Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for malate production
Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for malate production
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鉴定来自浅青紫链霉菌 TK54 的新型延胡索酸酶 C，作为苹果酸生产的良好候选者
DOI：
10.1007/s11033-013-2885-8
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发表时间：
2014-01-01
影响因子：
2.8
通讯作者：
Wang, Wen
中科院分区：
文献类型：
--
作者：
Su, Rui-Rui;Wang, Ao;Wang, Wen
富马酸酶是三羧酸循环中催化富马酸可逆水合为l-苹果酸的关键酶。该反应已广泛用于生产L-苹果酸的工业应用。本研究从变铅青链霉菌TK 54中克隆了一个丝氨酸蛋白酶C基因（slFumC），并在大肠杆菌中表达了融合蛋白（SlFumC）。经SDS-PAGE测定，SlFumC的分子量约为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 +/-A0.81mM）至8.0（57.0 +/-A1.12mM）下增加约8.5倍，这高于一些已知的脱氢酶。催化效率（k（cat））和比活性分别从pH6.5的79 U/mg增加到pH8.0的752 U/mg，从pH6.5的65 s（-1）增加到pH8.0的8.0（620 s（-1）），增加了9.5倍。因此，SlFumC可以通过增加pH以部分补偿底物亲和力的损失来获得强催化能力。该酶还表现出底物抑制现象，这是pH依赖性的。SlFumC的比活性随着磷酸盐浓度的增加而逐渐增强。然而，在高浓度的磷酸根离子下没有观察到抑制，这与其他II类酶的情况明显不同。在工业过程中，生产l-苹果酸的反应温度通常设定在40至60摄氏度之间。重组SlFumC在45 ℃时酶活最高，40 ℃培养48 h后酶活仍保持在85%以上，比链霉菌的其他产酶酶更耐热，是一种工业生产L-苹果酸的高效酶。
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 +/- A 0.81 mM) to 8.0 (57.0 +/- A 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 A degrees C. The recombinant SlFumC displayed maximal activity at 45 A degrees C and remained over 85 % of original activity after 48 h incubation at 40 A degrees 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.