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 and 4.96±0.25Umg, respectively, whereas with BNAH it displayed 17.01±0.74Umg (following BNAH oxidation) and 7.16±0.06Umg (following MR reduction). Binding of BNAH to AzoRo was determined with a K of 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.

Alternative coenzymes for biocatalysis

“…Interestingly, a non-flavin reductase from Nicotina tabacum (NtDBR) could catalyse the reduction of cinnamaldehyde with mimics BNAH, BAPH, CO 2 NAH and BuNAH, albeit with low conversions [20 ]. An azoreductase AzoRo was used with BNAH, displaying a higher activity for the degradation of the dye methyl red at neutral pH [25].…”

Flavoenzyme‐mediated Regioselective Aromatic Hydroxylation with Coenzyme Biomimetics

“…NCBs are synthetic truncated versions of NAD(P)H with variable substituents (Figure ) . The biocatalytic applications of NCBs were demonstrated with dehydrogenases, ene reductases, azoreductases, cytochrome P450s, NADH oxidases, and two‐component flavoprotein monooxygenases (group E and F) . NCBs are attractive to study the impact of redox potential and mode of coenzyme binding in oxidoreductases in order to elucidate their mechanism, and to further apply these enzymes in large scale reactions …”

Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

“…Synthetic nicotinamide coenzyme biomimetics (NCBs) have been shown to replace NAD(P)H in flavin-dependent enzymes such as nitroreductase and NAD(P)H quinone oxidoreductase [5][6][7], a cytochrome P450 BM3 variant [8], ene-reductases [9][10][11][12][13][14][15][16], and styrene monooxygenase [17]. In all cases described, a flavin was involved as an electron mediator [18][19][20][21][22].…”

“…Synthetic nicotinamide coenzyme biomimetics (NCBs) have been shown to replace NAD(P)H in flavin-dependent enzymes such as nitroreductase and NAD(P)H quinone oxidoreductase [5-7], a cytochrome P450 BM3 variant [8], ene-reductases [9][10][11][12][13][14][15][16], and styrene monooxygenase [17]. In all cases described, a flavin was involved as an electron mediator [18][19][20][21][22].Jones pioneered the use of synthetic cofactor analogues with horse liver ADH (HLADH) [23][24][25][26], followed by Fish [27] and others [28,29]. A dehydrogenase from the aldo-reductase superfamily from Pyrococcus furiosus (AdhD) was engineered to increase catalytic efficiency towards nicotinamide mononucleotide (NMN, Figure 1A) [30,31].…”