普通烟草NtASAT2的结构预测、克隆表达及功能验证

韩丽; 董滋强; 王丽娇; 李文钦; 王晨辉; 肖成志; 毛多斌

普通烟草NtASAT2的结构预测、克隆表达及功能验证

1. 郑州轻工业大学食品与生物工程学院, 河南郑州 450001;
2. 郑州轻工业大学烟草工业生物技术重点实验室, 河南郑州 450001

作者简介: 韩丽(1983—),女,内蒙古自治区牙克石市人,郑州轻工业大学副教授,博士,主要研究方向为酶工程。E-mail:han_l@zzuli.edu.cn;

基金项目: 中国烟草总公司重点研发项目(110202202006)
中图分类号: TS41⁺3;Q786

Structural prediction, cloning expression and functional verification of NtASAT2 from Nicotiana tabacum L.

1. College of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou 450001, China;
2. Key Laboratory of Biotechnology in Tobacco Industry, Zhengzhou University of Light Industry, Zhengzhou 450001, China
Received Date: 2024-05-10
Accepted Date: 2024-05-27
Available Online: 2025-04-16
CLC number: TS41⁺3;Q786

摘要: 为获得具有活性的普通烟草酰基糖酰基转移酶(NtASAT2),采取生物信息学方法对其序列及结构进行分析和预测,并对其基因进行克隆、原核表达与纯化,通过酶催化反应对获得的重组蛋白进行功能验证。结果表明:在NtASAT2二级结构中,α-螺旋及无规则卷曲占比较大,分别为39.04%和41.13%,且NtASAT2的氨基酸序列与粘毛烟草NacASAT2高度相似;在原核细胞BL21(DE3)表达的重组蛋白中,可溶性NtASAT2含量较少,且NtASAT2与镍柱的亲和力较弱,纯化后仅得到少量目标蛋白;在含有底物的酶催化反应体系中,NtASAT2表现出酶活性并催化生成蔗糖二酯。该研究结果可为NtASAT2在酶催化合成蔗糖酯方面的应用提供理论参考。
- 普通烟草 /
- 酰基糖酰基转移酶 /
- 蔗糖酯 /
- 克隆 /
- 表达与纯化
Abstract: To obtain functional acyl sugar acyltransferase NtASAT2 of Nicotiana tabacum L., bioinformatic methods were applied to analyze and predict the sequences and structure of NtASAT2. The gene of NtASAT2 was cloned, prokaryotic expressed, and then purified. T prokaryotic expressed, and then purified. The function of the recombinant protein was verified through enzymatic catalysis. The results showed that in the secondary structure of NtASAT2, α-helices and random coils account for a large proportion with 39. 04% and 41. 13%, respectively. The amino acid sequence of NtASAT2 was highly similar to that of sticky tobacco NacASAT2. The content of soluble NtASAT2 in the recombinant protein expressed by prokaryotic cell BL21 (DE3) was relatively low, and the affinity of NtASAT2 to nickel column was weak. After purification, only a small amount of the target protein was obtained. In enzymatic reaction systems containing substrates, NtASAT2 showed enzyme activity and catalyzed the formation of sucrose diesters. The research results could provide a theoretical basis for the application of acyl sugar acyltransferase in enzymatic synthesis of sucrose esters.
- Nicotiana tabacum L. /
- acylsugar acyltransferase /
- sucrose ester /
- cloning /
- expression and purification
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沈阳化工大学材料科学与工程学院沈阳 110142

普通烟草NtASAT2的结构预测、克隆表达及功能验证

作者简介: 韩丽(1983—),女,内蒙古自治区牙克石市人,郑州轻工业大学副教授,博士,主要研究方向为酶工程。E-mail:han_l@zzuli.edu.cn;

Structural prediction, cloning expression and functional verification of NtASAT2 from Nicotiana tabacum L.

计量

通讯作者: 陈斌, bchen63@163.com

普通烟草NtASAT2的结构预测、克隆表达及功能验证

作者简介:韩丽(1983—),女,内蒙古自治区牙克石市人,郑州轻工业大学副教授,博士,主要研究方向为酶工程。E-mail:han_l@zzuli.edu.cn

English Abstract

Structural prediction, cloning expression and functional verification of NtASAT2 from Nicotiana tabacum L.

目录

普通烟草NtASAT2的结构预测、克隆表达及功能验证

作者简介: 韩丽(1983—),女,内蒙古自治区牙克石市人,郑州轻工业大学副教授,博士,主要研究方向为酶工程。E-mail:han_l@zzuli.edu.cn;

Structural prediction, cloning expression and functional verification of NtASAT2 from Nicotiana tabacum L.

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通讯作者: 陈斌, bchen63@163.com

普通烟草NtASAT2的结构预测、克隆表达及功能验证

作者简介:韩丽(1983—),女,内蒙古自治区牙克石市人,郑州轻工业大学副教授,博士,主要研究方向为酶工程。E-mail:han_l@zzuli.edu.cn

English Abstract

Structural prediction, cloning expression and functional verification of NtASAT2 from Nicotiana tabacum L.

目录