响应面法优化磁性壳状固定化脂肪酶制备工艺研究

Process optimization of magnetic shell immobilized lipase by response surface methodology

中文关键词:  固定化脂肪酶  响应面法  制备工艺  荷叶  活性成分  快速筛选

英文关键词:immobilized lipase  response surface methodology  preparation process  lotus leaf  active ingredients  rapid screening

中文摘要:

      目的 优化磁性壳状脂肪酶固定化工艺, 并利用磁性固定化脂肪酶快速筛选荷叶中具有脂肪酶抑制作用的活性成分。方法 采用一锅法制备Fe3O4-壳聚糖作为固定化载体, 采用响应面设计法优化以聚乙二醇二缩水甘油醚为交联剂的脂肪酶固定化工艺, 确定反应pH、温度、加酶量和时间, 并进行固定化脂肪酶表征与活性测定; 固定化脂肪酶与荷叶样品液反应30 min后, 磁分离, 甲醇洗脱后采用高效液相色谱法(high performance liquid chromatography, HPLC)分析。结果 以固载率、酶活、酶活回收率为响应值, 确定最佳制备工艺为pH 8.1、反应温度40℃、酶质量浓度2.55 mg/mL、反应时间2.3 h, 固载率均值为85%、酶活均值为5.44 U/mg、酶活回收率均值为81%; 利用固定化脂肪酶快速筛选发现荷叶中荷叶碱与2-羟基-1-甲氧基阿朴啡具有较高的脂肪酶抑制活性。结论 本研究优选的脂肪酶固定化制备流程简捷, 脂肪酶利用率提高, 易分离, 有助于荷叶等药食两用原料中脂肪酶抑制成分的快速筛选研究。

英文摘要:

      Objective To optimize the immobilization process of magnetic shell lipase, and to screen the active ingredients with lipase inhibition in lotus leaves rapidly by magnetic immobilized lipase. Methods Lipase was immobilized using Fe3O4-chitosan as immobilize carrier prepared by one pot method and polyethylene glycol as crosslinking agent. Response surface design method was used to optimize the immobilized lipase process. The pH, temperature, amount of enzyme and reaction time were optimized. The immobilized lipase was characterized and its activity was determined. After the immobilized lipase reacted with lotus leaf sample solution for 30 minutes, the immobilized lipase was separated by magnetic separation, eluted by methanol, and analyzed by high performance liquid chromatography (HPLC). Results The optimal preparation conditions were pH 8.1, reaction temperature 40℃, enzyme mass concentration 2.55 mg/mL, reaction time 2.3 h. The mean fixation rates, activities and recoveries of immobilized lipase were 85%, 5.44 U/mg and 81%, respectively. Rapid screening of immobilized lipase affinity showed that nuciferine and 2-hydroxy-1-methoxy-apomorphine had higher lipase inhibition activity in lotus leaves. Conclusion The optimized lipase immobilized preparation process is simple, the utilization rate of lipase is improved, and the lipase is easy to be separated, which is helpful for the rapid screening of lipase inhibitors in dual-use raw materials such as lotus leaf.