TAILIEUCHUNG - Báo cáo khoa học: Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide

In two cycles of an error-prone PCR process, variants of formate dehy-drogenase from Candida boidiniiwere created which revealed an up to (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using sin-gle precursor molecules of polyacrylamide instead of the complete gel for selection. | ễFEBS Journal Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide Marion B. Ansorge-Schumacher1 Heike Slusarczyk2 Julia Schumers1 and Dennis Hirtz1 1 Department of Biotechnology RWTH Aachen University Germany 2 Institute of Enzyme Technology Heinrich-Heine-University Dusseldorf Research Center Julich Germany Keywords directed evolution entrapment formate dehydrogenase polyacrylamide stabilization Correspondence M. B. Ansorge-Schumacher Department of Biotechnology RWTH Aachen University D-52056 Aachen Germany Fax 49 241 8022387 Tel 49 241 8026620 E-mail Website http biokat In two cycles of an error-prone PCR process variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 440 higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine glutamic acid and cysteine residues remote from the active site which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid but decreased due the exchange of cysteine. Overall the variants reveal very suitable properties for application in a technical synthetic process enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method. Received 4 April2006 revised 19 June 2006 accepted 26 June 2006 doi Entrapment in polymeric matrices has long since become an important technique to improve recycling handling and mechanical strength of delicate biocatalysts during application in large-scale organic synthesis. It also enables reactions with otherwise instable or cofactor-dependent enzymes in organic solvents 1 2 .

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