摘要
目的:烟草酰基糖酰基转移酶(Nicotiana tabacum acylsugar acyltransferase,NtASAT1)可催化短支链脂肪酸与蔗糖形成蔗糖单酯。利用大肠杆菌原核表达系统分析NtASAT1的表达,并对其进行纯化及功能验证。方法:首先利用生物信息学软件对烟草NtASAT1的理化性质、二级结构及同源性进行分析;之后从烟草腺毛的cDNA中克隆NtASAT1基因,并构建表达载体,研究其在BL21(DE3)中的表达情况;最后利用镍柱对NtASAT1进行纯化,将纯化后得到的目标蛋白进行酶反应,通过液相色谱-质谱法(liquid chromatography-mass spectrometry,LC-MS)检测产物并验证其功能活性。结果:C端截短93个氨基酸之后的NtASAT1能够在BL21(DE3)中表达。表达后的蛋白质大部分以不可溶状态存在于细胞中,不同的诱导剂浓度、诱导时间及诱导温度对于蛋白质可溶性表达影响不明显。利用镍柱对其纯化得到目标蛋白,加入底物进行酶反应,通过LC-MS检测到产物蔗糖单酯的存在。结论:通过克隆及纯化得到重组蛋白trNtASAT1,加入底物进行酶反应后可检测到产物,证明纯化后的NtASAT1具有一定功能,为ASAT类酶的纯化及应用奠定基础。
Objective:It is reported that NtASAT1(Nicotiana tabacum acylsugar acyltransferase)from tobacco can transform sucrose and short branched chain fatty acids to sucrose monoester.Thus,we tried to use the prokaryotic expression system of Escherichia coli to analyze the expression and purification condition of NtASAT1 and further verify the function of purified NtASAT1.Methods:First,the physical and chemical properties,secondary structure and homology of tobacco NtASAT1 were analyzed by bioinformatics software.Then,the gene NtASAT1 was cloned from the cDNA of tobacco glandular hairs and constructed into the expression vector so as to study its expression in BL21(DE 3).Finally,NtASAT1 was purified by nickel column and the activity of purified target protein was then analyzed by enzyme reaction.The product of the enzymatic reaction was analyzed by liquid chromatography-mass spectrometry(LC-MS).Results:NtASAT1,which was truncated by 93 amino acids at the C-terminal,could be expressed in BL21(DE3).Most of the expressed protein existed in an insoluble state and different concentrations of inducer,induction time and induction temperature had no obvious effect on the soluble expression of the protein trNtASAT1.The target protein was purified by nickel column and after enzyme reaction by adding substrate,the product sucrose monoester could be detected by LC-MS.Conclusion:The recombinant protein trNtASAT1 was cloned and purified and the enzymatic product sucrose monoester was detected by LC-MS,which proved that the purified NtASAT1 was functional.This study laid the foundation for purification and further application of enzyme ASAT in industry.
作者
韩丽
王丽娇
肖成志
董滋强
杜悦
何培新
HAN Li;WANG Li-jiao;XIAO Cheng-zhi;DONG Zi-qiang;DU Yue;HE Pei-xin(School of Food and Biological Engineering,Zhengzhou University of Light Industry,Zhengzhou 450000,China;Collaborative Innovation Center for Food Production and Safety,Zhengzhou University of Light Industry,Zhengzhou 450000,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2023年第4期30-40,共11页
China Biotechnology
基金
河南省重点研发与推广专项(202102310869)。
关键词
烟草
酰基糖酰基转移酶
表达
纯化
Nicotiana tabacum
Acylsugar acyltransferase
Expression
Purification
作者简介
通讯作者:韩丽,电子信箱:han_l@zzuli.edu.cn。
