Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy
Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy
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基于拉曼光谱的几丁质酶水解结晶几丁质的无标记监测
DOI：
10.1039/d1an00581b
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Fujita Katsumasa
中科院分区：
文献类型：
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作者：
Ando Jun;Kawagoe Hiroyuki;Nakamura Akihiko;Iino Ryota;Fujita Katsumasa
我们展示了一种通过拉曼光谱法无标记监测结晶甲壳素降解酶（甲壳素酶）水解活性的方法。我们发现，结晶甲壳素在2995 cm−1处显示出一个特征性的拉曼峰，这在反应产物N，N′-二乙酰基壳二糖中没有出现。我们用这个拉曼峰作为结晶几丁质降解的标志来监测几丁质酶的水解活性。当将结晶几丁质悬浮液和几丁质酶混合在一起时，结晶几丁质在2995 cm-1处的峰强度随温育时间线性降低。峰强度的降低与释放的N，N′-二乙酰壳二糖的量的增加呈负相关，所述量通过用碱性铁氰化物的常规比色测定来测量。我们的研究结果，在这里，提供了一种新的方法，简单，原位，和标记的几丁质酶活性的监测。
We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm−1, which did not appear in the reaction product, N,N′-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm−1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N′-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.