Improving biocatalytic properties of an azoreductase \(\it via\) the \(\it N\)-terminal fusion of formate dehydrogenase
- Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows \(NAD(P)^{+}\) recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of \(\textit {Rhodococcus opacus}\) 1CP and FDH (\(FDH_{C23S}\) and \(FDH_{C23SD195QY196H}\)) of \(\textit {Candida boidinii}\) in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the \(\it N\)-terminus of the fusion protein. \(FDH_{C23S}\)+ AzoRo was the most active construct and was further characterized. Enzymatic activities of \(FDH_{C23S}\)+ AzoRo decreased compared to parental enzymes but showed improved substrate scope – accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.
Author: | Anna Christina R. NgoGND, Fabian Peter Josef SchultesGND, Artur MaierGND, Simon Niklas Herman HadewigGND, Dirk TischlerORCiDGND |
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URN: | urn:nbn:de:hbz:294-88687 |
DOI: | https://doi.org/10.1002/cbic.202100643 |
Parent Title (English): | ChemBioChem |
Publisher: | Wiley-VCH |
Place of publication: | Weinheim |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2022/04/29 |
Date of first Publication: | 2022/01/26 |
Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
Tag: | Brilliant Black BN; NADH regeneration systems; azoreductases; diazo dyes; fusion proteins |
Volume: | 23 |
Issue: | 6, Article e202100643 |
First Page: | e202100643-1 |
Last Page: | e202100643-10 |
Institutes/Facilities: | Lehrstuhl für Pflanzenphysiologie, Nachwuchsgruppe Mikrobielle Biotechnologie |
Lehrstuhl für Pflanzenphysiologie | |
Nachwuchsgruppe Mikrobielle Biotechnologie | |
open_access (DINI-Set): | open_access |
faculties: | Fakultät für Biologie und Biotechnologie |
Licence (English): | Creative Commons - CC BY-NC-ND 4.0 - Attribution-NonCommercial-NoDerivatives 4.0 International |