柑橘香橙素生成途径关键酶黄烷酮-3-羟化酶特性分析及原核表达
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Characteristic Analysis and Prokaryotic Expression of Key Enzyme Flavanone-3-Hydroxylase in Formation Pathway of Citrus Aromadendrin
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    摘要:

    香橙素是柑橘类黄酮的重要组成,黄烷酮-3-羟化酶是香橙素生成途径中的关键作用酶。对黄烷酮-3-羟化酶的特点进行总结与分析并在大肠杆菌中进行表达。首先从NCBI数据库筛选了来自柑橘、陆地棉、玉米、水母雪莲和欧芹的5种f3h,对其进行理化性质、蛋白质二三级结构、序列比对等生物信息学分析并以大肠杆菌为宿主进行表达。发现5种来源的f3h,其基因开放阅读框为1032~1119bp,编码343~372个氨基酸,蛋白分子质量为38.89~41.45kDa,理论等电点在5.43~5.96,且不同物种F3H的理化性质、结构域和蛋白质结构也有一定的相似性。通过构建大肠杆菌表达体系发现:黄烷酮-3-羟化酶均能进行原核表达,除去来源于水母雪莲的F3H未检测到香橙素的生成,其余4种F3H均能将柚皮素转化为香橙素且产物生成量不同,其中ZmF3H生成量最高达20.12mg/L。

    Abstract:

    Aromadendrin is an important component of citrus flavonoids, and flavanone-3-hydroxylase (F3H) plays a key role in the formation pathway of aromadendrin. In this study, we summarized and analyzed the characteristics of F3H, and expressed them in Escherichia coli BL21. First, 5 kinds of f3h from Citrus reticulata Banco, Gossypium hirsutum, Zea mays, Saussurea medusa and Petroselinum crispumin NCBI database were chosen. Then, physicochemical properties, secondary and tertiary structure of protein, sequence alignment and other bioinformatics analysis were carried out. At last, recombinant expression system were constructed and expressed with E.coli BL21. The open reading frame of f3h gene was 1032-1119bp, encoding 343-372 amino acids. The molecular weight of F3H was 38.89-41.45kDa, and the theoretical isoelectric point was 5.43-5.96. The physical and chemical properties, domains and protein structures of F3H from different species were also similar.By constructing the expression system of E.coli BL21,we found that all F3H could be prokaryotic expression. Except the F3H from Saussurea medusi, the other 4 kinds of F3H could convert naringin to aromadendrin with different production amounts, and the highest production amount of ZmF3H was 20.12mg/L.

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黄旭,田苗苗,肖妃垚,刘娟,王振,单杨.柑橘香橙素生成途径关键酶黄烷酮-3-羟化酶特性分析及原核表达[J].食品科学技术学报,2021,39(2):65-72.

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  • 收稿日期:2021-01-08
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  • 在线发布日期: 2021-04-01
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