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

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. A screening approach for azoreductases was developed, including cell‐free protein synthesis of an enzyme library and a multi‐well microplate assay. Applicability to known and novel enzymes was demonstrated, and preferred co‐substrates for each enzyme were evaluated. Five new and yet uncharacterized azoreductases were identified. Furthermore, it was shown that all azoreductases accept the co‐substrate BNAH as an electron donor.