Qi Jingxian, Paul Caroline E, Hollmann Frank, Tischler Dirk
Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Str. 29, Freiberg 09599, Germany.
Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
Enzyme Microb Technol. 2017 May;100:17-19. doi: 10.1016/j.enzmictec.2017.02.003. Epub 2017 Feb 9.
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.
源自不透明红球菌1CP的对氧不敏感的偶氮还原酶AzoRo在低pH值（约4）和高温（约50°C）下活性最高。由于稳定性问题，AzoRo在低pH值下使用时不是一种高效的生物催化剂。为了克服这个问题，我们发现AzoRo接受替代电子供体1-苄基-1,4-二氢烟酰胺（BNAH），这使得它在中性pH值下能够快速周转。为了筛选这种烟酰胺辅酶类似物作为电子来源，在中性条件下进行了AzoRo催化的反应，在这种条件下，通常观察到NADH的反应速率较慢。偶氮还原酶还原1个偶氮键需要2摩尔烟酰胺辅酶。AzoRo对甲基红（MR）的还原活性，使用NADH时为5.49±0.14Umg，使用NADPH时为4.96±0.25Umg，而使用BNAH时，其活性为17.01±0.74Umg（BNAH氧化后）和7.16±0.06Umg（MR还原后）。测定BNAH与AzoRo的结合常数，BNAH氧化时K为18.75±2.45μM，MR还原时K为12.45±0.47μM。为了证明该系统的适用性，使用78.6μg纯化的AzoRo在1小时反应内转化2.96μmol的MR（总反应体积：40ml）进行了放大反应。
