Mekasha Sophanit, Toupalová Hana, Linggadjaja Eka, Tolani Harish A, Anděra Ladislav, Arntzen Magnus Ø, Vaaje-Kolstad Gustav, Eijsink Vincent G H, Agger Jane W
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
Apronex s.r.o., Nad Safinou II/365, Vestec, 252 42 Jesenice u Prahy, Czech Republic.
Carbohydr Res. 2016 Oct 4;433:18-24. doi: 10.1016/j.carres.2016.07.003. Epub 2016 Jul 4.
Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).
壳聚糖是一种由D-葡萄糖胺（GlcN）和N-乙酰-D-葡萄糖胺（GlcNAc）组成的β-(1,4)-连接的聚阳离子多糖，其酶促解聚为利用富含几丁质的生物质提供了一条可能的途径。壳聚糖完全转化为单糖需要内切和外切壳聚糖酶的协同作用。在本研究中，我们开发了一种高效且经济高效的壳聚糖降解酶混合物，其中仅包含两种酶，一种来自天蓝色链霉菌的内切攻击型细菌壳聚糖酶ScCsn46A，以及一种来自嗜热栖热菌KOD1的外切攻击型葡糖胺特异性β-葡糖胺酶Tk-Glm。此外，我们开发了一种快速、可靠的定量方法，用于使用高效阴离子交换色谱-脉冲安培检测（HPAEC-PAD）分析GlcN。该方法的灵敏度很高，易于检测到小于50 pmol的量，这比基于折射率的更常用检测方法的灵敏度高出约1000倍。我们还通过电喷雾电离质谱（ESI-MS）对酶促降解反应过程中的产物形成进行了定性分析。
