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

Abstract

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(K_m) and catalytic constant(k_cat) 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.

CLC Number:

O643
Q559.1

TrendMD:

LIU Ming, GAO Yi, CAO Zhu-An. Catalytic Kinetics of Nitrile Hydratase Measured in Real-time by Ultraviolet Spectrometry[J]. Chem. J. Chinese Universities, 2005, 26(12): 2284.

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

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