【摘要】 针对目前国产食品级亮氨酸氨肽酶的工业生产产量不高的现状,将米曲霉、黑曲霉和酱油曲霉的5个亮氨酸氨肽酶基因(lapA、lap1O、lap2、lap1S、lap1N)在低蛋白背景的无孢黑曲霉HL-1中进行重组表达,通过信号肽替换对其编码区进行改造,利用杂合启动子PnaII及营养缺陷标记pyrG构建表达载体,并基于规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/Cas9工具设计亮氨酸氨肽酶的基因组定点整合策略,获得高活力的亮氨酸氨肽酶表达菌株,重组菌株LapA-C的亮氨酸氨肽酶活力达到11 701.2 U/mL,比未利用CRISPR工具的LapA-T重组表达菌株的酶活力(2 476.0 U/mL)提高了约3.7倍。此外,通过融合6×His标签实现了重组亮氨酸氨肽酶LapA的纯化,并对其进行了酶学性质研究:蛋白质大小约为35.0 kDa,重组亮氨酸氨肽酶LapA最适pH值为8.5,最适温度为65℃。综上所述,本研究基于CRISPR策略成功实现了亮氨酸氨肽酶在黑曲霉中的高效重组表达。更多还原

【Abstract】 Considering that the industrial production of food-grade leucine aminopeptidase is low in China, in this study, we investigated the recombinant expression of the five leucine aminopeptidase encoding genes(lapA, lap1 O, lap2, lap1 S and lap1 N) from Aspergillus oryzae, A. sojae and A. niger in aconidial A. niger strain HL-1 with a low-background of protein secretion. The encoding region was improved via signal peptide replacement, and the expression vector was constructed with the hybrid promoter PnaII and the auxotroph marker pyrG. The homologous recombination method combined with the clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 system was used to improve gene integration efficiency and protein expression. The LapA activity of the recombinant strain was 11 701.2 U/mL, about 4.7 times higher than that(only 2 476.0 U/mL) obtained without using CRISPR. The recombinant enzyme was purified using the 6 × His tag for enzymatic characterization. The molecular mass of the purified LapA was shown to be 35.0 kDa. The optimum reaction temperature and pH for the recombinant LapA were 65 ℃ and 8.5, respectively. Overall, the recombinant LapA was successfully overexpressed in A. niger.更多还原