新颖阿魏酸酯酶在黑曲霉中异源表达及其应用

The Heterologous Expression of the Novel Feruloyl Esterase in Aspergillus niger and Its Application

本研究依托桧状青霉的基因组学和蛋白组学数据,发现一种新型的阿魏酸酯酶。利用PCR技术成功扩增得到桧状青霉阿魏酸酯酶的基因,构建真菌表达盒通过原生质体的方法将其转化到黑曲霉中。通过SDS-PAGE检测和酶活力检测验证该阿魏酸酯酶成功地在黑曲霉胞外分泌表达。根据氨基酸序列相似性和底物特异性分析都显示该阿魏酸酯酶属于C型的阿魏酸酯酶,与常见的A型、B型阿魏酸酯酶性质区别较大。将成功表达阿魏酸酯酶的黑曲霉胞外酶液复配里氏木霉酶液后,水解不同生物质材料,纤维素水解效率均有大幅程度地提高,玉米秸秆,麦麸,玉米芯,木薯酒糟的水解效率分别提高68.8%,38.6%,15.6%和20.0%。该研究为新颖阿魏酸酯酶应用以及今后里氏木霉纤维素酶酶系复配提供新的思路。

In this study, a novel feruloyl esterase was found based on the genomic and proteomic data of Penicillium piceum. The gene of feruloyl esterase from Penicillium piceum was successfully amplified by PCR.The fungal expression box was constructed and transformed into Aspergillus niger through the method of protoplast. The extracellular expression of this feruloyl esterase in A. niger was proven to be successful by SDS-PAGE and enzymatic activity detection. The amino acid sequence and substrate specificity profiling of this novel feruloyl esterase revealed that it was a type C feruloyl esterase, which had different characteristics with the common type A and type B feruloyl esterases. The extracellular enzyme solution of Aspergillus niger which expressed the feruloyl esterase successfully with Trichoderma reesei enzyme solution to hydrolyze different biomass materials. The result showed that the hydrolysis efficiency of cellulose was greatly improved. The hydrolysis efficiencies of corn stover, wheat bran, corn cob, and cassava stillage residues was improved by 68.8%,38.6%, 15.6%, and 20.0%, respectively. This research would provide new insights for the application of the novel feruloyl esterase and the enzymatic mixing of cellulase in Trichoderma reesei enzyme.