北京棒杆菌天冬氨酸激酶五突变体的构建及酶学性质表征

Q789; 目的:天冬氨酸激酶(aspartate kinase,AK)是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸(Lys)与苏氨酸(Thr)对AK的协同反馈抑制.方法:在获得的北京棒杆菌四突变体T379N/A380C/G171I/Y198NAK(NCINAK)的基础上,发现G295位点与抑制剂Thr通过氢键相连且高度保守,对G295位点进行定点饱和突变,提取质粒,转入大肠杆菌BL21感受态细胞中诱导表达,采用高通量筛选获得酶活力显著提高的突变株,对野生型(Wild type,WT)、五突变体T379N/A380C/G171I/Y198N/G295...

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Published in食品工业科技 Vol. 42; no. 16; pp. 112 - 118
Main Authors 王哲人, 刘晓婷, 樊占青, 王亚南, 魏贞, 高欣, 方丽, 闵伟红
Format Journal 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 01.08.2021
Subjects
天冬氨酸激酶
酶学性质
酶动力学
五突变体
北京棒杆菌
Online AccessGet full text
ISSN1002-0306
DOI10.13386/j.issn1002-0306.2021010071

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Abstract Q789; 目的:天冬氨酸激酶(aspartate kinase,AK)是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸(Lys)与苏氨酸(Thr)对AK的协同反馈抑制.方法:在获得的北京棒杆菌四突变体T379N/A380C/G171I/Y198NAK(NCINAK)的基础上,发现G295位点与抑制剂Thr通过氢键相连且高度保守,对G295位点进行定点饱和突变,提取质粒,转入大肠杆菌BL21感受态细胞中诱导表达,采用高通量筛选获得酶活力显著提高的突变株,对野生型(Wild type,WT)、五突变体T379N/A380C/G171I/Y198N/G295LAK(NCINL AK)进行酶动力学研究和酶学性质表征.结果:成功构建五突变体NCINL AK,最大反应速率Vmax为259 U/(mg·min).与野生型相比,米氏常数Km值由3.44 mmol/L减小到0.93 mmol/L,酶与底物结合更加紧密;希尔系数n值由2.73降为1.21,正协同性降低;最适反应温度由25℃提高到28℃,最适pH由8.0升至8.5,半衰期由4.7 h延长至5.2 h;五突变体NCINL AK较野生型WT AK,不同底物抑制剂浓度在0.2、1.0、5.0、10.0mmol/L时,抑制作用被不同程度的减弱,甚至Lys抑制作用被完全解除,且在10 mmol/L Thr条件下有激活作用.结论:本实验获得酶活力提高87.20倍的五突变体NCINL AK,酶学性质得到明显改善,在一定抑制剂浓度范围内,基本解除Lys对AK的反馈抑制作用,Thr的反馈抑制得到一定缓解,提高了积累大量天冬氨酸族氨基酸的可能性,为构建高产天冬氨酸族氨基酸菌株提供参考,使甲硫氨酸(Met)等氨基酸的无污染、高效生产成为可能.
AbstractList Q789; 目的:天冬氨酸激酶(aspartate kinase,AK)是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸(Lys)与苏氨酸(Thr)对AK的协同反馈抑制.方法:在获得的北京棒杆菌四突变体T379N/A380C/G171I/Y198NAK(NCINAK)的基础上,发现G295位点与抑制剂Thr通过氢键相连且高度保守,对G295位点进行定点饱和突变,提取质粒,转入大肠杆菌BL21感受态细胞中诱导表达,采用高通量筛选获得酶活力显著提高的突变株,对野生型(Wild type,WT)、五突变体T379N/A380C/G171I/Y198N/G295LAK(NCINL AK)进行酶动力学研究和酶学性质表征.结果:成功构建五突变体NCINL AK,最大反应速率Vmax为259 U/(mg·min).与野生型相比,米氏常数Km值由3.44 mmol/L减小到0.93 mmol/L,酶与底物结合更加紧密;希尔系数n值由2.73降为1.21,正协同性降低;最适反应温度由25℃提高到28℃,最适pH由8.0升至8.5,半衰期由4.7 h延长至5.2 h;五突变体NCINL AK较野生型WT AK,不同底物抑制剂浓度在0.2、1.0、5.0、10.0mmol/L时,抑制作用被不同程度的减弱,甚至Lys抑制作用被完全解除,且在10 mmol/L Thr条件下有激活作用.结论:本实验获得酶活力提高87.20倍的五突变体NCINL AK,酶学性质得到明显改善,在一定抑制剂浓度范围内,基本解除Lys对AK的反馈抑制作用,Thr的反馈抑制得到一定缓解,提高了积累大量天冬氨酸族氨基酸的可能性,为构建高产天冬氨酸族氨基酸菌株提供参考,使甲硫氨酸(Met)等氨基酸的无污染、高效生产成为可能.
Author 王亚南
樊占青
闵伟红
高欣
方丽
刘晓婷
魏贞
王哲人
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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FANG Li
FAN Zhanqing
LIU Xiaoting
MIN Weihong
WANG Yanan
GAO Xin
WEI Zhen
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Keywords 天冬氨酸激酶
酶学性质
酶动力学
五突变体
北京棒杆菌
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Snippet Q789; 目的:天冬氨酸激酶(aspartate kinase,AK)是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸(Lys)与苏氨酸(Thr)对AK的协同...
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Title 北京棒杆菌天冬氨酸激酶五突变体的构建及酶学性质表征
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