Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

Abstract Molecular chaperones in aqueous‐organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self‐renaturing enzyme‐chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous‐organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme‐chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous‐methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems. Biotechnol. Bioeng. 2009;102: 1316–1322. © 2009 Wiley Periodicals, Inc.