黑曲霉脂肪酶基因的克隆及其在黑曲霉中的同源表达

Cloning of the gene encoding lipase from Aspergillus niger and its homologous expression in Aspergillus niger

旨在研究黑曲霉脂肪酶基因在黑曲霉中的同源表达情况。利用黑曲霉基因组作为模板克隆8种脂肪酶基因,与双元表达载体pCAMBIA连接构建8个表达载体。通过原生质体-PEG转化法,实现8个脂肪酶基因在黑曲霉中同源表达,其中转化子MA-H3-PglaA-SglaA脂肪酶活性最高。对该酶进行分离纯化和酶学性质表征,酶最适温度45℃,最适pH 7.5,K^(+)、Mg^(2+)等金属离子对脂肪酶有显著激活作用。酶底物特异性实验发现,该酶对底物pNPA亲和力最高,脂肪酶ANL-H3的K_(m)为1.893 mmol/L,V_(max)为1.610 mmol/(L·min)。该研究为黑曲霉脂肪酶的同源表达提供了有效的策略和数据支持,可为食品级脂肪酶的开发和应用提供理论基础。

The purpose of this study to investigate the homologous expression of Aspergillus niger lipase gene in A.niger.Eight lipase genes from A.niger were cloned by using A.niger genome as a template,which linked to the dual expression vector pCAMBIA to construct eight expression vectors.The homologous expression of eight lipase target genes in A.niger was achieved by protoplast-PEG transformation method,and the transformant MA-H3-PglaA-SglaA had the highest lipase activity.The properties of recombinant lipase were characterized after purification.The optimum temperature of the enzyme was 45 ℃,and the optimum pH was 7.5.Metal ions such as K^(+) and Mg^(2+) could significantly activate the lipase ANL-H3.The enzyme substrate specificity assay found that the enzyme had the highest affinity to pNPA.The K_(m) of ANL-H3 was 1.893 mmol/L,and the V_(max) was 1.610 mmol/(L·min).This study provides effective strategies and data support for the homologous expression of A.niger lipase,and provides theoretical basis for the further development and application of food-grade lipase.