生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 54-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 054

• 研究报告 • 上一篇    下一篇

一种高活性乙烯合成酶的鉴定和性质研究

  

  1. 1.天津科技大学生物工程学院,天津300457; 2.中国科学院天津工业生物技术研究所化学生物学中心,天津300308
  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 刘海萍,博士,副研究员,主要从事生化与分子生物学相关研究,E-mail: liu_hp@tib.cas.cn
  • 作者简介:苏宾宾,硕士,主要从事生物工程相关研究,E-mail:subb@tib.cas.cn
  • 基金资助:
    中国科学院青年创新促进会课题基金项目(2015137, to H. L. );天津市合成生物技术创新能力提升行动专项(TSBICIP-KJGG-002-02)

Identification and characterization of ahighly activeethylene for mingenzyme

  1. 1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China;2. Center for Chemical Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy ofSciences, Tianjin 300308, China
  • Online:2021-08-18 Published:2021-08-18

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摘要：

对不同微生物中乙烯合成酶(EFE) 同源基因的筛选,成功获得了一种Neofusicoccumparvum( NpEFE) 来 源的高活性乙烯合成酶。以pET28a 为载体成功实现了这种高活性efe基因在大肠杆菌BL21(DE3) 中的异源表达,并且对其纯酶酶学性质进行了研究分析。结果显示,该种属乙烯合成酶的酶比活力为(730.7±56.9) U /mg, 催化反应最适pH 值为7. 5。酶动力学研究表明,对底物α-酮戊二酸,其Km 为155. 7 μmol / L,kcat 为51. 4 /min, 对底物L-精氨酸,其Km 为139. 7 μmol / L,kcat 为53. 4 /min。与目前研究较为广泛的来源于Pseudomonassyringae(PsEFE) 的乙烯合成酶相比,在较高浓度的底物条件下,NpEFE 酶活性约是PsEFE 的2 倍,并且表现出较弱的底物抑制作用, 这在未来生物乙烯生产的工业化应用中具有很大的潜力。

关键词: 乙烯合成酶, 乙烯, 底物抑制, α-酮戊二酸依赖酶

Abstract:

The development of ethylene biosynthesis is essential. By screening ethylene forming enzyme (EFE) homologue genes in sev-eral microorganisms, an EFE protein with high activity from Neofusicoccumparvum(NpEFE) was identified. The heterogenous expression of this efegene in E.coliBL21(DE3) was successfully realized with pET28a as the vector and its enzymatic properties were analyzed. The specific activity of NpEFEwas(730.7±56.9)U /mgandtheoptimumpHforcatalysiswas7.5. Undertheactivityassayconditions, NpEFE exhibited a Km of 155.7μmol /L and a kcat of 51. 4 / min for 2-oxoglutarate, and a Km of139.7μmol /L and a kcat of 53. 4/ min for arginine. Compared to the extensively studied EFE from Pseudomonassyringae(PsEFE), NpEFE showed less substrate inhibition and al-most two-fold higher activity than PsEFE at high substrate concentrations. Therefore, NpEFE would be more potential in future ethylene industrial bioproduction.

Key words: ethylene forming enzyme, ethylene, substrate inhibition, 2-oxoglutarate dependent protein

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