谷氨酸棒状杆菌高丝氨酸脱氢酶单突变体酶学性质表征

Q517; 通过定点突变技术,提高高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)的催化活性,减少通路中代谢产物对其产生的反馈抑制和阻遏作用.对HSD与底物高丝氨酸分子进行对接,通过其空间结构分析,选择Asp61和Gly25两个关键位点进行定点饱和突变,通过酶活力筛选,发现突变体A61L和G25G较野生型(wild type,WT)酶活力显著提高.对这两个突变体进行酶动力学和酶学性质研究,结果显示:相较于WT,A61L和G25G的Km值降低,底物亲和力增强,酶活力分别提高到1.21、1.35倍;n值减小,正协同性增加;A61L和G25G最适温度与WT相同均为40 ℃;...

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Published in食品科学 Vol. 44; no. 18; pp. 110 - 116
Main Authors 江泽沅, 柳羽哲, 高欣, 曾琦, 闵伟红
Format Magazine Article
LanguageChinese
Published 吉林农业大学食品科学与工程学院,小麦和玉米深加工国家工程实验室,吉林长春 130118 2023
Subjects
homoserine dehydrogenase
enzymatic properties
酶学性质
高丝氨酸脱氢酶
Corynebacterium glutamicum
定点突变
酶动力学
谷氨酸棒状杆菌
Online AccessGet full text
ISSN1002-6630
DOI10.7506/spkx1002-6630-20220830-353

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Abstract Q517; 通过定点突变技术,提高高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)的催化活性,减少通路中代谢产物对其产生的反馈抑制和阻遏作用.对HSD与底物高丝氨酸分子进行对接,通过其空间结构分析,选择Asp61和Gly25两个关键位点进行定点饱和突变,通过酶活力筛选,发现突变体A61L和G25G较野生型(wild type,WT)酶活力显著提高.对这两个突变体进行酶动力学和酶学性质研究,结果显示:相较于WT,A61L和G25G的Km值降低,底物亲和力增强,酶活力分别提高到1.21、1.35倍;n值减小,正协同性增加;A61L和G25G最适温度与WT相同均为40 ℃;A61L最适pH值与WT相同为8.0,G25G最适pH值为8.5较WT提高;A61L和G25G酶活力半衰期较WT分别延长1h和减少0.5 h;低浓度K+、Mg2+、Ca2+对突变体和WT有激活作用;不同体积分数甲醇、乙醇、乙腈和二甲基亚砜对突变体和WT有明显抑制作用;在1～25 mmol/L抑制剂浓度下,突变体受抑制作用较WT明显减弱.本研究获得酶活力提高、别构抑制减弱的突变体G25G和A61L,为优化HSD合成代谢途径和构建高产蛋氨酸、苏氨酸和异亮氨酸菌株提供了参考.
AbstractList Q517; 通过定点突变技术,提高高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)的催化活性,减少通路中代谢产物对其产生的反馈抑制和阻遏作用.对HSD与底物高丝氨酸分子进行对接,通过其空间结构分析,选择Asp61和Gly25两个关键位点进行定点饱和突变,通过酶活力筛选,发现突变体A61L和G25G较野生型(wild type,WT)酶活力显著提高.对这两个突变体进行酶动力学和酶学性质研究,结果显示:相较于WT,A61L和G25G的Km值降低,底物亲和力增强,酶活力分别提高到1.21、1.35倍;n值减小,正协同性增加;A61L和G25G最适温度与WT相同均为40 ℃;A61L最适pH值与WT相同为8.0,G25G最适pH值为8.5较WT提高;A61L和G25G酶活力半衰期较WT分别延长1h和减少0.5 h;低浓度K+、Mg2+、Ca2+对突变体和WT有激活作用;不同体积分数甲醇、乙醇、乙腈和二甲基亚砜对突变体和WT有明显抑制作用;在1～25 mmol/L抑制剂浓度下,突变体受抑制作用较WT明显减弱.本研究获得酶活力提高、别构抑制减弱的突变体G25G和A61L,为优化HSD合成代谢途径和构建高产蛋氨酸、苏氨酸和异亮氨酸菌株提供了参考.
Author 高欣
江泽沅
曾琦
闵伟红
柳羽哲
AuthorAffiliation 吉林农业大学食品科学与工程学院,小麦和玉米深加工国家工程实验室,吉林长春 130118
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GAO Xin
JIANG Zeyuan
MIN Weihong
ZENG Qi
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DocumentTitle_FL Characterization of Enzymatic Properties of Single Mutants of Homoserine Dehydrogenase from Corynebacterium glutamicum
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Issue 18
Keywords homoserine dehydrogenase
enzymatic properties
酶学性质
高丝氨酸脱氢酶
Corynebacterium glutamicum
定点突变
酶动力学
谷氨酸棒状杆菌
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Snippet Q517; 通过定点突变技术,提高高丝氨酸脱氢酶(homoserine dehydrogenase,HSD)的催化活性,减少通路中代谢产物对其产生的反馈抑制和阻遏作用.对HSD与底物高丝氨酸分子进行对...
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StartPage 110
Title 谷氨酸棒状杆菌高丝氨酸脱氢酶单突变体酶学性质表征
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