烟草肠杆菌芳香族氨基酸转氨酶的结构预测、克隆表达及酶学性质研究
Structural prediction, cloning, expression and enzymatic propertity of aromatic amino acid transaminase from Enterobacter tabaci
-
摘要: 为提升芳香族氨基酸转氨酶(AAT)在合成生长素、促进作物生长方面的潜在应用价值,采用生物信息学方法对烟草肠杆菌β7(Enterobacter tabaci strain β7) AAT的结构进行分析和预测,利用大肠杆菌对该酶进行异源克隆表达和纯化,并测定重组AAT的酶学性质。结果表明:AAT含有396个氨基酸,该酶活性中心关键氨基酸残基为Lys246;由大肠杆菌异源表达的重组AAT的分子质量约为43 kDa,其最适底物为色氨酸,最适反应温度为50℃、pH值为8.0;重组AAT对乙醇具有较好的耐受性,且Cu2+对该酶活性有一定的促进作用。该研究结果可为烟草肠杆菌AAT的酶分子改造和高产生长素工程菌的构建提供理论基础。Abstract: To enhance the potential application of aromatic amino acid transaminase (AAT) in the synthesis of auxin (IAA) and promotion of crop growth, bioinformatics methods were applied to analyze and predict the structure of AAT from Enterobacter tabaci strain β7. Then, this AAT was cloned, expressed and purified in Escherichia coli, and the enzymatic properties of recombinant AAT were determined. The bioinformatics analysis results revealed that the length of this AAT protein is composed of 396 amino acids. The key residue in the active center of this AAT was Lys246. The molecular weight of the recombinant AAT expressed in E.coli was approximately 43 kDa. The optimal substrate for recombinant AAT was tryptophan, at a temperature of 50 ℃ and a pH value of 8.0. Cu2+ had a certain activating effect on enzyme activity, and recombinant AAT showed good tolerance to the organic solvent ethanol. These results provided a theoretical basis for the AAT enzyme modification and the construction of high-yield auxin engineering bacteria.
-
-
[1]
贾利霞,齐艳华.生长素代谢、运输及信号转导调控水稻粒型研究进展[J].植物学报,2022,57(3):263-275.
-
[2]
黄申,刘丹阳,张增辉,等.肠杆菌和不动杆菌混菌发酵提升山东烟叶品质机制研究[J].轻工学报,2023,38(1):45-53.
-
[3]
KUMAR G P,SEN S K,KANTI M T.Production and metabolism of IAA by Enterobacter spp.(Gammapro-teobacteria) isolated from root nodules of a legume Abrus precatorius L.[J].Biocatalysis and Agricultural Biotechnology,2015,4(3): 296-303.
-
[4]
王威,THINZAR T,张亚,等.一株烟草肠杆菌(Enterobacter tabaci)S4菌株的鉴定及其效果测定[J].农业资源与环境学报,2020,37(3):407-412.
-
[5]
周亚男,韩小斌,魏可可,等.烟草根际可培养微生物多样性及防病促生菌的筛选[J].微生物学通报,2021,48(12):4649-4663.
-
[6]
EMAMI S,ALIKHANI H A,POURBABAEI A A,et al.Assessment of the potential of indole-3-acetic acid producing bacteria to manage chemical fertilizers application [J].International Journal of Environmental Research,2019,13(4):603-611.
-
[7]
朱夜琳.大肠杆菌中吲哚丙酮酸的代谢工程研究[D].合肥:中国科学技术大学,2017.
-
[8]
KOGA J,SYONO K,ICHIKAWA T,et al.Involvement of L-tryptophan aminotransferase in indole-3-acetic acid biosynthesis in Enterobacter cloacae [J].Biochimicaet Biophysica Acta,1994,1209(2):241-247.
-
[9]
STEPANOVA A N,ROBERTSON-HOYT J,YUN J,et al.TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development [J].Cell, 2008,133(1):177-191.
-
[10]
TAO Y,FERRER J L,LJUNG K,et al.Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants [J].Cell,2008,133(1):164-176.
-
[11]
DUCA D,LORV J,PATTEN C L,et al.Indole-3-acetic acid in plant-microbe interactions [J].Antonie Van Leeuwenhoek,2014,106(1):85-125.
-
[12]
黄泰.色氨酸代谢产物吲哚-3-丙酮酸通过芳香烃受体通路调节Th17细胞/Treg细胞平衡缓解类风湿关节炎[D].合肥:安徽医科大学,2023.
-
[13]
杨旭,李晨飞,张志平,等.烟草肠杆菌(Enterobacter tabaci)β7发酵优化及菌剂制备[J].食品与发酵工业,2024,50(17)97-102.
-
[14]
杨何宝,郭世奇,尹守亮,等.利用强启动子对纤维二糖酶基因在里氏木霉中进行异源表达[J].纤维素科学与技术,2022,30(4):39-48
,67. -
[15]
徐亚军,李珂,刘珂珂,等.大豆根瘤内生菌全细胞可溶性蛋白SDS-PAGE电泳图谱分析[J].食品研究与开发,2019,40(11):159-165.
-
[16]
宋凤艳,周超君.蜂蜜中蛋白质及氨基酸含量的测定[J].现代食品,2016,22(20):108-110.
-
[17]
NADEEM M S,KHAN J A, ANWAR F. Molecular cloning,E.coli expression and characterization of thermostable alanine aminotransferase from Pyrococcus abyssi [J].Pakistan Journal of Zoology,2023,55(2):871-880.
-
[18]
KOPER K,HATAYA S,HALL A G,et al.Biochemical characterization of plant aromatic aminotransferases [J].Methods in Enzymology,2023,680:35-83.
-
[19]
鞠文君.转氨酶yhxA的可溶表达及其催化性能的改造[D].长春:吉林大学,2019.
-
[1]
-
点击查看大图
计量
- PDF下载量: 18
- 文章访问数: 4094
- 引证文献数: 0
下载:
