Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, PR China.
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, PR China.
Colloids Surf B Biointerfaces. 2018 Sep 1;169:280-288. doi: 10.1016/j.colsurfb.2018.04.066. Epub 2018 May 16.
In this study, a novel and efficient synchronously immobilization of double enzymes (DE, alcalase and trypsin) were developed by using the chitosan and sodium tripolyphosphate-coated magnetic nanoparticles (FeO@CS-TPP) as support. The physicochemical properties of DE-FeO@CS-TPP NPs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and magnetic measurements. The preparation conditions and stabilities were also optimized and assessed. Results showed that the optimum preparation conditions were as follows: ratio of alcalase and trypsin of 3:1, enzyme concentration of 6% (w/w), temperature of 50 °C and pH of 9.0, the enzyme concentration absorbed by FeO@CS-TPP NPs of 6 mg mL, immoblization time of 4 h, and glutaraldehyde content of 3% (w/w). The K and V values were determined as 0.5035 mg mL, 6.900 mg mL min for the immobilized enzymes, respectively. DE-FeO@CS-TPP NPs was more stable than the free enzyme above 40 °C. The activity of DE-FeO@CS-TPP NPs was preserved 86% after 35 days storage and retained more than 60% of its initial activity after ten times of successive reuse. Application of DE-FeO@CS-TPP NPs in various protein hydrolysis showed favorable degrees of hydrolysis, yields and antioxidant activity. The results demonstrated that the DE-FeO@CS-TPP NPs were suitable for application in food protein hydrolysis.
在这项研究中，开发了一种新型且有效的双酶（DE、碱性蛋白酶和胰蛋白酶）同步固定化方法，使用壳聚糖和三聚磷酸钠包覆的磁性纳米粒子（FeO@CS-TPP）作为载体。通过傅里叶变换红外光谱（FTIR）、X 射线衍射（XRD）、透射电子显微镜（TEM）和磁测量对 DE-FeO@CS-TPP NPs 的物理化学性质进行了表征。还优化和评估了制备条件和稳定性。结果表明，最佳制备条件如下：碱性蛋白酶和胰蛋白酶的比例为 3:1，酶浓度为 6%（w/w），温度为 50°C，pH 值为 9.0，FeO@CS-TPP NPs 吸附的酶浓度为 6 mg/mL，固定化时间为 4 h，戊二醛含量为 3%（w/w）。固定化酶的 K 和 V 值分别为 0.5035 mg/mL 和 6.900 mg/mL·min。在 40°C 以上，DE-FeO@CS-TPP NPs 比游离酶更稳定。在 35 天储存后，DE-FeO@CS-TPP NPs 的酶活保留了 86%，重复使用十次后仍保留了初始活性的 60%以上。DE-FeO@CS-TPP NPs 在各种蛋白质水解中的应用显示出良好的水解度、产率和抗氧化活性。结果表明，DE-FeO@CS-TPP NPs 适合用于食品蛋白质水解。
