摘要
谷氨酸脱氢酶(glutamate dehydrogenase,GDH)催化L-谷氨酸可逆氧化脱氨生成α-酮戊二酸,为研究其催化特性,在大肠杆菌中克隆表达了枯草芽孢杆菌来源的谷氨酸脱氢酶(BsGDH).酶学特征表明,BsGDH具有氧化和还原双向酶活性且最适催化温度分别为60℃和65℃,最适催化pH均为弱碱性(分别为pH 8.0和pH 7.5),但在弱碱环境下(pH 7.0-8.0)该酶稳定性较差.为提高BsGDH在催化合成α-酮戊二酸应用中的经济性,基于蛋白表面电荷工程,对该酶进行分子改造,以期将其催化最适pH调至其稳定性较高的中性环境.首先利用ROSETTA软件选择7个突变候选位点,结果发现突变体N16D和K218D催化谷氨酸合成α-酮戊二酸的最适pH由8.0下调至7.0,同时在pH 7.0条件下酶活较野生型分别提高2.9倍和5.4倍,且逆反应还原方向酶活降低.另外,突变体N16D和K218D热稳定性都得到显著改善,50℃下放置3 h后,突变体N16D可以保持54%左右的酶活,K218D保持40%左右的酶活,而野生型酶活此时完全丧失.本研究成功将BsGDH最适催化pH范围下调至其稳定性良好的pH范围.(图10表3参23)
Glutamate dehydrogenases catalyze the reversible oxidative deamination of L-glutamate toα-ketoglutarate(α-KG).The gene encoding glutamate dehydrogenase from Bacillus subtilis(BsGDH)was cloned and expressed in Escherichia coli.The ROSETTA supercharge software was used to reset the surface charge of BsGDH to adjust its pH range.Characterization of the molecular properties of wild-type BsGDH and its mutants showed that BsGDH could catalyze oxidative deamination and reductive amination reactions.The optimum temperatures for the oxidative deamination and reductive amination of BsGDH were 60℃and 65℃,respectively,and the optimal pH values were recorded at a slightly alkaline pH of 8.0 and 7.5,respectively.However,the enzyme exhibited poor stability at pH values above 7.0.These results suggest that the optimal pH for activity is not consistent with the optimal stability.We selected seven mutation sites using ROSETTA supercharge software analysis.Among all mutants,compared with the wild-type BsGDH,the optimal pH for oxidative deamination activities of N16D and K218D were reduced from 8.0 to 7.0,and the activities were increased 2.9 folds and 5.4 folds,respectively.Furthermore,the thermal stability of N16D and K218D was significantly improved.After 3 h of incubation at 50℃,N16D retained more than 54%of its residual activity and K218D retained more than 40%,while the wild-type BsGDH was completely deactivated.In this study,we successfully adjusted the optimal pH for the catalysis of BsGDH to be consistent with the optimal stability.
作者
武文慧
刘菲
王雅玲
杨套伟
饶志明
WU Wenhui;LIU Fei;WANG Yaling;YANG Taowei;RAO Zhiming(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2023年第1期7-14,共8页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(21778024)
江苏高校优势学科建设工程项目和江苏高校品牌专业建设工程项目资助
关键词
谷氨酸脱氢酶
酶学性质
最适PH
表面电荷工程
定点突变
glutamate dehydrogenase
enzymatic property
optimum pH
surface charge engineering
sitedirected mutagenesis
作者简介
通信作者:杨套伟,E-mail:yangtw@jiangnan.edu.cn
