Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Cairo, Egypt.
J Microbiol Biotechnol. 2010 Feb;20(2):332-9.
The lipase from Mucor racemosus NRRL 3631 was partially purified by fractional precipitation using 60% ammonium sulfate, which resulted in a 8.33-fold purification. The partially purified lipase was then immobilized using different immobilization techniques: physical adsorption, ionic binding, and entrapment. Entrapment in a 4% agar proved to be the most suitable technique (82% yield), as the immobilized lipase was more stable at acidic and alkaline pHs than the free enzyme, plus 100% of the original activity was retained owing to the thermal stability of the immobilized enzyme after heat treatment for 60 min at 45 degrees Celsius. The calculated half-lives (472.5, 433.12, and 268.5 min at 50, 55, and 60 degrees Celsius, respectively) and the activation energy (9.85 kcal/mol) for the immobilized enzyme were higher than those for the free enzyme. Under the selected conditions, the immobilized enzyme had a higher K(m) (11.11 mM) and lower V(max) (105.26 U/mg protein) when compared with the free enzyme (8.33 mM and 125.0 U/mg protein, respectively). The operational stability of the biocatalyst was tested for both the hydrolysis of triglycerides and esterification of fatty acids with glycerol. After 4 cycles, the immobilized lipase retained approximately 50% and 80% of its original activity in the hydrolysis and esterification reactions, respectively.
米根霉 NRRL 3631 的脂肪酶经 60%硫酸铵分步沉淀法部分纯化，酶活提高了 8.33 倍。然后采用物理吸附、离子结合和包埋法对部分纯化的脂肪酶进行固定化。结果表明，琼脂包埋法（产率 82%）是最适合的技术，因为固定化脂肪酶在酸性和碱性 pH 值下比游离酶更稳定，并且由于固定化酶的热稳定性，经 45℃热处理 60min 后，仍保留了 100%的原始酶活。固定化酶的半衰期（分别为 50、55 和 60℃时为 472.5、433.12 和 268.5min）和活化能（9.85kcal/mol）均高于游离酶。在选定的条件下，与游离酶相比，固定化酶的 K(m)（11.11mM）更高，V(max)（105.26U/mg 蛋白）更低（分别为 8.33mM 和 125.0U/mg 蛋白）。对该生物催化剂的水解和酯化两种反应的操作稳定性进行了测试。经过 4 个循环，固定化脂肪酶在水解和酯化反应中分别保留了约 50%和 80%的原始酶活。
