灵芝来源UDP-葡萄糖脱氢酶的发酵优化及酶学性质探究
首发时间:2022-11-17
摘要:UDP-葡萄糖脱氢酶(EC 1.1.1.22)可催化UDP-葡萄糖(UDP-Glc)生成UDP-葡糖醛酸(UDP-GlcA),UDP-GlcA不仅是灵芝多糖合成中糖供体途径的产物之一,也是UDP-木糖(UDP-Xyl)合成的主要前体。本文对来源于灵芝的UDP-葡萄糖脱氢酶(UGD)在大肠杆菌中进行异源表达、发酵优化以及酶学性质分析,经过对培养基初始pH、发酵温度、诱导剂添加策略及诱导时间等展开优化,酶产量提高25.4%。酶学性质探究结果表明,灵芝UGD最适反应温度为30℃,最适反应pH为9,Mg2+、Ca2+对灵芝UGD活性有着明显促进作用,其Km值为0.14 mM,反应最大速率Vmax为1428.6 μmol/(minoL)。灵芝UDP-葡萄糖脱氢酶的研究进一步拓展了食药用菌来源UGD的酶学性质信息,同时也为开展灵芝多糖合成机制的研究提供理论支持。
关键词: UDP-葡萄糖脱氢酶 异源表达 酶学性质 发酵优化
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Fermentation optimization of UDP-glucose dehydrogenase from Ganoderma lucidum and its enzymatic property
Abstract:UDP-glucose dehydrogenase (EC 1.1.1.22) can catalyze the conversion of UDP glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is not only a product of sugar donor pathway in Ganoderma lucidum polysaccharide synthesis, but also the main precursor for UDP-xylose synthesis. The encode gene of UDP-glucose dehydrogenase (UGD) were cloned from G. lucidum and expressed in E. coli in this work, by optimizing the fermentation conditions of recombinant E. coli, and analyzing the enzymatic properties of the purified UGD. The enzyme yield was increased by 25.4% after optimizing the initial pH of culture medium, fermentation temperature, inducer addition strategy and induction time. The results of enzymatic properties revealed that the optimum reaction temperature and pH of G. lucidum UGD was 30 ℃ and 9, respectively. In addition, Mg2+ and Ca2+ both greatly promoted the activity of G. lucidum UGD, Km value was 0.14 mM and the maximal reaction rate Vmax was 1428.6 μmol/(minoL). The study on UDP-glucose dehydrogenase from G. lucidum expands the knowledge of UGD enzymatic properties from edible and medicinal fungi, and it also provides a theoretical reference for the study of G. lucidum polysaccharide synthesis mechanism.
Keywords: UDP-glucose dehydrogenase heterologous expression enzymatic properties fermentation optimization
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灵芝来源UDP-葡萄糖脱氢酶的发酵优化及酶学性质探究
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