摘要/Abstract
基于硫代糖苷类化合物对糖苷酶的水解抗性，设计并合成了以对硝基苯酚为紫外吸收基团的二糖底物分子4-硝基苯基N，N'-二乙酰基-4-硫基-β-壳二糖苷（pNP-TCB），并分别研究了两个糖苷水解酶第18家族（GH18）几丁质酶和第20家族（GH20）β-N-乙酰己糖胺酶对pNP-TCB的降解响应.结果显示底物pNP-TCB几乎不被β-N-乙酰己糖胺酶降解，却能够被几丁质酶降解并释放出对硝基苯酚，该分子可在405nm处通过酶标仪检出.表明底物pNP-TCB具有良好的特异性，从而实现细胞水平GH18几丁质酶的性质研究且不受己糖胺酶的干扰，为GH18几丁质酶和GH20 β-N-乙酰己糖胺酶的筛选分离提供良好的检测手段.
关键词: 对硝基苯酚, 二糖底物, 壳二糖苷, 几丁质酶, 紫外吸收
Based on the resistance of glycosidase to the hydrolysis of glycosidase, the probe molecule p-nitrophenyl N,N'-diacetyl-4-thio-β-chitobioside (pNP-TCB) was designed and synthesized. The effects of pNP-TCB on the degradation of glycoside hydrolase family 18 (GH18) chitinase and 20 (GH20) β-N-acetylhexosaminidase were studied. The results showed that when pNP-TCB was combined with β-N-acetylhexosaminidase, the disaccharide substrate was hardly degraded. But when it reacted with chitinase, the pNP-TCB was degraded by enzyme to release p-nitrophenol group, and the corresponding ultraviolet absorption value can be detected by enzyme-labeled instrument at 405 nm. It indicates that the substrate molecule exhibits good specificity, which can be used to study the properties of GH18 chitinase at the cellular level without interference of β-N-acetylhexosaminidase. The pNP-TCB can also provide a good detection method for screening and isolation between the GH18 chitinase and the GH20 β-N-acetyl hexose.
Key words: p-nitrophenol, disaccharide substrate, chitobioside, chitinase, UV absorption
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