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

Jie Zhou; Jianhao Chen; Nisha Zhuang; Alei Zhang; Kequan Chen; Ning Xu; Fengxue Xin; Wenming Zhang; Weiliang Dong; Min Jiang

doi:10.3389/fbioe.2020.00579

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

Front Bioeng Biotechnol. 2020 Jun 12:8:579. doi: 10.3389/fbioe.2020.00579. eCollection 2020.

Authors

Jie Zhou¹, Jianhao Chen¹, Nisha Zhuang¹, Alei Zhang^{1

2}, Kequan Chen^{1

2}, Ning Xu¹, Fengxue Xin^{1

2}, Wenming Zhang^{1

2}, Weiliang Dong^{1

2}, Min Jiang^{1

2}

Affiliations

¹ State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
² Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing, China.

Abstract

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.

Keywords: affinity chromatography; chitin-binding domain; enzyme conversion; fusion tag; protein immobilization; protein purification.