Zhu Shiyu, Cong Xin, Sun Zheng, Chen Zhe, Chen Xu, Zhu Zhenzhou, Li Shuyi, Cheng Shuiyuan
National R&D Center for Se-rich Agricultural Products Processing, Wuhan Polytechnic University Wuhan 430023 China
School of Modern Industry for Selenium Science and Engineering, Wuhan Polytechnic University Wuhan 430205 PR China.
RSC Adv. 2022 Aug 10;12(34):22082-22090. doi: 10.1039/d2ra03765c. eCollection 2022 Aug 4.
Enzymatic synthesis of selenium (Se)-enriched peptides is vital for their application in supplementing organic Se. However, the poor stability and reusability of the free enzyme impedes the reaction. In this work, a highly stable immobilized Alcalase was synthesized by immobilizing Alcalase on tannic acid (TA) and polyethyleneimine (PEI) modified FeO nanoparticles (NPs). The optimal immobilization conditions for immobilized Alcalase were found at a TA/PEI (v/v) ratio of 1 : 1, pH of 10, and temperature of 40 °C, and the results from scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform Infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) characterization confirmed the successful immobilization of Alcalase. The results of an enzyme property test showed that immobilized Alcalase had higher thermal and pH stability than free Alcalase, and retained 61.0% of the initial enzyme activity after 10 repetitions. Furthermore, the organic Se content of Se-enriched peptide prepared through enzymatic hydrolysis of (CV) protein with immobilized Alcalase was 2914 mg kg, and the molecular weight was mainly concentrated in 924.4 Da with complete amino acid components. Therefore, this study proposes the feasibility of immobilized enzymes for the production of Se-enriched peptides.
酶法合成富硒肽对于其在补充有机硒方面的应用至关重要。然而，游离酶稳定性差且不可重复使用阻碍了反应进行。在本研究中，通过将碱性蛋白酶固定在单宁酸（TA）和聚乙烯亚胺（PEI）修饰的FeO纳米颗粒（NPs）上，合成了一种高度稳定的固定化碱性蛋白酶。固定化碱性蛋白酶的最佳固定条件为TA/PEI（v/v）比例1∶1、pH值10以及温度40℃，扫描电子显微镜（SEM）、透射电子显微镜（TEM）、傅里叶变换红外光谱（FTIR）和X射线光电子能谱（XPS）表征结果证实了碱性蛋白酶的成功固定。酶性质测试结果表明，固定化碱性蛋白酶比游离碱性蛋白酶具有更高的热稳定性和pH稳定性，重复使用10次后仍保留61.0%的初始酶活性。此外，用固定化碱性蛋白酶对（CV）蛋白进行酶解制备的富硒肽有机硒含量为2914 mg/kg，分子量主要集中在924.4 Da，氨基酸组成完整。因此，本研究提出了固定化酶用于生产富硒肽的可行性。
