+,Ca^2+and Mg^2+,and inhibited by Cu^2+and Fe^3+significantly.High performance liquid chromatography (HPLC)analysis shows that feather enzymolysis solution mainly contains valine,isoleucine and phenylalanine,also includes more tyrosine and cystine.The concentration of free amino acid is 14.59μmol/mL.
作者
辛青龙
刘业学
何光明
王兴吉
曹珊
李玉
XIN Qinglong;HU Yexue;HE Guangming;WANG Xingji;CAO Shan;LI Yu(The Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education,Tianjin University of Science &Technology,Tianjin300457,China;Shandong Longkete Enzyme Preparation Co.Ltd.,Yishui 276400,China;College of Chemical Engineering and Material Science,Tianjin University of Science &Technology,Tianjin300457,China)
出处
《中国皮革》
CAS
2018年第12期28-34,共7页
China Leather
基金
国家重点研发计划,项目编号:2017YFB0308401
