高等学校化学学报

高等学校化学学报 ›› 2005, Vol. 26 ›› Issue (12): 2284.

• 研究论文 • 上一篇    下一篇

紫外光谱法实时测定腈水合酶的催化动力学

刘铭,高毅,曹竹安   

  1. 清华大学化学工程系生物化工研究所,北京,100084
  • 收稿日期:2004-10-22 出版日期:1905-03-14 发布日期:1905-03-14
  • 通讯作者: 曹竹安(1940年出生)，男，教授，博士生导师，主要从事工业生物催化与生物转化研究. E-mail:cza-dce@tsinghua.edu.cn
  • 基金资助:

    国家重点基础研究发展计划(“九七三”计划)项目(批准号：2003CB716007)资助.

Catalytic Kinetics of Nitrile Hydratase Measured in Real-time by Ultraviolet Spectrometry

LIU Ming, GAO Yi, CAO Zhu-An*   

  1. nstitute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
  • Received:2004-10-22 Online:1905-03-14 Published:1905-03-14
  • Contact: CAO Zhu-An;E-mail:cza-dce@tsinghua.edu.cn

被引次数

20

摘要：

采用紫外光谱法研究了腈水合酶催化丙烯腈水合的过程,在不同丙烯腈初始浓度下,测定了催化过程中275nm紫外吸光度的变化,计算出丙烯酰胺的生成速率.用Michaelis-Menten方程对不同丙烯腈浓度下的Nocardiasp.腈水合酶催化速率进行了拟合,得到该酶以丙烯腈为底物的米氏常数(Km)为8.46mmol/L,单位质量腈水合酶的催化速率常数(kcat)为2398μmol/(min·mg).

关键词: 腈水合酶;紫外光谱;催化动力学;丙烯酰胺;Nocardiasp., 腈水合酶, 紫外光谱, 催化动力学, 丙烯酰胺, Nocardiasp.

Abstract:

Nitrile hydratase(NHase), which catalyzes the hydration of nitriles to amides, is applied to industrial production of acrylamide. Kinetics of acrylonitrile hydration catalyzed by NHase is essential for the investigation of enzymatic properties and industrial production of acrylamide. The key in the study of enzymatic kinetics is to determine the real-time amount of acrylamide formed in the hydration, and then to calculate the initial reaction rate. A novel analytical method was developed to monitor the kinetics of acrylonitrile hydration catalyzed by purified Nocardia sp. NHase. The amount of acrylamide formed in the process was measured on-line by ultraviolet spectrometry with 275 nm wavelength. Compared with the conventional method of gas chromatography, UV spectroscopy is more sensitive and rapid for analyzing acrylamide quantitatively in real-time. The reaction rates in a series of initial concentrations of acrylonitrile were measured and calculated from the time-varying ultraviolet absorption. The kinetic correlation between reaction rate and acrylonitrile concentration was fitted with Michaelis-Menten equation. Michaelis constant(Km) and catalytic constant(kcat) were calculated to be 8.46 mmol/L and 2398 μmol/(min·mg), respectively.

Key words: Nitrile hydratase; UV Spectroscopy; Catalytic kinetics; Acrylamide; Nocardia sp., Nitrile hydratase, UV Spectroscopy, Catalytic kinetics, Acrylamide, Nocardia sp.

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