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+、Mg2+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Km为1.893 mmol/L,Vmax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 Mg2+ could significantly activate the lipase ANL-H3. The enzyme substrate specificity assay found that the enzyme had the highest affinity to pNPA. The Km of ANL-H3 was 1.893 mmol/L, and the Vmax 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.
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