Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase

Ngo, Anna; Qi, Jingxian; Juric, Cindy; Bento, Isabel; Tischler, Dirk

doi:10.1016/j.abb.2022.109123

Cited by 11 publications

(6 citation statements)

References 65 publications

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“…Both enzymes were individually produced as N ‐terminal His 10 ‐tagged proteins demonstrating the functionality of this small fusion tag [11,28,29] . Several studies now demonstrate the importance of finding the right linkers when fusing two proteins [21,30,31] .…”

Section: Resultsmentioning

confidence: 99%

“…This AzoRo operates better on acidic pH and the enzyme activity drops from neutral to basic pH. [ 11 , 28 , 29 ]…”

Section: Resultsmentioning

confidence: 99%

“…Meanwhile, the azoreductase AzoRo used from Rhodococcus opacus 1CP shows a limited substrate scope, only accepting (1) (Figure 2) as a substrate but can use both NADH and NADPH as electron donor (co‐substrate) to reduce (1) . This AzoRo operates better on acidic pH and the enzyme activity drops from neutral to basic pH [11,28,29] …”

Section: Resultsmentioning

confidence: 99%

“…This AzoRo operates better on acidic pH and the enzyme activity drops from neutral to basic pH. [11,28,29] Both enzymes were individually produced as N-terminal His 10 -tagged proteins demonstrating the functionality of this small fusion tag. [11,28,29] Several studies now demonstrate the importance of finding the right linkers when fusing two proteins.…”

Section: Fusion Protein Construction Expression Optimization and Prot...mentioning

confidence: 99%

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Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase

et al. 2022

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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 Rhodococcus opacus 1CP and FDH (FDH C23S and FDH C23SD195QY196H ) of 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 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 scopeaccepting 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.

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Section: Resultsmentioning

confidence: 99%

“…This AzoRo operates better on acidic pH and the enzyme activity drops from neutral to basic pH. [ 11 , 28 , 29 ]…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Fusion Protein Construction Expression Optimization and Prot...mentioning

confidence: 99%

See 2 more Smart Citations

Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase

et al. 2022

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“…Some have even shown the potential to completely degrade azo dyes. They are also more environmentally friendly and produce less sludge [117][118][119][120][121].…”

Section: Biological Treatment Of Dyes Using Bacteriamentioning

confidence: 99%

Microbial Degradation of Azo Dyes: Approaches and Prospects for a Hazard-Free Conversion by Microorganisms

Ngo

Tischler

2022

IJERPH

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Azo dyes have become a staple in various industries, as colors play an important role in consumer choices. However, these dyes pose various health and environmental risks. Although different wastewater treatments are available, the search for more eco-friendly options persists. Bioremediation utilizing microorganisms has been of great interest to researchers and industries, as the transition toward greener solutions has become more in demand through the years. This review tackles the health and environmental repercussions of azo dyes and its metabolites, available biological approaches to eliminate such dyes from the environment with a focus on the use of different microorganisms, enzymes that are involved in the degradation of azo dyes, and recent trends that could be applied for the treatment of azo dyes.

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Cell‐Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor

et al. 2022

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Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.

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Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase

Cited by 11 publications

References 65 publications

Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase

Improving Biocatalytic Properties of an Azoreductase via the N‐Terminal Fusion of Formate Dehydrogenase

Microbial Degradation of Azo Dyes: Approaches and Prospects for a Hazard-Free Conversion by Microorganisms

Cell‐Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor

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