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

壳寡糖由2-10个氨基葡萄糖单元组成，通过β-1，4-糖苷键结合。它们的高级寡糖(五聚体或较大的寡聚体)具有多种生理活性，如抗菌、抗肿瘤和免疫增强活性。因此，为了利用壳寡糖作为功能食品材料和药品，需要含有较高比例的低聚糖的产品。壳聚糖可通过部分水解制得壳寡糖。由于五聚体和六聚体等较高级的低聚糖是壳聚糖水解的中间产物，因此要想高产率地生产这些低聚糖，就必须控制水解反应。使用固定化形式的酶将有效地获得较高的寡糖，原因如下：1.由于固定化在不溶物质上的酶可以立即从反应混合物中分离出来，因此在目标较高的低聚糖的产率由于进一步的降解而变得较低之前，可以很容易地终止反应；2.无需任何化学品或加热即可停止反应；以及3.酶催化剂可以回收并重复使用。正是基于这一点，我们对壳聚糖酶的固定化进行了研究，提出了一种利用多点附着法在直链淀粉包被的磁性纳米颗粒上获得稳定的固定化壳聚糖酶的方法，并利用固定化后的酶生产壳寡糖。将固定化壳聚糖酶用于生产具有良好生物活性的壳寡糖五聚体和六聚体。在该固定化壳聚糖酶间歇水解壳聚糖的过程中，成功地获得了目标低聚糖的高得率(按重量计为所用壳聚糖的40%)。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.