Abstract：
Flavin reductases play a vital role in catalyzing the reduction of flavin through NADH or NADPH oxidation. The gene encoding flavin reductase from the thermophilic bacterium Geobacillus mahadii Geo-05 (GMHpaC) was cloned, overexpressed in Escherichia coli BL21 (DE3) pLysS, and purified to homogeneity. The purified recombinant GMHpaC (Class II) contains chromogenic cofactors, evidenced by maximal absorbance peaks at 370 nm and 460 nm. GMHpaC stands out as the most thermostable and pH-tolerant flavin reductase reported to date, retaining up to 95 % catalytic activity after incubation at 70 °C for 30 min and maintaining over 80 % activity within a pH range of 2-12 for 30 min. Furthermore, GMHpaC\'s catalytic activity increases by 52 % with FMN as a co-factor compared to FAD and riboflavin. GMHpaC, coupled with 4-hydroxyphenylacetate-3-monooxygenase (GMHpaB) from G. mahadii Geo-05, enhances the hydroxylation of 4-hydroxyphenylacetate (HPA) by 85 %. The modeled structure of GMHpaC reveals relatively conserved flavin and NADH binding sites. Modeling and docking studies shed light on structural features and amino acid substitutions that determine GMHpaC\'s co-factor specificity. The remarkable thermostability, high catalytic activity, and general stability exhibited by GMHpaC position it as a promising enzyme candidate for various industrial applications.
摘要：
黄素还原酶在通过NADH或NADPH氧化催化黄素还原中起着至关重要的作用。克隆了来自嗜热细菌mahadiiGeo-05（GMHpaC）的编码黄素还原酶的基因，在大肠杆菌BL21（DE3）pLysS中过表达，并纯化至同质。纯化的重组GMHpaC（II类）含有显色辅因子，由370nm和460nm处的最大吸收峰证明。GMHpaC是迄今为止报道的最耐热和耐pH的黄素还原酶，在70°C孵育30分钟后保留高达95％的催化活性，并在2-12的pH范围内保持超过80％的活性30分钟。此外，与FAD和核黄素相比,以FMN作为辅因子的GMHpaC的催化活性增加了52%。GMHpaC,与来自G.mahadiiGeo-05的4-羟基苯基乙酸酯-3-单加氧酶（GMHpaB）偶联，可将4-羟基苯基乙酸酯（HPA）的羟基化提高85％。GMHpaC的建模结构揭示了相对保守的黄素和NADH结合位点。建模和对接研究揭示了确定GMHpaC辅因子特异性的结构特征和氨基酸取代。非凡的热稳定性，高催化活性，GMHpaC表现出的一般稳定性将其定位为各种工业应用的有希望的候选酶。
