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Simultaneous refolding, purification, and immobilization of recombinant Fibrobacter succinogenes 1,3-1,4-β-D-glucanase on artificial oil bodies

Authors :
Yi-Fang Zeng
Ying-Jing Hung
Je-Ruei Liu
Jason T.C. Tzen
Ming-Ju Chen
Chi-Chung Peng
Source :
Journal of Chemical Technology & Biotechnology. 84:1480-1485
Publication Year :
2009
Publisher :
Wiley, 2009.

Abstract

BACKGROUND: 1,3-1,4-β-D-glucanase (1,3-1,4-β-D-glucan 4-glucanohydrolase; EC 3.2.1.73) has been used in a range of industrial processes. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of 1,3-1,4-β-D-glucanase was investigated. RESULTS: A 1,3-1,4-β-D-glucanase gene from Fibrobacter succinogenes was overexpressed in Escherichia coli as a recombinant protein fused to the N terminus of oleosin, a unique structural protein of seed oil bodies. With the reconstitution of the artificial oil bodies (AOBs), refolding, purification, and immobilization of active 1,3-1,4-β-D-glucanase was accomplished simultaneously. Response surface modeling (RSM), with central composite design (CCD), and regression analysis were successfully applied to determine the optimal temperature and pH conditions of the AOB-immobilized 1,3-1,4-β-D-glucanase. The optimal conditions for the highest immobilized 1,3-1,4-β-D-glucanase activity (7.1 IU mg−1 of total protein) were observed at 39 °C and pH 8.8. Furthermore, AOB-immobilized 1,3-1,4-β-D-glucanase retained more than 70% of its initial activity after 120 min at 39 °C, and it was easily and simply recovered from the surface of the solution by brief centrifugation; it could be reused eight times while retaining more than 80% of its activity. CONCLUSIONS: These results indicate that the AOB-based system is a comparatively simple and effective method for simultaneous refolding, purification, and immobilization of 1,3-1,4-β-D-glucanase. Copyright © 2009 Society of Chemical Industry

Details

ISSN :
02682575
Volume :
84
Database :
OpenAIRE
Journal :
Journal of Chemical Technology & Biotechnology
Accession number :
edsair.doi...........f0e6bfc6c3c24e245f6619d80d36cd5c
Full Text :
https://doi.org/10.1002/jctb.2205