高产高纯度脯氨酰内肽酶黑曲霉工程菌的构建

利用基因工程技术，构建过表达黑曲霉来源带有自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-protAS和过表达以黑曲霉内源高效分泌的葡糖淀粉酶信号肽以及α-淀粉酶信号肽代替自身信号肽的脯氨酰内肽酶黑曲霉重组菌株TH2-SglaA-protA和TH2-SamyA-protA。SDS-PAGE结果显示，脯氨酰内肽酶在重组菌株中分泌表达，但是存在不同程度的糖基化。酶活检测结果表明，重组菌株TH2-protAS、TH2-SglaA-protA和TH2-SamyA-protA的最高酶活分别为1.70、2.19、1.91 U/mL。在重组菌株TH2-SglaA-protA的基础上，利用基因敲除技术，敲除了主要的背景蛋白酸稳定的α-淀粉酶，结合发酵条件优化，获得了高纯度的脯氨酰内肽酶。综合上述结果可得出结论:可在黑曲霉中同源高效分泌表达脯氨酰内肽酶；glaA信号肽和amyA信号肽明显增加黑曲霉中脯氨酰内肽酶的分泌表达量，且glaA信号肽的效果优于amyA信号肽；将基因敲除和发酵调控相结合，可以有效去除分泌的背景蛋白，获得高产高纯度脯氨酰内肽酶的菌株，该重组菌株具有明确的工业应用潜力。

Construction of Aspergillus niger Engineering Bacteria with High Yield and High Purity Prolyl Endopeptidase

Using genetic engineering technology,the recombinant strain TH2-protAS of Aspergillus niger which overexpressed the prolyl endopeptidase derived from Aspergillus niger with its own signal peptide and the glucoamylase signal peptide andα-Amylase signal peptide replaces its own signal peptide prolyl endopeptidase Aspergillus niger recombinant strains TH2-SglaA-protA and TH2-SamyA-protA.The results of SDS-PAGE showed that prolyl endopeptidase was secreted and expressed in the recombinant strain,but there were different degrees of glycosylation.The enzyme activity test results showed that the highest enzyme activities of recombinant strains TH2-protAS,TH2-SglaA-protA and TH2-SamyA-protA were 1.70,2.19,1.91 U/mL,respectively.On the basis of the recombinant strain TH2-SglaA-protA,gene knockout technology was used to knock out the main background protein acid-stableα-amylase,combined with optimization of fermentation conditions,to obtain high-purity prolyl endopeptidase.Based on the above results,it could conclude that prolyl endopeptidase could be secreted and expressed efficiently in Aspergillus niger.glaA signal peptide and amyA signal peptide significantly increased the secretion and expression of prolyl endopeptidase in Aspergillus niger,and the effect of signal peptide glaA was better than that of amyA signal peptide.The combination of gene knockout and fermentation regulation could effectively remove the secreted background protein and obtain a strain with high-yield and high-purity prolyl endopeptidase.This recombinant strain had a clear industrial application potential.