枯草芽孢杆菌克隆与表达微小毛霉凝乳酶基因  

Cloning and expression of chymosin gene from Mucor pusillus cDNA library in Bacillus subtilis

作　　者：李自刚[1,2,3] 李凤娟[2] 云丽[2] 吴坤[2] 赵跃进 屈凌波[4] 

机构地区：[1]郑州牧业工程高等专科学校,河南郑州450011 [2]河南农业大学,河南郑州450001 [3]三门峡龙飞生物工程有限公司,河南三门峡472100 [4]河南工业大学,河南郑州450001

出　　处：《中国酿造》2012年第10期68-72,共5页China Brewing

基　　金：河南省重大公益科研招标项目资金资助(HNZB[2008]N84)

摘　　要：从自制的酒酿利用酪蛋白培养基分离纯化到了一株产凝乳酶的微小毛霉菌株(ZZMZ-19),从ZZMZ-19菌株的cDNA文库筛选到了两个凝乳酶基因ch1和ch2并实现了凝乳酶基因ch1和ch2在枯草芽孢杆菌菌株(ZZMZ-01)中的的克隆与表达。ch1和ch2阳性克隆菌株在酪蛋白培养基中发酵30h左右的时间凝乳酶酶活达到最大,分别为48.27SU/mL和41.02SU/mL,相比出发菌株发酵时间缩短了15h。用交联葡聚糖凝胶柱G100分离纯化其发酵上清液后,用十二烷基硫酸钠聚丙烯酰胺凝胶电泳方法测得CHⅡ和CHⅢ分子量分别为32ku和30ku。A high chymosinproducing Mucorpusillus strains （ZZMZ-19） was isolated from home-made Jiu-Niang using casein medium, and two novel chymosin genes chl and ch2 were cloned from cDNA library of ZZMZ-19 and expressed in Bacillus subtilis strains （ZZMZ-01）. The recombined ZZMZ-01 strain was cultured in the casein medium. The results showed that the maximum activities of recombined chymosin were obtained after 30h fermentation, which was 15h earlier than that of the original strain Mucor pusillus ZZMZ-19. The values were 48.27SU/ml and 41.02SU/ml, respec- tively. The expressed chymosin were purified using cross-linking glucan gel column and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that the molecular weights of CH1 and CH2 enzymes were 32 ku and 30 ku, respectively.

关 键 词：酒酿 微小毛霉 凝乳酶 枯草芽孢杆菌 克隆 表达 

分 类 号：Q93-331[生物学—微生物学]