State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.
Appl Microbiol Biotechnol. 2014 Feb;98(4):1621-30. doi: 10.1007/s00253-013-4996-5. Epub 2013 May 29.
The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s(-1), and 2.83 mM(-1) s(-1), respectively. The K m for NAD(+) was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.
聚多曲霉（Ogataea parapolymorpha）DL-1 甲酸脱氢酶（FDH）的 cDNA 基因被克隆并在大肠杆菌中表达。重组酶通过镍亲和层析进行纯化，并被鉴定为由两个相同亚基组成的同源二聚体，每个亚基约 40 kDa。该酶表现出广泛的催化活性 pH 最优值，从 pH 6.0 到 7.0。它的最适温度相对较高，在 40、50、55 和 60°C 下分别暴露 4 小时后，保留了初始活性的 93%、88%、83%和 71%，表明该酶具有良好的热稳定性。此外，该酶还表现出对有机溶剂如二甲基亚砜、正丁醇和正己烷的显著耐受能力。该酶对底物甲酸钠的米氏常数（K(m)）、转换数（k(cat)）和催化效率（k(cat)/K(m)）值分别估计为 0.82 mM、2.32 s(-1)和 2.83 mM(-1) s(-1)。对 NAD(+)的 K m 值为 83 μM。由于其广泛的 pH 最优值、良好的热稳定性和高有机溶剂耐受性，聚多曲霉 FDH 可能是一种很好的 NADH 再生催化剂候选物。
