北京棒杆菌Corynebacterium pekinense天冬氨酸激酶P184M酶动力学分析及酶学性质表征

目的研究北京棒杆菌天冬氨酸激酶（aspartate kinase,AK）184位点脯氨酸（proline,P）突变为蛋氨酸（DL-methionine,M）对酶动力学及酶学性质的影响。方法对北京棒杆菌AK家族同源序列比对,选择出高度保守且远离底物结合位点的Pro184位点,对其进行饱和定点突变,成功构建突变体P184M。结果动力学和酶学性质研究表明：P184M AK Vmax比WT（wide type）提高了5.06倍;n值为0.19,突变体由WT的正协同性变为负协同性,同时Km（mol/L）值增大;突变体AK最适pH为6.5,低于野生型最适pH 7.0;最适反应温度28℃,低于WT的30℃,...

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Bibliographic Details
Published in食品安全质量检测学报 Vol. 8; no. 5; pp. 1750 - 1756
Main Author 王锐楠 张芷睿 方丽 闵伟红 刘春雷
Format Journal Article
LanguageChinese
Published 小麦和玉米深加工国家工程实验室/吉林农业大学食品科学与工程学院,长春,130118 2017
Subjects
北京棒杆菌
天冬氨酸激酶
突变株
酶学性质
aspartate kinase
enzymatic properties
天冬氨酸激酶
酶学性质
突变株
Corynebacterium pekinense
mutant
北京棒杆菌
Online AccessGet full text
ISSN2095-0381

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Summary:目的研究北京棒杆菌天冬氨酸激酶（aspartate kinase,AK）184位点脯氨酸（proline,P）突变为蛋氨酸（DL-methionine,M）对酶动力学及酶学性质的影响。方法对北京棒杆菌AK家族同源序列比对,选择出高度保守且远离底物结合位点的Pro184位点,对其进行饱和定点突变,成功构建突变体P184M。结果动力学和酶学性质研究表明：P184M AK Vmax比WT（wide type）提高了5.06倍;n值为0.19,突变体由WT的正协同性变为负协同性,同时Km（mol/L）值增大;突变体AK最适pH为6.5,低于野生型最适pH 7.0;最适反应温度28℃,低于WT的30℃,适宜温度区间变窄;半衰期由WT的2.1 h降为1.2 h;金属离子、有机溶剂和抑制剂Thr＋Met、Lys＋Met、Thr＋Lys＋Met对突变株AK均表现出激活作用。结论突变体天冬氨酸激酶P184M的酶动力学性质和酶学性质改变显著,为构建高产蛋氨酸菌株提供了一定的参考依据。
Bibliography:11-5956/TS
WANG Rui-Nan, ZHANG Zhi-Rui, FANG Li, MIN Wei-Hong, LIU Chun-Lei （National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China）
Objective To investigate the effects of site 184 proline （P） of aspartate kinases （AK） mutationed to DL-methionine （M） on enzyme kinetics and enzymatic properties.Methods Mutant P184M was successfully constructed by saturation site-directed mutagenesis of highly conserved amino acid and far away from inhibitors binding site P184 which was selected by sequence homology analysis of aspartate kinase family.Results The kinetics and enzymatic results showed that the Vmax of P 184M AK was 5.06 times higher than that of wide type （WT）.The n value was 0.19 indicating that mutant changed to negative synergistic reaction from synergistic reaction and Km value increased simultaneously.The optimum pH of P184M was 6.5,which was lower than that of WT （pH 7.0）.The optimum temperature of
ISSN:2095-0381