海藻酸钠固定化根霉脂肪酶的制备及其性质

Preparation and Properties of Sodium Alginate-Immobilized Rhizopus delemar Lipase

研究了以海藻酸钠为载体,用包埋法制备固定化德氏根霉(Rhizopus delemar)脂肪酶的条件.将酶粉和海藻酸钠溶于pH 5.0的HAc-NaAc缓冲溶液,用注射器将此混合液滴入到0.05 mol/L无菌CaCl2溶液中,静置固化45 min,经过滤、洗涤和干燥后得到球状固定化酶.固定化酶的活力回收约为34.1%.酶学性质研究表明,此固定化酶的热稳定性较好.游离酶在60℃下保温1 h已完全丧失活力,而固定化酶在100℃下保温1 h仅损失36.2%的活力,在100℃下保温6 h仍可保持46.8%的酶活力.酶经固定化后,其橄榄油水解反应的最适温度由40℃上升至90℃,Km值由13.8 mg/ml下降为8.1 mg/ml.常见有机溶剂对固定化酶的活力影响较小.将该固定化脂肪酶用于非水溶剂中正戊酸异戊酯的合成,重复使用6次后,固定化酶仍保持95%的酶活力.

The immobilization of Rhizopus delemar lipase in sodium alginate gel spheres was studied. Enzyme powder and sodium alginate were dissolved in an HAc-NaAc buffer at pH 5.0, and then the mixture was added dropwise into a 0.05 mol/L CaCI2 solution. After solidification in the solution for 45 min followed by filtration, rinsing and drying, immobilized enzyme beads were gained with a 34.1% activity recovery of the total enzyme added. The thermal stability of the immobilized enzyme was tested. There was only 36.2 % loss of enzyme activity when the immobilized lipase was heated at 100 ℃ for 1 h, while the free enzyme lost all its activity when heated at 60℃ for 1 h. After incubation at 100℃ for 6 h, the immobilized enzyme still kept 46.8% of the original activity. The optimal reaction temperature of the immobilized enzyme for the olive oil hydrolysis was 90℃ compared to 40℃ of the free lipase. The Michaelis constant of the immobilized lipase was 8.1 mg/ml, and that of the free lipase was 13.8 mg/ml. The immobilized lipase was employed to catalyze the esterification in a non-aqueous solution, and it maintained 95 % of the activity after consecutive use 6 times.