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Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for L-malate production

Su, Rui-Rui, Wang, Ao, Hou, Song-Tao, Gao, Peng, Zhu, Guo-Ping, Wang, Wen
Molecular biology reports 2014 v.41 no.1 pp. 497-504
Escherichia coli, Streptomyces lividans, catalytic activity, genes, industrial applications, molecular weight, pH, polyacrylamide gel electrophoresis, temperature, thermal stability, tricarboxylic acid cycle
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 Kₘvalue 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 (kcₐₜ) 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.