Abstract：
NAD+ -dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s-1 at 25°C compared to 7.7 ± 1.0 s-1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2 O2 inactivation, showing a 200-fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2 O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H2 O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2 O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.
摘要：
NAD+依赖性甲酸脱氢酶(FDH)催化甲酸和NAD+的转化以产生二氧化碳和NADH。该反应在生物技术上很重要，因为FDH广泛用于各种酶合成中的NADH再生。然而,这种多功能酶在工业应用中的主要缺点是活性低，要求其大量利用以达到最佳的工艺条件。这里,与来自假单胞菌属的FDH相比，来自单一芽孢杆菌（BsFDH）的FDH的生化和催化特性得到了表征。101(PsFDH),在各种生物催化反应中常用的FDH。数据显示，BsFDH具有较高的甲酸氧化活性，在25°C时的kcat值为15.3±1.9s-1，而PsFDH为7.7±1.0s-1。在最佳温度（60°C）下，BsFDH表现出比PsFDH高6倍的活性。BsFDH表现出更高的pH稳定性和对叠氮化钠和H2O2失活的优异耐受性，显示与PsFDH相比，叠氮化物抑制的Ki值高200倍，并且在0.5％H2O2存在下保持稳定。BsFDH作为辅因子再生系统的应用，通过HadA的反应对4-硝基苯酚进行脱毒，证明了其产生H2O2副产物。使用BsFDH的生物催化级联表现出明显优异的转化活性,因为该系统良好地耐受H2O2。总之,数据表明，BsFDH是一种稳健的酶，适合未来在工业生物技术中的应用。
