Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy
Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy
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DOI：
10.1016/j.ab.2004.06.025
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发表时间：
2004-10-15
影响因子：
2.9
通讯作者：
Curto, MJM
中科院分区：
文献类型：
--
作者：
Karmali, K;Karmali, A;Curto, MJM
通过傅里叶变换红外光谱研究了黑曲霉和尼崎青霉葡萄糖氧化酶 (EC 1.1.3.4) 的简单直接测定方法。该酶在氧化氘 ((H2O)-H-2) 的磷酸盐缓冲液中催化碳 1 处的 D-葡萄糖氧化成 D-葡萄糖酸-1,5-内酯和过氧化氢。测量由于C-O伸缩振动而在1212cm(-1)处的D-葡萄糖酸-1,5-内酯带最大值随时间变化的强度，以研究D-葡萄糖氧化的动力学。测得D-葡萄糖酸-1,5-内酯的消光系数ε为1.28mM(-1)cm(-1)。通过使用来自黑曲霉和尼崎假单胞菌的葡萄糖氧化酶作为无细胞提取物或作为纯化的酶制剂，初始速度与酶浓度成正比。 Lineweaver-Burk图测定的动力学常数（V-max、K-m、k(cat)和k(cat)/K-m）分别为433.78 +/- 59.87 U mg(-1)蛋白质、10.07 +/- 1.75 mM、1095.07 +/- 151.19 s(-1)和108.74 s(-1)分别为毫米（-1）。这些数据与使用基于水介质中辣根过氧化物酶的连锁测定的分光光度法获得的结果一致：470.36 +/- 42.83 U mg(-1) 蛋白质、6.47 +/- 0.85 mM、1187.77 +/- 108.16 s(-1) 和 183.58 s(-1) mM(-1)分别为 V-max、K-m、k(cat) 和 k(cat)/K-m。因此，这种光谱方法非常适合使用无细胞提取物或纯化的酶制剂来测定葡萄糖氧化酶活性及其动力学参数，并具有实时测量葡萄糖氧化酶活性的额外优点。 (C) 2004 Elsevier Inc. 保留所有权利。
A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation Of D-glucose at carbon 1 into D-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((H2O)-H-2). The intensity of the D-glucono-1,5-lactone band maximum at 1212cm(-1) due to C-O stretching vibration was measured as a function of time to study the kinetics of D-glucose oxidation. The extinction coefficient epsilon of D-glucono-1,5-lactone was determined to be 1.28mM(-1) cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V-max, K-m, k(cat), and k(cat)/K-m) determined by Lineweaver-Burk plot were 433.78 +/- 59.87 U mg(-1) protein, 10.07 +/- 1.75 mM, 1095.07 +/- 151.19 s(-1), and 108.74 s(-1) mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36 +/- 42.83 U mg(-1) protein, 6.47 +/- 0.85 mM, 1187.77 +/- 108.16 s(-1), and 183.58 s(-1) mM(-1) for V-max, K-m, k(cat), and k(cat)/K-m, respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity. (C) 2004 Elsevier Inc. All rights reserved.