摘要
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(wildtype,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0.5h;低浓度K^(+)、Mg^(2+)、Ca^(2+)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1~25mmol/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。
Site-directed mutagenesis was used to improve the catalytic activity of homoserine dehydrogenase(HSD)to reduce its feedback inhibition and repression by metabolites in the pathway.HSD was docked with the substrate homoserine,and its spatial structure was analyzed.Two key sites,Gly25 and Asp61,were selected for site-directed saturation mutation.The activity screening showed that the mutants A61L and G25G had significantly increased enzyme activity when compared to the wild type(WT).The kinetics and enzymatic properties of these two mutants were studied.It was found that compared to the WT enzyme,the Km values of G25G and A61L decreased,the substrate affinity increased,and the enzyme activity increased by 1.21 and 1.35 times,respectively;the n value decreased,and the positive synergy increased.The optimum temperature for A61L and G25G was 40℃,the same as that for WT;the optimum pH for A61L and WT was 8.0,which was lower than that(8.5)for G25G.The half-lives of A61L and G25G were 1 h longer and 0.5 h shorter than that of WT,respectively.Low concentrations of K^(+),Mg^(2+),and Ca^(2+)could activate the mutants and WT,while different concentrations of methanol,ethanol,acetonitrile and dimethyl sulfoxide had significantly inhibitory effects on the mutants and WT.At inhibitor concentrations of 1-25 mmol/L,the inhibitory effect was significantly weaker on the mutants than on WT.The mutants G25G and A61L showed improved enzyme activity and weakened allosteric inhibition.This study provides a reference for optimizing the biosynthetic pathway of HSD and constructing strains capable of producing high yield of methionine,threonine and isoleucine.
作者
江泽沅
柳羽哲
高欣
曾琦
闵伟红
JIANG Zeyuan;LIU Yuzhe;GAO Xin;ZENG Qi;MIN Weihong(National Engineering Laboratory for Wheat and Corn Further Processing,College of Food Science and Engineering,Jilin Agricultural University,Changchun 130118,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2023年第18期110-116,共7页
Food Science
基金
“十四五”国家重点研发计划重点专项(2021YFD2101000,2021YFD2101002)
国家自然科学基金面上项目(31771967)。
