El-Shishtawy Reda M, Al Angari Yasser M, Alotaibi Maha M, Almulaiky Yaaser Q
Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
Int J Biol Macromol. 2023 Apr 1;233:123539. doi: 10.1016/j.ijbiomac.2023.123539. Epub 2023 Feb 4.
An innovative approach for immobilizing α-amylase was used in this investigation. The acrylic fabric was first treated with hexamethylene diamine (HMDA) and then coated with copper ions that were later reduced to copper nanoparticles (CuNPs). The corresponding materials obtained, Cu(II)@HMDA-TA and CuNPs@HMDA-TA, were employed as carriers for α-amylase, respectively. The structural and morphological characteristics of the produced support matrices before and after immobilization were assessed using various techniques, including FTIR, SEM, EDX, TG/DTG, DSC, and zeta potential. The immobilized α-amylase exhibited the highest level of activity at pH 7.0, with immobilization yields observed for CuNPs@HMDA-TA (81.7 %) (60 unit/g support) followed by Cu(II)@HMDA-TA (71.7 %) (49 unit/g support) and 75 % and 61 % of activity yields, and 91.7 % and 85 % of immobilization efficiency, respectively. Meanwhile, biochemical characterizations of the activity of the soluble and immobilized enzymes were carried out and compared. Optimal temperature, pH, kinetics, storage stability, and reusability parameters were optimized for immobilized enzyme activity. The optimal pH and temperature were recorded as 6.0 and 50 °C for soluble α-amylase while the two forms of immobilized α-amylase exhibit a broad pH of 6.0-7.0 and optimal temperature at 60 °C. After recycling 15 times, the immobilized α-amylase on CuNPs@HMDA-TA and Cu(II)@HMDA-TA preserved 63 % and 52 % of their activities, respectively. The two forms of immobilized α-amylase displayed high stability when stored for 6 weeks and preserved 85 % and 76 % of their activities, respectively. Km values were calculated as 1.22, 1.39, and 1.84 mg/mL for soluble, immobilized enzymes on CuNPs@HMDA-TA, and Cu(II)@HMDA-TA, and Vmax values were calculated as 36.25, 29.68, and 21.57 μmol/mL/min, respectively. The total phenolic contents of maize kernels improved 1.4 ± 0.01 fold after treatment by two immobilized α-amylases.
本研究采用了一种创新方法来固定化α-淀粉酶。首先用六亚甲基二胺（HMDA）处理丙烯酸织物，然后用铜离子进行包覆，随后铜离子被还原为铜纳米颗粒（CuNPs）。所得到的相应材料Cu(II)@HMDA-TA和CuNPs@HMDA-TA分别用作α-淀粉酶的载体。使用包括傅里叶变换红外光谱（FTIR）、扫描电子显微镜（SEM）、能谱仪（EDX）、热重/微商热重曲线（TG/DTG）、差示扫描量热法（DSC）和zeta电位等多种技术，对固定化前后所制备载体基质的结构和形态特征进行了评估。固定化α-淀粉酶在pH 7.0时表现出最高活性水平，CuNPs@HMDA-TA的固定化产率为81.7%（60单位/克载体），其次是Cu(II)@HMDA-TA的固定化产率为71.7%（49单位/克载体），活性产率分别为75%和61%，固定化效率分别为91.7%和85%。同时，对可溶性酶和固定化酶的活性进行了生化表征并进行比较。针对固定化酶活性优化了最佳温度、pH、动力学、储存稳定性和可重复使用性参数。可溶性α-淀粉酶的最佳pH和温度记录为6.0和50℃，而两种形式的固定化α-淀粉酶的pH范围较宽，为6.0 - 7.0，最佳温度为60℃。在循环使用15次后，CuNPs@HMDA-TA和Cu(II)@HMDA-TA上的固定化α-淀粉酶分别保留了其63%和52%的活性。两种形式的固定化α-淀粉酶在储存6周时表现出高稳定性，分别保留了其85%和76%的活性。可溶性、CuNPs@HMDA-TA上的固定化酶和Cu(II)@HMDA-TA的米氏常数（Km）值分别计算为1.22、1.39和1.84毫克/毫升，最大反应速度（Vmax）值分别计算为36.25、29.68和21.57微摩尔/毫升/分钟。用两种固定化α-淀粉酶处理后，玉米粒的总酚含量提高了1.4±0.01倍。
