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

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