黑曲霉阿魏酸酯酶A的克隆、表达及快速酶活检测体系的建立

Cloning and Expression of Feruloyl Esterase A from Aspergillus niger,and Establishment of Fast Activity Detection Methods

阿魏酸酯酶是参与半纤维素降解的重要酶类.为了提高阿魏酸酯酶的活性和其它性能,本文作者采用RT-PCR技术从黑曲霉CIB423.1中克隆了编码阿魏酸酯酶A的cDNA,构建了外泌表达的质粒,并将其转化到毕赤酵母GS115进行表达.通过检测培养液上清中的酶活,表明阿魏酸酯酶已成功在这一体系表达.同时,本文还建立了快速、稳定的酶活检测体系,为后续对阿魏酸酯酶酶突变体活性进行高通量筛选奠定了基础.

Feruloyl esterases （FAEs） play an important role in hemicellulose degradation. In order to apply the directed molecular evolution method to improve the feruloyl esterase＇s activity and performances, the cDNA encoding feruloyl esterase A （FAEA） was cloned from Aspergillus niger CIB 423.1 using RT-PCR. The recombinant plasmid encoding FAEA was constructed and transformed into Pichia pastoris GS115. The extracellular expression was proven to be successful by enzymatic activity detection of the culture supernatant. Meanwhile, a fast and stable platform for feruloyl esterase assay was established, which made it possible to screen FAEA mutants in a high-throughput fashion in upcoming studies. Fig 5, Ref 17