灵芝UDP-葡萄糖4-差向异构酶酶学性质研究及其应用

Expression and characterization of the UDP-glucose 4-epimerase from Ganoderma lingzhi

【背景】灵芝多糖是灵芝的重要活性物质之一。UDP-葡萄糖4-差向异构酶(UDP-glucose 4-epimerase,UGE,EC 5.1.3.2)是灵芝多糖合成途径中糖供体生成的重要酶,其参与了UDP-葡萄糖与UDP-半乳糖的相互转化,与多糖中半乳糖残基含量密切相关。【目的】通过对来源于灵芝的UGE基因进行异源表达,丰富灵芝多糖糖供体合成途径重要酶的酶学特性信息,深入了解灵芝多糖代谢合成途径。【方法】以灵芝菌株(Ganoderma lingzhi)CGMCC 5.26的cDNA为模板,克隆得到UGE基因GL30389,并在Escherichia coli BL21(DE3)中诱导表达,产物纯化后进行酶学性质、酶动力学、底物专一性及转化率的研究。【结果】灵芝UGE的分子量为45 kDa。最适反应pH值为6.0,在pH 7.0−9.0范围内有较好的稳定性;最适反应温度为30℃,温度在40℃时稳定性最好。Fe^(2+)和Mg^(2+)对UGE有激活作用。以UDP-葡萄糖为底物时,Km为0.824 mmol/L,V_(max)为769.230μmol/(L·min),k_(cat)为1.333 s^(−1), k_(cat)/K_(m)为1.618 L/(mmol·s)。灵芝UGE对D-葡萄糖、半乳糖醛酸及N-乙酰葡萄糖胺有催化活性。通过优化pH、温度、底物与酶的配比、添加金属离子将转化率从16.0%提升至39.4%。【结论】灵芝UGE与植物来源的UGE酶学性质较为相似,其催化效率优于大部分细菌来源的UGE。本研究丰富了灵芝多糖糖供体合成途径重要酶的酶学特性信息,有利于深入了解灵芝多糖代谢合成途径。

[Background]Ganoderma lingzhi polysaccharides(GLP)are the main active component of this mushroom.UDP-glucose 4-epimerase(UGE,EC 5.1.3.2),an essential enzyme in the generation of sugar donor in synthesis of GLP,catalyzes the interconversion of UDP-glucose and UDP-galactose and thus is closely related to the content of galactose residue in GLP.[Objective]Through heterogeneous expression of the UGE gene from G.lingzhi,this study aims to further explore the enzymatic properties of important enzymes in the generation of sugar donor in GLP synthesis and gain a clearer insight into the synthesis pathway of GLP.[Methods]With the cDNA of G.lingzhi CGMCC 5.26 as template,UGE gene was cloned and then expressed in Escherichia coli BL21(DE3).The yielded recombinant enzyme was then purified and the enzymatic properties and kinetics,substrate specificity,and conversion rate of the purified enzymes were investigated.[Results]The recombinant enzyme was 45 kDa.The optimum reaction pH was 6.0,and it was stable at pH 7.0−9.0.The optimum temperature was 30℃,and it was most stable at 40℃.Fe^(2+)and Mg^(2+)could activate UGE.With UDP-glucose as substrate,the enzyme had the Km of 0.824 mmol/L,V_(max) of 769.230μmol/(L·min),kcat of 1.333 s^(−1),and k_(cat)/K_(m) of 1.618 L/(mmol·s).It catalyzed D-glucose,galacturonic acid,and N-acetylglucosamine.The conversion rate was increased from 16.0%to 39.4%at the optimal pH,temperature,and ratio of substrate with the addition of metal ions.[Conclusion]UGE from G.lingzhi has similar enzymatic properties to plants-derived UGE,and the catalytic efficiency was higher than most of the UGEs from bacteria.This study enriches the enzymatic properties of important enzymes involved the sugar donor synthesis in GLP production,which helps enhance the understanding of GLP synthesis pathway.