title: 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.
authors.affiliation: Department of Chemical Engineering, University of California, 201 Gilman Hall, Berkeley, California 94720, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Bergeron
authors.forename: Lizabeth
authors.initials: L
authors.lastname: Gomez
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Whitehead
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Clark
mesh_terms: D000658:Amoxicillin / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D004795:Enzyme Stability; D007700:Kinetics; D000432:Methanol; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014867:Water
chemical_list: D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014867:Water; D000658:Amoxicillin; D010399:Penicillin Amidase; D000432:Methanol