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.