微波法高效制备固定化胰蛋白酶

High-Efficient Preparation of Immobilized Trypsin with Microwave

DOI：10.3969/j.issn.1673-1689.2019.05.013

中文关键词: 微波固定化 高效 树脂 固定化胰蛋白酶

英文关键词: microwave-assisted immobilization,high efficiency,resins,immobilized trypsin

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中文摘要:

为探讨高效制备固定化酶的方法，研究采用微波固定化的方法来提高共价固定化过程的效率。以D151树脂为载体，采用中心组合设计响应面法优化制备固定化胰蛋白酶的条件。通过二次多项式回归方程的拟合，最终确定了制备固定化胰蛋白酶的最佳条件为：吸附时间1.5 h、戊二醛质量分数1.0%、加酶量8.2 mg/mL、pH 5.0、交联温度29.0 ℃及交联时间3.5 min。制备的固定化胰蛋白酶在25～45 ℃的温度范围内、pH在4.0～5.0和9.0～10.0范围内具有良好的稳定性；重复使用8次后，酶活力保留了初始酶活力的73.1%；储藏5周后酶活力仍保留了71.1%。进一步使用固定化胰蛋白酶水解酪蛋白，其效率比使用游离胰蛋白酶的水解效率更高。结果表明，采用微波辅助固定化胰蛋白酶是一种值得尝试的高效方法，制备的固定化胰蛋白酶也具有良好的催化性能。

英文摘要:

To find the efficient preparation method of the immobilized trypsin，the microwave-assisted method was applied to improve the efficiency of the covalent enzyme immobilization process. The conditions of preparing immobilized trypsin under microwave irradiation with D151 resins as the carrier were optimized by use of central composite design-response surface method （CCD-RSM）. Through fitting quadratic polynomial regression equations，the optimum immobilization conditions were obtained as follows：adsorption time 1.5 h，glutaraldehyde concentration 1.0%，addition of trypsin 8.2 mg/mL，pH 5.0，crosslinking temperature 29.0 ℃，and crosslinking time 3.5 min. The immobilized trypsin was stable under the condition of 25～45 ℃ as well as pH range was 4.0～5.0 or 9.0～10.0. The immobilized trypsin still could retain 73.1% of its initial activity after reused for 8 cycles. The immobilized trypsin could keep 71.1% of its initial activity with storage at 4 ℃ after 5 weeks. Furthermore，the immobilized trypsin had higher efficiency to hydrolyze casein than free trypsin. In conclusion，the proposed microwave-assisted method is an available and efficient method to produce the immobilized trypsin with excellent catalytic properties.

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