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AssigneeDegussa AG
InventorsHeike Slusarczyk, Stephan Felber, Maria-Regina Kula, Martina Pohl
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Novel improved enzymes prepared by recombination, especially to rec-FDHs. Directed evolution has made it possible to generate catalytically more active and more stable muteins which can preferably be used in an industrial process for the preparation of e.g. amino acids. The invention further relates to the nucleic acids coding for these enzymes, to vehicles containing these nucleic acids and to advantageous primers for the preparation of the nucleic acids by means of PCR. The invention additionally relates to a process for the preparation of further improved rec-FDHs, and a method of screening more stable and/or more active dehydrogenases is claimed.
A mutant of the wild-type rec-FDH from Candida boidinii which is more stable and/or more active as compared to said wild-type rec-FDH from Candida boidinii, wherein the mutant contains (a) the amino acid exchange C23S or C23S/C262A and (b) one or more amino acid exchanges selected from the group consisting of E18D, K35R, D149E, E151D, R178S, R178G, K206R, F285Y, F285S, T315N and K356E.
A mutant of the native wild-type FDH from Candida boidinii which is more stable and/or more active as compared to said wild-type FDH from Candida boidinii, wherein the mutant contains (a) the amino acid exchange C23S or C23S/C262A and (b) one or more amino acid exchanges selected from the group consisting of E18D, K35R, D149E, E151D, R178S, R178G, K206R, F285Y, F285S, T315N and K356E.
An amino acid sequence having FDH activity which is more stable and/or more active as compared to the wild-type rec-FDH and the native wild-type enzyme from Candida boidinii and which contain one or more of the following amino acid exchanges:
A primer selected from the group consisting of SEQ ID NO:
A cell containing a cloned gene for a dehydrogenase and a cloned gene for a rec-FDH.
A method of identifying more active mutants of an NAD- or NADP-dependent dehydrogenase, comprising a quantitative screening method for determination of the activity, said method consisting of the following steps:
Dependencies are inferred from the claim text.