紫外光谱法实时测定腈水合酶的催化动力学

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

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275 nm紫外吸光度的变化,计算出丙烯酰胺的生成速率.用M ichaelis-M enten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8.46 mmol/L,单位质量腈水合酶的催化速率常数(kcat)为2 398μmol/(m in.mg).

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 realtime. 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 2398μmoL/（ min·mg）, respectively.