TY - JOUR
T1 - Changing the electron donor improves azoreductase dye degrading activity at neutral pH
AU - Qi, Jingxian
AU - Paul, Caroline E.
AU - Hollmann, Frank
AU - Tischler, Dirk
N1 - Accepted Author Manuscript
PY - 2017
Y1 - 2017
N2 - 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 2 mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49 ± 0.14 U mg−1 and 4.96 ± 0.25 U mg−1, respectively, whereas with BNAH it displayed 17.01 ± 0.74 U mg−1 (following BNAH oxidation) and 7.16 ± 0.06 U mg−1 (following MR reduction). Binding of BNAH to AzoRo was determined with a Km of 18.75 ± 2.45 μM (BNAH oxidation) and 12.45 ± 0.47 μM (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6 μg of purified AzoRo to convert 2.96 μmol of MR (total reaction volume: 40 ml) within a 1 h reaction.
AB - 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 2 mol nicotinamide coenzyme are needed. AzoRo displayed Methyl Red (MR) reduction activities with NADH and NADPH of 5.49 ± 0.14 U mg−1 and 4.96 ± 0.25 U mg−1, respectively, whereas with BNAH it displayed 17.01 ± 0.74 U mg−1 (following BNAH oxidation) and 7.16 ± 0.06 U mg−1 (following MR reduction). Binding of BNAH to AzoRo was determined with a Km of 18.75 ± 2.45 μM (BNAH oxidation) and 12.45 ± 0.47 μM (MR reduction). In order to show applicability of this system an upscaled reaction was performed using 78.6 μg of purified AzoRo to convert 2.96 μmol of MR (total reaction volume: 40 ml) within a 1 h reaction.
KW - 1-benzyl-1,4-dihydronicotinamide
KW - Azo dyes
KW - Azoreductase
KW - Methyl red degradation
KW - Nicotinamide cofactor mimics
KW - Rhodococcus
UR - http://resolver.tudelft.nl/uuid:023e0c18-7509-4709-b2bf-e5edd58c2fe5
UR - http://www.scopus.com/inward/record.url?scp=85012155335&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2017.02.003
DO - 10.1016/j.enzmictec.2017.02.003
M3 - Article
AN - SCOPUS:85012155335
SN - 0141-0229
VL - 100
SP - 17
EP - 19
JO - Enzyme and Microbial Technology: biotechnology research and reviews
JF - Enzyme and Microbial Technology: biotechnology research and reviews
ER -