Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide. 2006

Marion B Ansorge-Schumacher, and Heike Slusarczyk, and Julia Schümers, and Dennis Hirtz
Department of Biotechnology, RWTH Aachen University, Germany. m.ansorge@biotec.rwth-aachen.de

In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (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.

UI MeSH Term Description Entries
D002175 Candida A genus of yeast-like mitosporic Saccharomycetales fungi characterized by producing yeast cells, mycelia, pseudomycelia, and blastophores. It is commonly part of the normal flora of the skin, mouth, intestinal tract, and vagina, but can cause a variety of infections, including CANDIDIASIS; ONYCHOMYCOSIS; VULVOVAGINAL CANDIDIASIS; and CANDIDIASIS, ORAL (THRUSH). Candida guilliermondii var. nitratophila,Candida utilis,Cyberlindnera jadinii,Hansenula jadinii,Lindnera jadinii,Monilia,Pichia jadinii,Saccharomyces jadinii,Torula utilis,Torulopsis utilis,Monilias
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D005560 Formate Dehydrogenases Flavoproteins that catalyze reversibly the reduction of carbon dioxide to formate. Many compounds can act as acceptors, but the only physiologically active acceptor is NAD. The enzymes are active in the fermentation of sugars and other compounds to carbon dioxide and are the key enzymes in obtaining energy when bacteria are grown on formate as the main carbon source. They have been purified from bovine blood. EC 1.2.1.2. Formate Dehydrogenase,Formate Hydrogenlyases,NAD-Formate Dehydrogenase,Dehydrogenase, Formate,Dehydrogenase, NAD-Formate,Dehydrogenases, Formate,Hydrogenlyases, Formate,NAD Formate Dehydrogenase
D000180 Acrylic Resins Polymers of high molecular weight which are derived from acrylic acid, methacrylic acid or other related compounds and are capable of being molded and then hardened to form useful components. Acrylic Resin,Resin, Acrylic,Resins, Acrylic
D012996 Solutions The homogeneous mixtures formed by the mixing of a solid, liquid, or gaseous substance (solute) with a liquid (the solvent), from which the dissolved substances can be recovered by physical processes. (From Grant & Hackh's Chemical Dictionary, 5th ed) Solution
D019020 Directed Molecular Evolution The techniques used to produce molecules exhibiting properties that conform to the demands of the experimenter. These techniques combine methods of generating structural changes with methods of selection. They are also used to examine proposed mechanisms of evolution under in vitro selection conditions. Evolution, Molecular, Directed,Molecular Evolution, Directed,In Vitro Molecular Evolution,Laboratory Molecular Evolution,Directed Molecular Evolutions,Evolution, Directed Molecular,Evolution, Laboratory Molecular,Evolutions, Directed Molecular,Evolutions, Laboratory Molecular,Laboratory Molecular Evolutions,Molecular Evolution, Laboratory,Molecular Evolutions, Directed,Molecular Evolutions, Laboratory
D019943 Amino Acid Substitution The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties. Amino Acid Substitutions,Substitution, Amino Acid,Substitutions, Amino Acid

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