Enzyme immobilization appears as a remarkable technique to safely attach enzymes for several applications and cryogels stand as promising support materials to be used in such investigations. In this work, papain enzyme was immobilized onto an interpenetrating network obtained by cryogelation of N,N′-methylenebisacrylamide cross-linked 2-hydroxyethyl methacrylate and glutaraldehyde cross-linked chitosan. Cryogels were modified with –NH2 functionality and glutaraldehyde in order to attach papain covalently. Immobilization was carried out at 25 ℃ in 0.1 M pH 7.0 phosphate buffer at 1.0 mg/ml enzyme concentration for 5 h. The amount of papain immobilized onto cryogel was calculated to be 15.2 ± 2.54 mg/g cryogel. Macroporous structure and surface area were determined by scanning electron microscopy and Brunauer–Emmett–Teller techniques, respectively. Energy dispersive X-ray analysis showed that papain was bound to the cryogel and cryogel structure was composed of 2-hydroxyethyl methacrylate, chitosan, and glutaraldehyde. Proteolytic activities of free and immobilized papain were measured using casein as substrate. Optimum pH values and temperatures were 8.0 and 65 ℃ for free and immobilized enzymes and kinetic parameters were calculated at these conditions. Reusability and storage stability results indicated that immobilization enhanced the stability of papain compared to free form. Efficiency of immobilized papain was demonstrated by apple juice clarification study as an industrial use of the enzyme. Phenolic compound, protein, total soluble solid contents, and viscosity of apple juice before and after clarification were determined.

中文翻译：

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木瓜蛋白酶共价固定在聚（甲基丙烯酸羟乙酯）-壳聚糖冷冻凝胶上用于苹果汁澄清

酶固定似乎是一种非凡的技术，可以安全地将酶用于多种应用，而冷冻凝胶则是用于此类研究的有前途的支持材料。在这项工作中，木瓜蛋白酶被固定在通过 N,N'-亚甲基双丙烯酰胺交联的甲基丙烯酸 2-羟乙酯和戊二醛交联的壳聚糖的冷冻凝胶化获得的互​​穿网络上。冷冻凝胶用 –NH2 官能团和戊二醛修饰，以共价连接木瓜蛋白酶。固定化在 25°C 下在 0.1 M pH 7.0 磷酸盐缓冲液中以 1.0 mg/ml 酶浓度进行 5 小时。固定在冷冻凝胶上的木瓜蛋白酶的量计算为 15.2 ± 2.54 毫克/克冷冻凝胶。大孔结构和表面积分别通过扫描电子显微镜和 Brunauer-Emmett-Teller 技术确定。能量色散 X 射线分析表明木瓜蛋白酶与冷冻凝胶结合，冷冻凝胶结构由甲基丙烯酸 2-羟乙酯、壳聚糖和戊二醛组成。使用酪蛋白作为底物测量游离和固定化木瓜蛋白酶的蛋白水解活性。游离和固定化酶的最佳 pH 值和温度分别为 8.0 和 65 ℃，并在这些条件下计算了动力学参数。可重复使用性和储存稳定性结果表明，与游离形式相比，固定化增强了木瓜蛋白酶的稳定性。作为酶的工业用途的苹果汁澄清研究证明了固定化木瓜蛋白酶的效率。测定了澄清前后苹果汁的酚类化合物、蛋白质、总可溶性固形物含量和粘度。