Construction and characterization of novel bifunctional fusion proteins composed of alcohol dehydrogenase and NADH oxidase with efficient oxidized cofactor regeneration.
Biotechnol Appl Biochem
; 69(4): 1535-1544, 2022 Aug.
Article
em En
| MEDLINE
| ID: mdl-34269481
ABSTRACT
To tune the efficiency of oxidized cofactor recycling between alcohol dehydrogenase (ADH) and NADH oxidase (NOX) for the production of aromatic chiral alcohols, we designed and constructed four novel bifunctional fusion proteins composed of thermostable ADH and NOX from Thermococcus kodakarensis KOD1. ADH was linked to the N- or C-terminus of NOX with a typical rigid linker (EAAAK)3 and a flexible linker (GGGGS)3 , respectively. Compared with the parental enzymes, the NOX moieties in the four fusion proteins exhibited higher specific activities (141%-282%), while the ADH moieties exhibited varying levels of specific activity (69%-167%). All fusion proteins showed decreased affinities toward the cofactors, with increased Km values toward NADH (159%-406%) and NAD+ (202%-372%). In the enantioselective oxidation of (RS)-1-phenylethanol coupled with cofactor regeneration, the four fusion proteins displayed different positive and negative effects on the recycling efficiency of the oxidized cofactor. The two fusion proteins composed of NOX at the N-terminus exhibited higher total turnover numbers than the corresponding mixtures of individual enzymes with equal activities, particularly at low cofactor concentrations. These findings suggest high cofactor recycling efficiencies of the fusion proteins with appropriate design and their potential application in the biosynthesis of chiral alcohols.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Álcool Desidrogenase
/
NADH NADPH Oxirredutases
Idioma:
En
Revista:
Biotechnol Appl Biochem
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
China