白曲霉酸性蛋白酶在黑曲霉中表达  

Expression of acid protease from Aspergillus kawachii in Aspergillus niger

作　　者：李杰[1] 吴婷[1] 马南[1] 王欣[1] 杨建乐[1] 李健友[1] 张会[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2016年第5期29-35,共7页Journal of Northeast Agricultural University

基　　金：黑龙江省应用技术研究与开发计划重大项目(GA15B203)

摘　　要：研究分析固态发酵酸性蛋白酶生产菌株及其产品,ITS序列鉴定结果表明,该菌株为曲霉属,质谱分析结果表明其产品为Aspergillus saitoi酸性蛋白酶Aspergillopepsin I（EC.3.4.23.18）。根据Aspergillus saitoi酸性蛋白酶Aspergillopepsin I基因序列pep1（GI：473517）设计引物,以固态发酵酸性蛋白酶生产菌株基因组DNA为模板,利用PCR技术扩增获得pep1基因。序列分析结果表明,扩增片段与白曲霉Aspergillus kawachii酸性蛋白酶基因组序列相似性为99%,其编码蛋白与白曲霉Aspergillus kawachii酸性蛋白酶（GAA90749.1）相似性为100%,将该基因命名为pep B。构建黑曲霉表达载体p SZHG-pep B,通过农杆菌介导法转化黑曲霉CICC2462,筛选得到在gla A位点发生同源重组纯合转化子。经摇瓶发酵后,对产物进行SDS-PAGE、酶活检测以及酸性蛋白酶酶学性质和酶稳定性研究。结果表明,纯合同源重组菌株经SDS-PAGE检测时在47 ku左右处有明显目的蛋白条带,其发酵产物酸性蛋白酶酶活达5 543 U·m L-1,为出发菌株152倍。对菌株所产酸性蛋白酶酶学性质研究发现,该酶最适反应温度为50℃,最适反应p H 3.0,在4℃和25℃条件下,酶在p H 3.0~4.0时较稳定。In this study, solid fermentation acid protease＇s producing strain from Zhaodong Richeng enzyme Ltd. and its products were analyzed, ITS sequence indicated that the strain belongs to the genus Aspergillus and mass spectrometric analysis showed that its product was acidic protease Aspergillopepsin I（EC.3.4.23.18） of Aspergillus saitoi. According to gene sequence of pep1（GI：473517） from acid protease Aspergillopepsin I of Aspergillus saitoi, primers were designed, producing strain＇s genomic DNA from Solid-state fermentation acid protease as the template, and by the method of PCR, pep1 gene was cloned. Sequence analysis showed that amplified fragment was 99% similar to acid protease＇s genome sequence of Aspergillus kawachii and encoding protein was 100% similar to acid protease of Aspergillus kawachii, and it was named as pep B. Furthermore Aspergilluse niger expression vector p SZHG-pep B was constructed and the homozygous transformation of homologous recombination at the gla A site was selected by Agrobacterium mediated transformation of Aspergillus niger CICC2462 By shaking flask fermentation, the product was studied by SDS-PAGE and the activity was detected, acid protease＇s characterization and stabilitywas studied. Experimental results showed that the pure homologous recombination strains had about 47 ku protein band which was found by SDS-PAGE and acid protease activity of fermentation product was 5 543 U · m L-1which is 152 times of the activity of starting strain. By studying the acid protease＇s characterization and stability of this strain,we found that the enzyme optimum reaction temperature is 50 ℃, the optimal p H was 3.0, what＇s more,enzyme activity is stable when the p H is between 3.0 and 4.0 meanwhile the temperature is 4 ℃ or 25 ℃.

关 键 词：酸性蛋白酶 黑曲霉 同源重组 分泌表达 

分 类 号：Q815[生物学—生物工程]