Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1

Frontiers in Bioengineering and Biotechnology

Min Jiang¹  Alei Zhang¹  Fengxue Xin¹  Wenming Zhang¹  Kequan Chen¹  Weiliang Dong¹  Jie Zhou²  Jianhao Chen²  Nisha Zhuang²  Ning Xu² 

[1] Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China;

关键词: chitin-binding domain;    fusion tag;    affinity chromatography;    protein purification;    protein immobilization;    enzyme conversion;   

DOI  :  10.3389/fbioe.2020.00579

来源: DOAJ

【 摘 要 】

A new protein immobilization and purification system has been developed based on the improved plasmid vectors, designated pETChBD-X, which contained the gene coding for two novel chitin-binding domains ChBD-AB, factor Xa cleavage site and adapted for gene fusions. The ChBD-AD from Chitinolyticbacter meiyuanensis SYBC-H1 was used as a novel affinity tag to anchor fusion proteins to chitin granules. The granules carrying the ChBD-AD fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble recombination protein can be obtained after Factor Xa cleavage. The efficiency of this system has been demonstrated by reaching 95% of protein absorbed to chitin within 30 min and recycling over 75% of interest protein after Factor Xa cleavage to separate interest protein and fusion tag. Furthermore, 65% L-glutamate oxidase with this fusion tag could be purified and immobilized within only one step and to be reused in converting L-glutamate to α-ketoglutaric acid directly, the average conversion rate kept above 65% even within four batches of enzyme conversion reaction.

【 授权许可】

Unknown