The NAD⁺-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme’s thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.

来自博伊假丝酵母的NAD ⁺依赖性甲酸脱氢酶（FDH; EC 1.2.1.2）（Cbo FDH）已广泛用于依赖NAD（H）的工业生物催化以及由二氧化碳生产可再生燃料和化学物质。在本工作中，通过定点诱变研究了氨基酸残基Phe285，Gln287和His311对结构稳定性的影响。克隆了野生型和突变型酶（Gln287Glu，His311Gln和Phe285Thr / His311Gln），并在大肠杆菌中表达。圆二色性（CD）光谱用于确定每个突变对热稳定性的影响。结果表明，Phe285，Gln287和His311在增强酶的热稳定性中起决定性作用。野生型和突变型酶Gln287Glu，His311Gln和Phe285Thr / His311Gln的解链温度分别为64、70、77和73°C。还研究了pH和温度对野生型和突变型酶催化活性的影响。有趣的是，突变酶His311Gln在基本pH范围（1个pH单位）下显示出最适pH值的较大变化，并且最适温度（25°C）大幅增加。本工作支持保守的残基Phe285，Gln287和His311的多功能作用，并进一步强调了它们作为蛋白质工程研究目标的关键作用。