[논문]Kinetics of Horseradish Peroxidase-Catalyzed Nitration of Phenol in a Biphasic System

Kong, Mingming; Zhang, Yang; Li, Qida; Dong, Runan; Gao, Haijun

doi:10.4014/jmb.1607.07039

Kinetics of Horseradish Peroxidase-Catalyzed Nitration of Phenol in a Biphasic System 원문보기

Journal of microbiology and biotechnology, v.27 no.2, 2017년, pp.297 - 305

Kong, Mingming (School of Life Science, Beijing Institute of Technology) , Zhang, Yang (School of Life Science, Beijing Institute of Technology) , Li, Qida (School of Life Science, Beijing Institute of Technology) , Dong, Runan (School of Life Science, Beijing Institute of Technology) , Gao, Haijun (School of Life Science, Beijing Institute of Technology)

Abstract ▼ AI-Helper

The use of peroxidase in the nitration of phenols is gaining interest as compared with traditional chemical reactions. We investigated the kinetic characteristics of phenol nitration catalyzed by horseradish peroxidase (HRP) in an aqueous-organic biphasic system using n-butanol as the organic solvent and ${NO_2}^-$ and $H_2O_2$ as substrates. The reaction rate was mainly controlled by the reaction kinetics in the aqueous phase when appropriate agitation was used to enhance mass transfer in the biphasic system. The initial velocity of the reaction increased with increasing HRP concentration. Additionally, an increase in the substrate concentrations of phenol (0-2 mM in organic phase) or $H_2O_2$ (0-0.1 mM in aqueous phase) enhanced the nitration efficiency catalyzed by HRP. In contrast, high concentrations of organic solvent decreased the kinetic parameter $V_{max}/K_m$. No inhibition of enzyme activity was observed when the concentrations of phenol and $H_2O_2$ were at or below 10 mM and 0.1 mM, respectively. On the basis of the peroxidase catalytic mechanism, a double-substrate ping-pong kinetic model was established. The kinetic parameters were ${K_m}^{H_2O_2}=1.09mM$, ${K_m}^{PhOH}=9.45mM$, and $V_{max}=0.196mM/min$. The proposed model was well fit to the data obtained from additional independent experiments under the suggested optimal synthesis conditions. The kinetic model developed in this paper lays a foundation for further comprehensive study of enzymatic nitration kinetics.

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문제 정의

Any variation in the structure or chemical nature of the enzyme upon hydration could change the constants of the kinetic constants [32]. The effect of phenol concentrations with varying concentrations of n-butanol on nitration efficiency was studied in this work. Both Michaelis-Menten and Lineweaver-Burk plots (Figs.

가설 설정

(ii) External mass transfer limitations were ignored. Experiments performed at different stirring speeds showed that 165 rpm was sufficient to avert mechanical damage of HRP and avoid external mass transfer resistances.
(iii) The concentration of phenol is much higher than that of the enzyme. This guarantees that the rate-determining step is determined by the enzymatic process.
In this paper, we investigated the kinetics of HRP-catalyzed nitration of phenol in an organic-aqueous biphasic system. The kinetic characteristics of HRP-catalyzed nitration largely depend on mass transfer between two phases and the concentrations of organic solvent, enzyme, and substrates.

제안 방법

Based on the comparison of the experimental and calculated data, we can conclude that the proposed kinetic model in this study is a reasonable representation of the HRP-catalyzed nitration process and can be used for reaction simulations. Additionally, the developed kinetic model provides a foundation for further comprehensive study of enzymatic nitration kinetics.
The effect of phenol concentrations with varying concentrations of n-butanol on nitration efficiency was studied in this work. Both Michaelis-Menten and Lineweaver-Burk plots (Figs. 2A and 2B) were constructed to estimate the maximum reaction rate (v_max) and Michaelis constant (K_m) for each assay. The Michaelis-Menten plots clearly indicated that the phenol nitration rate increased as the phenol concentration increased.
The performance of this prediction model was assessed by additional independent experiments performed according to the suggested reaction conditions of initial buffer pH 7.0, reaction temperature 25ºC, 40% (v/v) n-butanol as organic phase, agitation speed of 165 rpm, and 5 µg/ml HRP.
To further understand the catalytic process of HRP, we investigated the qualitative aspects of the reaction and a series of tests was performed (Fig. 4). The process of HRPcatalyzed nitration using phenol as the substrate was well depicted by typical Michaelis–Menten curves (Fig.

대상 데이터

The samples taken from the above systems were analyzed on a Shimadzu LC-15C HPLC instrument (Shimadzu, Japan) equipped with a SPD-15C detector (detection wavelength, 254 nm) [24]. The separation was performed on a Kromasil 100-5C18 column (250 × 4.

이론/모형

Km and vmax were determined by the Lineweaver-Burk plot method.
This indicated that the initial reaction rate depends on the substrate concentration. Then, we applied the Lineweaver-Burk plot method to examine the kinetic behavior of the catalytic reaction of HRP. As shown in Figs.

성능/효과

6. Overall, the model fits the data well, with a relative error that is under 8%, which confirms that the ping-pong model without enzyme inhibition by the substrates that is presented in this work is appropriate to illuminate the kinetic behavior of HRP-catalyzed nitration of phenol.

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