Microbial Biotechnology, Faculty of Biology and Biotechnology, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780, Bochum, Germany.
Chembiochem. 2022 Mar 18;23(6):e202100643. doi: 10.1002/cbic.202100643. Epub 2022 Feb 10.
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 and FDH ) 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 +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDH +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting 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.
偶氮还原酶还原偶氮染料需要 NAD(P)H，但 NAD(P)H 的高成本限制了其应用。甲酸脱氢酶 (FDH) 可以使 NAD(P) 循环再生，因此，这两种生物催化剂的融合似乎很有前途。本研究探讨了融合蛋白中涉及节杆菌 1CP 偶氮还原酶 (AzoRo) 和毕赤酵母 FDH (FDH 和 FDH) 的变化，这些融合蛋白的不同位置带有 His 标签作为连接物。位置会影响酶活性，因为当 AzoRo 位于融合蛋白的 N 端时，其活性降低了 20 倍。FDH+AzoRo 是最活跃的构建体，并进一步进行了表征。与亲本酶相比，FDH+AzoRo 的酶活性降低，但显示出改善的底物范围，可接受更大的染料。此外，pH 对融合蛋白的稳定性和活性有影响，因为在 pH 6（FDH 的最适 pH 以下）时，染料还原降低了 50%以上，这可能归因于 AzoRo 部分 NADH 供应受损。
