Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, People's Republic of China.
Mol Biol Rep. 2014 Jan;41(1):497-504. doi: 10.1007/s11033-013-2885-8. Epub 2013 Dec 5.
Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to L-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing L-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K m value of SlFumC for L-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s(-1)) to 8.0 (620 s(-1)) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for L-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of L-malate.
延胡索酸酶是一种关键酶，可催化三羧酸循环中反式乌头酸到 L-苹果酸的可逆水合作用。该反应已广泛应用于生产 L-苹果酸的工业应用中。在这项研究中，从链霉菌 lividans TK54（slFumC）中克隆并表达了一种融合蛋白（SlFumC）的延胡索酸酶 C 基因。SlFumC 的分子量通过 SDS-PAGE 测定约为 49 kDa。动力学研究表明，SlFumC 对 L-苹果酸的 K m 值在 pH 6.5（6.7 ± 0.81 mM）增加约 8.5 倍至 8.0（57.0 ± 1.12 mM），高于某些已知的延胡索酸酶。在 pH 6.5（65 s(-1))增加约 9.5 倍至 8.0（620 s(-1))时，催化效率（k cat）和比活性分别增加，并且从 pH 6.5 时的 79 U/mg 增加到 pH 8.0 时的 752 U/mg。因此，SlFumC 可以通过增加 pH 值来获得较强的催化能力，从而部分补偿底物亲和力的丧失。该酶还表现出底物抑制现象，这是 pH 值依赖性的。随着磷酸盐浓度的增加，SlFumC 的比活性逐渐增强。然而，在高浓度的磷酸盐离子下没有观察到抑制作用，这与其他 II 类延胡索酸酶明显不同。在工业过程中，L-苹果酸生产的反应温度通常在 40 到 60°C 之间。重组 SlFumC 在 45°C 下显示出最大活性，在 40°C 下孵育 48 小时后仍保持原始活性的 85%以上，这比来自链霉菌的其他延胡索酸酶更耐热，使其成为用于工业生产 L-苹果酸的有效酶。
