河北大学学报(自然科学版) ›› 2017, Vol. 37 ›› Issue (6): 605-613.DOI: 10.3969/j.issn.1000-1565.2017.06.008

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黄连对羟苯基丙酮酸双加氧酶的原核表达和酶学性质

梁玉玲1,2,佐藤文彦3   

  • 收稿日期:2017-05-26 发布日期:2017-11-25
  • 作者简介:梁玉玲(1965—),女,河北定州人,河北大学教授,主要从事植物分子生物学与基因工程研究. E-mail: yuling_liang@163.com
  • 基金资助:
    河北省自然科学基金资助项目(C2013201126)

Prokaryotic expression and characterization of ρ-hydroxyphenylpyruvate dioxygenase from cultured Coptis japonica cells

LIANG Yuling1,2,SATO Fumihiko3   

  1. 1.College of Life Sciences, Hebei University, Baoding 071002, China; 2.Biological Engineering Technology Research Center in Hebei Province, Baoding 071002, China; 3.Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
  • Received:2017-05-26 Published:2017-11-25

摘要: 为了研究黄连对羟苯基丙酮酸双加氧酶(CjHPPD)的功能和性质,在E.coli中异源表达其编码cDNA得到了重组的CjHPPD. 经HPLC和LC-MS检测确定重组CjHPPD具有催化对羟苯基丙酮酸形成尿黑酸的酶活性.酶学特性分析表明,CjHPPD催化尿黑酸形成的最适pH值为6.5,产物尿黑酸的积累至少在反应开始10 min内随时间呈线形增长,最适反应温度为30 ℃.以ρ-HPP为底物进行底物亲和性分析表明,CjHPPD符合Michaelis-Menten动力学曲线,Km值为43.2 μmol/L.结果表明,CjHPPD的Km值明显高于其他已报道的植物HPPD的Km值,高Km使得CjHPPD具有较强除草剂抗性.

关键词: 黄连, 对羟苯基丙酮酸双加氧酶, 原核表达, 酶学性质, Km

Abstract: To characterize the ρ-hydroxyphenylpyruvate dioxygenase from cultured Coptis japonica cells(CjHPPD),recombinant protein was produced in E.coli. When the enzyme activity of recombinant CjHPPD was measured in vitro,the formation of homogentisate was determined by HPLC and confirmed by LC-MS analysis. Enzyme assays indicated that the optimum pH for the reaction of HPP transfer to homogentiaste was approximately 6.5. A time course study showed that the accumulation of homogentisate was linear with time for at least 10 min in the presence of 10 μL(37.18 μg)of desalted enzyme and 200 μmol/L hydroxyphenlpyruvate. The optimum temperature was 30 ℃. Characterization of the substrate affinity of recombinant CjHPPD using HPP as the substrate indicated that CjHPPD also followed Michaelis-Menten-type kinetics and Km was estimated to be 43.2 μmol/L. The Km of CjHPPD was much higher than the reported values for the HPPD enzyme from other plants. Since HPPD inhibitors are known to compete for the substrate for binding,the higher Km value of HPP might affect herbicide tolerance.- DOI:10.3969/j.issn.1000-1565.2017.06.008黄连对羟苯基丙酮酸双加氧酶的原核表达和酶学性质梁玉玲1,2,佐藤文彦3(1.河北大学 生命科学学院,河北 保定 071002;2.河北省生物工程技术研究中心,河北 保定 071002;3. 京都大学 大学院,日本 京都 606-8502)摘 要:为了研究黄连对羟苯基丙酮酸双加氧酶(CjHPPD)的功能和性质,在E.coli中异源表达其编码cDNA得到了重组的CjHPPD. 经HPLC和LC-MS检测确定重组CjHPPD具有催化对羟苯基丙酮酸形成尿黑酸的酶活性.酶学特性分析表明,CjHPPD催化尿黑酸形成的最适pH值为6.5,产物尿黑酸的积累至少在反应开始10 min内随时间呈线形增长,最适反应温度为30 ℃.以ρ-HPP为底物进行底物亲和性分析表明,CjHPPD符合Michaelis-Menten动力学曲线,Km值为43.2 μmol/L.结果表明,CjHPPD的Km值明显高于其他已报道的植物HPPD的Km值,高Km使得CjHPPD具有较强除草剂抗性.关键词:黄连;对羟苯基丙酮酸双加氧酶;原核表达;酶学性质;Km值中图分类号:Q786 文献标志码:A 文章编号:1000-1565(2017)06-0605-09Prokaryotic expression and characterization of ρ-hydroxyphenylpyruvatedioxygenase from cultured Coptis japonica cellsLIANG Yuling1,2,SATO Fumihiko3(1.College of Life Sciences,Hebei University,Baoding 071002,China;2.Biological Engineering Technology Research Center in Hebei Province,Baoding 071002,China;3.Graduate School of Biostudies,Kyoto University,Kyoto 606-8502,Japan)Abstract: To characterize the ρ-hydroxyphenylpyruvate dioxygenase from cultured Coptis japonica cells(CjHPPD),recombinant protein was produced in E.coli. When the enzyme activity of recombinant CjHPPD was measured in vitro,the formation of homogentisate was determined by HPLC and confirmed by LC-MS analysis. Enzyme assays indicated that the optimum pH for the reaction of HPP transfer to homogentiaste was approximately 6.5. A time course study showed that the accumulation of homogentisate was linear with time for at least 10 min in the presence of 10 μL(37.18 μg)of desalted enzyme and 200 μmol/L hydroxyphenlpyruvate. The optimum temperature was 30 ℃. Characterization of the substrate affinity of recombinant CjHPPD using HPP as the substrate indicated that CjHPPD also followed Michaelis-Menten-type kinetics and Km was estimated to be 43.2 μmol/L. The Km of CjHPPD was much higher than the reported values for the HPPD enzyme from other plants. Since HPPD inhibitors are known to compete for the substrate for binding,the higher Km value of HPP might affect herbicide tolerance.- 收稿日期:2017-05-26 基金项目:河北省自然科学基金资助项目(C2013201126) 第一作者:梁玉玲(1965—),女,河北定州人,河北大学教授,主要从事植物分子生物学与基因工程研究.E-mail: yuling_liang@163.com第6期梁玉玲等:黄连对羟苯基丙酮酸双加氧酶的原核表达和酶学性质

Key words: Coptis japonica, ρ -hydroxyphenylpyruvate dioxygenase, prokaryotic expression, characterization, Km value

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