灵芝多糖糖供体合成途径中关键酶的异源表达及其酶学性质

Heterologous expression and characterization of the key enzymes involved in sugar donor synthesis of polysaccharide in Ganoderma lucidum

摘要:

【背景】灵芝多糖(Ganoderma lucidum polysaccharide，GLP)是一种具有多种生物活性的大分子物质，已有研究者通过发酵调控优化或菌种改良使灵芝多糖产量得到一定的提高。但由于灵芝多糖糖供体合成途径并未完全明晰，其中关键酶特性解析也不完善，使得灵芝多糖产量的大幅度提高仍存在瓶颈。【目的】通过异源表达大量制备灵芝中含量较少的磷酸葡萄糖变位酶(Phosphoglucomutase，PGM)、UDP-葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase，UGPG)和磷酸甘露糖异构酶(Phosphomannose isomerase，PMI)，并分别探究和比较其酶学性质，深入了解灵芝多糖的糖供体合成途径中关键酶的特性信息，为灵芝多糖合成发酵策略的高效制定提供依据。【方法】以灵芝菌株CGMCC 5.26的cDNA为模板，克隆得到关键酶基因gl-pgm、gl-ugpg和gl-pmi，分别在E. coli BL21(DE3)中诱导表达，产物通过Co-NTA树脂纯化后进行酶学性质研究。【结果】纯化酶GLpgm、GLugpg和GLpmi均在E. coli中实现大量表达。GLpgm的最适反应pH为8.5，GLugpg和GLpmi的最适pH同为7.5；GLpgm、GLugpg和GLpmi最适反应温度依次为35、40和30 °C；1 mmol/L的Ag+和Cu2+对3种酶均具有强烈抑制作用，Mn2+、Mg2+对GLpgm和GLpmi均有激活作用，其中Mn2+对GLpgm的激活作用高达2.7倍。GLpgm、GLugpg和GLpmi的kcat/Km值分别为196.08、818.60和1 105.22 mmol/(L·s)。【结论】在最适反应pH、温度及金属离子作用方面，GLpgm、GLugpg和GLpmi与植物及真菌来源的这3种酶较为相似，对底物的催化效率相对其他来源的酶高，为基于灵芝多糖合成途径的调控提供了更完善的信息。

Abstract:

[Background] Ganoderma lucidum polysaccharide (GLP), as a macromolecular substance with various biological activities, has attracted a lot of attention in recent years. Some researchers have improved the yield of GLP through optimization of fermentation regulation or strain improvement for G. lucidum. However, it is a fact that the biosynthesis pathway of GLP is still unclear completely, and the understanding about characteristics of key enzymes involved in GLP synthesis are insufficient. Thus, there is a bottleneck on substantially improving the yield of GLP. [Objective] Through heterologous expression of the genes of phosphoglucomutase (PGM), UDP-glucose pyrophosphorylase (UGPG), and phosphomannose isomerase (PMI) cloned from G. lucidum in E. coli, decent amounts of the purified enzymes were obtained. We characterized their enzymatic properties and compared them with other sources, to provide insights into the characteristic information of them, and build an efficient strategy for the synthetic of GLP on fermentation. [Methods] Genes of gl-pgm, gl-ugpg and gl-pmi were cloned from G. lucidum strain CGMCC 5.26 and expressed in E. coli, the recombinases were purified with Co-NTA resin respectively. Then, the enzymatic properties of the purified enzymes were studied. [Results] GLpgm, GLugpg and GLpmi, were successfully expressed in E. coli. The optimum reaction pH of GLpgm was 8.5, and it was 7.5 for GLugpg and GLpmi. The optimum reaction temperature of GLpgm, GLugpg and GLpmi were 35, 40 and 30 °C, respectively. For them, 1 mmol/L of Ag+ and Cu2+ had strong inhibitory effects, while Mn2+ and Mg2+ had activation effects on GLpgm and GLpmi, especially the use of Mn2+ could increase the GLpgm activity by 2.7 times. The kcat/Km values of GLpgm, GLugpg and GLpmi were 196.08, 818.60 and 1 105.22 mmol/(L·s), respectively. [Conclusion] GLpgm, GLugpg and GLpmi were similar to the sources of plant and other fungi in terms of optimum pH, temperature and metal ions action, while the catalytic efficiency was relatively higher than them. Their properties could provide more informantion for the regulation of GLP based on it’s pathway.