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

壳聚糖低聚糖由2到10个氨基葡萄糖单元组成，这些氨基葡萄糖单元通过- 1,4 -糖苷键结合。它们的高寡糖（五聚体或更大的低聚体）具有多种生理活性，如抗菌、抗肿瘤和免疫增强活性。因此，为了利用壳聚糖低聚糖作为功能性食品原料和药品，高低聚糖产品的高含量含量是很有必要的。壳聚糖部分水解可制得壳聚糖低聚糖。由于五聚体和六聚体等高级低聚糖是壳聚糖水解的中间产物，因此要高产出它们，必须控制水解反应。以固定化形式使用酶可以有效地获得较高的低聚糖，原因如下：1。由于固定在不溶性物质上的酶可以立即从反应混合物中分离出来，在目标高寡糖的产率因进一步降解而降低之前，水解反应可以很容易地终止；2. 无需任何化学药品或加热即可停止反应；和3。酶催化剂可回收再利用。考虑到这些问题，我们对壳聚糖酶的固定化进行了研究，并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，然后利用固定化酶制备壳聚糖低聚糖。利用固定化壳聚糖酶制备了具有良好生物活性的壳聚糖五聚体和六聚体。利用固定化壳聚糖酶批量水解壳聚糖，成功地获得了高得率的目标低聚糖（以重量为基础，占所用壳聚糖的40%）。固定化壳聚糖酶可重复回收利用。

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.