内蒙古农业大学学报（自然科学版）2026，Vol.47Issue(4)：55-62,8.DOI:10.16853/j.cnki.1009-3575.2026.04.007
蒺藜苜蓿苯甲醇O-苯甲酰转移酶MtBEBT的原核表达、纯化及功能预测
Prokaryotic Expression,Purification and Functional Prediction of Benzyl Alcohol O-Benzoyltransferase MtBEBT from Medicago Truncatula
摘要
Abstract
This study successfully constructed a prokaryotic expression system for the benzyl alcohol O-benzoyltransferase MtBEBT from Medicago truncatula.After optimization of induction conditions,low-temperature induction was performed at 16℃for 14 h with 0.2 mmol/L IPTG,and the soluble production of recombinant protein was increased to 32.7 mg/L culture medium.A two-step purification strategy involving Ni²⁺-NTA affinity chromatography combined with Superdex 200 size-exclusion chromatography was employed,re-sulting in a recombinant protein with over 95%purity(purification fold of 8.2,recovery rate of 64.3%).The bioinformatics analy-sis revealed that MtBEBT was a basic hydrophilic protein(pI=7.08),with its secondary structure predominantly composed of ran-dom coils(47.93%)and α-helices(35.08%).The tertiary structure model quality parameter GMQE value reached 0.92.Molecu-lar docking simulations revealed that its affinity for benzoyl-CoA(binding energy-7.8 kcal/mol)was significantly higher than that for benzyl alcohol(-5.2 kcal/mol).The key active sites,Ala168,Asn290,and Arg372,stabilized substrate binding through a hy-drogen bond network.The expression and purification system established in this study provided a material basis for the crystal struc-ture analysis of MtBEBT.The predicted active sites offered a theoretical foundation for research on enzyme functional mechanisms and plant metabolic engineering.关键词
蒺藜苜蓿/BAHD酰基转移酶/分离纯化/分子对接/活性位点Key words
Medicago truncatula/BAHD acyltransferase/Separation and purification/Molecular docking/Active site分类
化学化工引用本文复制引用
李琦,杨晶,王桂花,韩晓东..蒺藜苜蓿苯甲醇O-苯甲酰转移酶MtBEBT的原核表达、纯化及功能预测[J].内蒙古农业大学学报（自然科学版）,2026,47(4):55-62,8.基金项目
内蒙古盐碱地饲草生产关键技术集成与推广应用项目(RH2500002255) （RH2500002255）
