阿魏酸酯酶处理固体底物木质纤维素时,难以实现游离酶与产物或固体残留的完全分离,增加了生产成本。该实验以1-乙基-3-(3-二甲氨基丙基)碳二亚胺1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐[1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,EDC]为交联剂,将阿魏酸酯酶共价固定在pH响应型聚合物Eudragit L-100上可以解决这样的问题。通过对交联剂浓度、pH、时间、温度等固定化酶条件的优化,确定了制备固定化酶的最佳条件。在最佳固定化条件(EDC 5 g/L、pH 6.0、固定化时间1 h、温度25 ℃)下,固定化酶表现出比游离酶更好的热稳定性,40 ℃反应1 h后,固定化酶和游离酶的残留活性分别约为50%和30%。此外,固定化酶的pH稳定性也显著提高。以脱淀粉麦麸为底物生产阿魏酸(ferulic acid, FA)时,5次反应后固定化酶残留活性仍保持在64%左右。该研究提出的采用pH响应型聚合物Eudragit L-100固定化酶对于不溶底物的酶法水解提供了一个非常简单有效的方法。
When ferulate esterase reacts with lignocellulose, it is difficult to completely separate the free enzyme from the product or residual solid substrate, which increases the production cost. However, feruloyl esterase was covalently immobilized onto a pH responsive polymer Eudragit L-100 with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as cross-linking agent, which could solve the problem. The parameters of immobilized enzyme such as crosslinking agent concentration, immobilization pH, immobilization time, and reaction temperature were optimized. Under the optimum conditions (EDC 5 g/L, pH 6.0, immobilization time 1 h, and immobilization temperature 25 ℃), the immobilized enzyme exhibited much better thermal stability compared with free enzyme. After 1 h of incubation at 40 ℃, the residual activity was about 50% and 30% for the immobilized enzyme and free enzyme, respectively. In addition, the immobilized enzyme also exhibited significantly improved pH stability and about 64% residual activity was retained after five runs when using destarched wheat bran as substrate to produce ferulic acid. The pH responsive polymer Eudragit L-100 immobilized enzyme proposed in this study offers an attractive approach for enzymatic hydrolysis of insoluble substrate.
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