期刊名称：Computational and Structural Biotechnology Journal
印刷版ISSN：2001-0370
出版年度：2014
卷号：9
期号：0
DOI：10.5936/csbj.201402005
语种：English
出版社：Computational and Structural Biotechnology Journal
摘要：Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)+ regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 193R194H showed comparable high rates and low Km values for NADPH (kcat 20 s-1, Km 6 µM) and NADH (kcat 25 s-1, Km 9 µM) with retention of 70 % of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2‑heptanone production.
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