Production of chitosan oligosaccharides using immobilized enzyme on polysaccharide-coated magnetic nanoparticles

壳寡糖由通过β-1，4-糖苷键结合的2至10个葡糖胺单元组成。它们的高级寡糖（五聚体或更大的低聚物）具有多种生理活性，如抗微生物、抗肿瘤和免疫增强活性。因此，为了利用壳寡糖作为功能性食品材料和药物，需要含有大部分高级寡糖的产品。壳寡糖可以由壳聚糖的部分水解获得。由于高级寡糖如五聚体和六聚体是壳聚糖水解的中间产物，因此有必要控制水解反应以高产率制备它们。使用固定化形式的酶将有效地获得更高的寡糖，原因如下：1.由于固定在不溶性材料上的酶可以立即从反应混合物中分离，因此水解反应可以在目标高级寡糖的产率由于进一步降解而变得较低之前容易地终止; 2.反应可以在没有任何化学品或加热的情况下停止;以及3.酶催化剂可以回收并重复使用。基于以上考虑，本文对壳聚糖酶的固定化进行了研究，提出了一种利用多点吸附法将壳聚糖酶固定在直链淀粉包覆的磁性纳米粒子上的方法，并利用该方法制备了壳寡糖。利用固定化壳聚糖酶制备具有生物活性的五聚体和六聚体壳寡糖。在分批水解壳聚糖使用固定化壳聚糖酶，目标寡糖的高产率（40%的使用壳聚糖，基于重量），成功地获得。固定化壳聚糖酶可回收重复使用。

Chitosan oligosaccharides consist of between 2 and 10 glucosamine units that are bound via beta-1, 4-glycoside linkages. Their higher oligosaccharides (pentamer or larger oligomers) possess various physiological activities such as antimicrobial, antitumor and immunoenhancing activities. Therefore, in order to utilize chitosan oligosaccharides as functional food materials and pharmaceuticals, products containing a large fraction of higher oligosaccharides are desirable. Chitosan oligosaccharides can be obtained from the partial hydrolysis of chitosan. As the higher oligosaccharides such as pentamers and hexamers are intermediate products in chitosan hydrolysis, it is necessary to control the hydrolysis reaction to produce them at high yield. The use of enzymes in an immobilized form would be effective at obtaining higher oligosaccharides for the following reasons: 1. As enzymes immobilized on insoluble materials can be separated immediately from the reaction mixture, hydrolysis reactions can be terminated easily before the yield of target higher oligosaccharides becomes lower due to further degradation; 2. The reaction can be stopped without any chemicals or heating; and 3. Enzyme catalysts can be recovered and used repeatedly. By considering these points, we studied the immobilization of chitosanase and presented a method for obtaining stable immobilized chitosanase on amylose-coated magnetic nanoparticles using the multipoint-attachment method, and then utilized the immobilized enzyme to produce chitosan oligosaccharides. The immobilized chitosanase was used to produce pentamers and hexamers of chitosan oligosaccharides, which possess beneficial biological activities. In batch hydrolysis of chitosan using the immobilized chitosanase, a high yield of target oligosaccharides (40% of the used chitosan, based on weight) was successfully obtained. The immobilized chitosanase could be recovered and reused repeatedly.