title: Changing the electron donor improves azoreductase dye degrading activity at neutral pH.
abstract: The oxygen-insensitive azoreductase AzoRo originating from Rhodococcus opacus 1CP was found to be most active at low pH (ca. 4) and high temperature (ca. 50°C). AzoRo is not an efficient biocatalyst when used at low pH due to stability problems. To overcome this issue, we discovered that AzoRo accepts an alternative electron donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), which allows fast turnover at neutral pH. In order to screen this nicotinamide coenzyme mimic as a source of electrons, AzoRo-catalysed reactions were run under neutral conditions, under which typically slow rates are observed with NADH. For the reduction of 1 azo bond by azoreductases 2mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49±0.14Umg-1and 4.96±0.25Umg-1, respectively, whereas with BNAH it displayed 17.01±0.74Umg-1(following BNAH oxidation) and 7.16±0.06Umg-1(following MR reduction). Binding of BNAH to AzoRo was determined with a Kmof 18.75±2.45μM (BNAH oxidation) and 12.45±0.47μM (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6μg of purified AzoRo to convert 2.96μmol of MR (total reaction volume: 40ml) within a 1h reaction.
authors.affiliation: Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Str. 29, Freiberg 09599, Germany. Electronic address: jingxianqi@gmail.com.
authors.forename: Jingxian
authors.initials: J
authors.lastname: Qi
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Caroline E
authors.initials: CE
authors.lastname: Paul
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Frank
authors.initials: F
authors.lastname: Hollmann
authors.affiliation: Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Str. 29, Freiberg 09599, Germany. Electronic address: dirk-tischler@email.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tischler
mesh_terms: D001391:Azo Compounds / Q000378:metabolism*; D055162:Biocatalysis; D003067:Coenzymes / Q000378:metabolism; D004396:Coloring Agents / Q000378:metabolism*; D004579:Electron Transport; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009601:Nitroreductases; D010084:Oxidation-Reduction; D012240:Rhodococcus / Q000201:enzymology
chemical_list: C445701:1-benzyl-1,4-dihydronicotinamide; D001391:Azo Compounds; D003067:Coenzymes; D004396:Coloring Agents; D009243:NAD; C008492:methyl red; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase
keywords: 1-benzyl-1,4-dihydronicotinamide; Azo dyes; Azoreductase; Methyl red degradation; Nicotinamide cofactor mimics; Rhodococcus