Real-Time Monitoring of Enzyme-Catalysed Reactions using Deep UV Resonance Raman Spectroscopy.
Real-Time Monitoring of Enzyme-Catalysed Reactions using Deep UV Resonance Raman Spectroscopy.
复制标题
DOI：
10.1002/chem.201701388
复制
发表时间：
2017-05-23
期刊：
影响因子：
--
通讯作者：
Goodacre R
中科院分区：
文献类型：
--
作者：
Westley C;Fisk H;Xu Y;Hollywood KA;Carnell AJ;Micklefield J;Turner NJ;Goodacre R
对于酶催化生物转化，连续原位检测方法最大限度地减少了样品操作的需要，最终实现更准确的底物和产物的实时动力学测定。我们首次建立了一种基于紫外共振拉曼（UVRR）光谱的在线实时定量方法来同时监测多个生物转化。为了例证该方法的通用性和多功能性，使用了多种底物和酶系统，包括腈水合酶（NHase）和黄嘌呤氧化酶（XO），这两种酶都具有工业和生物学意义，并结合了多步酶促转化。 UVRR 光谱的多变量数据分析，包括多元曲线分辨率交替最小二乘法 (MCR-ALS)，用于实现底物和产物的绝对定量； UVRR 与 MCR-ALS 相结合的 HPLC 重复基准测试证实了出色的重现性。
For enzyme‐catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real‐time kinetic determinations of substrate(s) and product(s). We have established for the first time an on‐line, real‐time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution‐alternating least squares (MCR‐ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR‐ALS by HPLC confirmed excellent reproducibility.