Quantitative at-line monitoring of enzymatic hydrolysis using benchtop diffusion nuclear magnetic resonance spectroscopy

IF 1.9 3区 化学 Q3 CHEMISTRY, MULTIDISCIPLINARY Magnetic Resonance in Chemistry Pub Date : 2024-01-18 DOI:10.1002/mrc.5427
Evan R. McCarney, Kenneth A. Kristoffersen, Kathryn E. Anderssen
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Abstract

Benchtop diffusion nuclear magnetic resonance (NMR) spectroscopy was used to perform quantitative monitoring of enzymatic hydrolysis. The study aimed to test the feasibility of the technology to characterize enzymatic hydrolysis processes in real time. Diffusion ordered spectroscopy (DOSY) was used to measure the signal intensity and apparent self-diffusion constant of solubilized protein in hydrolysate. The NMR technique was tested on an enzymatic hydrolysis reaction of red cod, a lean white fish, by the endopeptidase alcalase at 50°C. Hydrolysate samples were manually transferred from the reaction vessel to the NMR equipment. Measurement time was approximately 3 min per time point. The signal intensity from the DOSY experiment was used to measure protein concentration and the apparent self-diffusion constant was converted into an average molecular weight and an estimated degree of hydrolysis. These values were plotted as a function of time and both the rate of solubilization and the rate of protein breakdown could be calculated. In addition to being rapid and noninvasive, DOSY using benchtop NMR spectroscopy has an advantage compared with other enzymatic hydrolysis characterization methods as it gives a direct measure of average protein size; many functional properties of proteins are strongly influenced by protein size. Therefore, a method to give protein concentration and average size in real time will allow operators to more tightly control production from enzymatic hydrolysis. Although only one type of material was tested, it is anticipated that the method should be applicable to a broad variety of enzymatic hydrolysis feedstocks.

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利用台式扩散核磁共振光谱对酶水解进行在线定量监测。
利用台式扩散核磁共振 (NMR) 光谱对酶水解过程进行定量监测。该研究旨在测试该技术实时表征酶水解过程的可行性。扩散有序光谱(DOSY)用于测量水解物中溶解蛋白质的信号强度和表观自扩散常数。核磁共振技术在红鳕鱼(一种瘦的白鱼)的酶水解反应中进行了测试,该反应是由内肽酶在 50°C 下进行的。水解物样品由人工从反应容器转移到 NMR 设备。每个时间点的测量时间约为 3 分钟。DOSY 实验的信号强度用于测量蛋白质浓度,表观自扩散常数被转换成平均分子量和估计的水解程度。将这些值绘制成时间函数图,就可以计算出溶解速率和蛋白质分解速率。与其他酶水解表征方法相比,使用台式核磁共振光谱的 DOSY 除了快速、非侵入性之外,还有一个优势,即它可以直接测量蛋白质的平均大小;蛋白质的许多功能特性受蛋白质大小的影响很大。因此,一种能实时提供蛋白质浓度和平均大小的方法将使操作人员能够更严格地控制酶水解的生产。虽然只测试了一种原料,但预计该方法应适用于多种酶水解原料。
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来源期刊
CiteScore
4.70
自引率
10.00%
发文量
99
审稿时长
1 months
期刊介绍: MRC is devoted to the rapid publication of papers which are concerned with the development of magnetic resonance techniques, or in which the application of such techniques plays a pivotal part. Contributions from scientists working in all areas of NMR, ESR and NQR are invited, and papers describing applications in all branches of chemistry, structural biology and materials chemistry are published. The journal is of particular interest not only to scientists working in academic research, but also those working in commercial organisations who need to keep up-to-date with the latest practical applications of magnetic resonance techniques.
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