Biochemical characterization of an azoreductase from Rhodococcus opacus 1CP possessing methyl red degradation ability

Qi, Jingxian; Schlömann, Michael; Tischler, Dirk

doi:10.1016/j.molcatb.2016.04.012

Cited by 44 publications

(42 citation statements)

References 43 publications

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“…Based on flavin cofactor content and nicotinamide dependence or preference, azoreductase superfamily can be classified into five major groups-(1) flavin-containing NADH-dependent azoreductase (Matsumoto et al 2010;Liu et al 2007), (2) flavin-containing NADPH-dependent azoreductase (Chen et al 2005;Wang et al 2007), (3) flavin-containing NAD(P)H-dependent azoreductase (Qi et al 2016(Qi et al , 2017aOturkar et al 2013), (4) flavinfree NAD(P)H-dependent azoreductase (Blumel et al 2002;Chen et al 2010;Misal et al 2011Misal et al , 2014Misal et al , 2015, and (5) flavin-containing NAD(P)H-dependent quinone Fig. 1 Azoreduction mechanism of amaranth dye by azoreductase enzyme: The UV-Visible spectrum demonstrating the complete reduction of amaranth dye in 24 h of incubation with purified azoreductase.…”

Section: Azoreductase Classificationmentioning

confidence: 99%

Azoreductase: a key player of xenobiotic metabolism

Misal

Gawai

2018

Bioresour. Bioprocess.

135

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Azoreductases are diverse flavoenzymes widely present among microorganisms and higher eukaryotes. They are mainly involved in the biotransformation and detoxification of azo dyes, nitro-aromatic, and azoic drugs. Reduction of azo bond and reductive activation of pro-drugs at initial level is a crucial stage in degradation and detoxification mechanisms. Using azoreductase-based microbial enzyme systems that are biologically accepted and ecofriendly demonstrated complete degradation of azo dyes. Azoreductases are flavin-containing or flavin-free group of enzymes, utilizing the nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a reducing equivalent. Azoreductases from anaerobic microorganisms are highly oxygen sensitive, while azoreductases from aerobic microorganisms are usually oxygen insensitive. They have variable pH, temperature stability, and wide substrate specificity. Azo dyes, nitro-aromatic compounds, and quinones are the known substrates of azoreductase. The present review gives an overview of recent developments in the known azoreductase enzymes from different microorganisms, its novel classification scheme, significant characteristics, and their plausible degradation mechanisms.

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Section: Azoreductase Classificationmentioning

confidence: 99%

Azoreductase: a key player of xenobiotic metabolism

Misal

Gawai

2018

Bioresour. Bioprocess.

135

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“…Enzyme production : The previously described expression vector pET_FOYE_01 was checked by sequencing with pET primers and freshly transformed into Escherichia coli BL21(DE3) for gene expression . A single colony was used to inoculate precultures (50 mL) consisting of lysogeny broth (LB) medium [tryptone (10 g L −1 ), yeast extract (5 g L −1 ), NaCl (10 g L −1 )] and appropriate antibiotics [ampicillin (100 μg mL −1 ) and chloramphenicol (50 μg mL −1 )].…”

Section: Methodsmentioning

confidence: 99%

Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

et al. 2020

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Ene‐reductases allow regio‐ and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene‐reductase FOYE‐1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE‐1 to produce valuable compounds. A maximum activity of (319.7±3.2) U mg−1 with NADPH or of (206.7±3.4) U mg−1 with 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) for the reduction of N‐methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE‐1 showed excellent performance in most systems with up to 20 vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200 mL biotransformation of 750 mg (R)‐carvone afforded 495 mg of (2R,5R)‐dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE‐1.

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“…Afterwards, the parental DNA was DpnI digested and the remaining pET16bP construct harboring the mutation was transformed into E. coli BL21 cells for gene expression. Successful mutation of the target genes was proven by sequencing of the plasmid using the pET16-check-fw/pET16-check-rev primer [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

et al. 2018

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Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1) of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

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Biochemical characterization of an azoreductase from Rhodococcus opacus 1CP possessing methyl red degradation ability

Cited by 44 publications

References 43 publications

Azoreductase: a key player of xenobiotic metabolism

Azoreductase: a key player of xenobiotic metabolism

Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

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