期刊论文

High-expression and characterization of a thermophilicα-galactosidase gene in Pichia pastoris

中文

生物技术

1004-311X

2019

29

2

116-121+115

10.16519/j.cnki.1004-311x.2019.02.0021

中国大洋矿产资源开发协会项目（“深海生物资源计划”,DY135-B2-07）；

本校为第一机构

[目的]实现耐热α-半乳糖苷酶在毕赤酵母中的高效表达, 并初步研究其酶学性质.[方法]克隆来源于埃默森篮状菌(Talaromyces emersonii) 的α-半乳糖苷酶基因TEgal, 构建p AO815-TEgal重组表达载体, 采用DNS法测定其水解活性及酶学性质;通过薄层层析研究其水解底物谱;并构建TEgal基因多拷贝表达框, 实现了该基因的高效表达.[结果]TEgal对棉籽糖水解活性最高9. 5 U/m L, 最适温度75℃, 最适pH值3. 5; Na+、K+对TEgal有促进作用, Mg2+、Co2+、Mn2+、Ca2+、Fe2+、Zn2+均能抑制酶活, 多拷贝重组表达菌株活性最高为22. 4 U/m L.[结论]成功构建耐热α-半乳糖苷酶高效表达菌株, 通过提升基因剂量将酶活和...

[Objective] To realize the high-efficiency expression of thermophilic α-galactosidase in Pichia pastoris and to study its enzymatic properties.[Method] A α-galactosidase gene TEgal from Talaromyces emersonii was cloned to construct p AO815-TEgal recombinant expression vector. The hydrolytic activity and characteristics were determined by DNS method, and the hydrolyzed substrate spectrum were studied by thin layer chromatography. A multi-copy expression cassette strategy of TEgal was used to increase the expression of the gene. [Result]Hydroly...