Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences (NMBU), P.O. Box 5003, 1432 Ås, Norway.
Carbohydr Polym. 2018 Apr 15;186:420-428. doi: 10.1016/j.carbpol.2018.01.059.
BsCsn46A, a GH family 46 chitosanase from Bacillus subtilis had been previously shown to have potential for bioconversion of chitosan to chito-oligosaccharides (CHOS). However, so far, in-depth analysis of both the mode of action of this enzyme and the composition of its products were lacking. In this study, we have employed size exclusion chromatography, H NMR, and mass spectrometry to reveal that BsCsn46A can rapidly cleave chitosans with a wide-variety of acetylation degrees, using a non-processive endo-mode of action. The composition of the product mixtures can be tailored by varying the degree of acetylation of the chitosan and the reaction time. Detailed analysis of product profiles revealed differences compared to other chitosanases. Importantly, BsCsn46A seems to be one of the fastest chitosanases described so far. The detailed analysis of preferred endo-binding modes using HO showed that a hexameric substrate has three productive binding modes occurring with similar frequencies.
BsCsn46A 是枯草芽孢杆菌的 GH 家族 46 壳聚糖酶，先前已被证明具有将壳聚糖生物转化为壳寡糖 (CHOS) 的潜力。然而，到目前为止，对该酶的作用模式和产物组成的深入分析还很缺乏。在这项研究中，我们使用排阻色谱、H NMR 和质谱来揭示 BsCsn46A 可以使用非连续的内切作用模式快速切割具有多种乙酰化程度的壳聚糖。通过改变壳聚糖的乙酰化程度和反应时间，可以调整产物混合物的组成。与其他壳聚糖酶相比，对产物谱的详细分析显示出差异。重要的是，BsCsn46A 似乎是迄今为止描述的最快的壳聚糖酶之一。使用 HO 对首选内切结合模式的详细分析表明，六聚体底物有三种具有相似频率的产生活性结合模式。
