Abstract

The characteristics of a purified lipase from Rhizopus oryzae ZAC3 (RoL-ZAC3) were investigated. RoL-ZAC3, a 15.8 kDa protein, which was optimally active at pH 8 and 55 °C had a half-life of 126 min at 60 °C. The kinetic parameters using p-nitrophenylbutyrate as substrate were 0.19 ± 0.02 mM, 126 ± 5.6 U/ml and 122 s⁻¹ for K_m, V_max and k_cat respectively. RoL-ZAC3 showed stability in methanol and isopropanol with Na⁺ enhancing the activity. p-nitrophenyloleate and castor oil were the best preferred substrates among the p-nitrophenyl esters and vegetable oils tested respectively. About 43% residual activity was observed after incubation for 30 min at 75 °C. Circular dichroism thermal scan showed that the lipase displayed intense negative ellipticities even at high temperature. Perturbation of the tertiary structure with increasing temperature caused the exposure of hydrophobic side chains to the aqueous environment as revealed by tryptophan fluorescence, with a t−T_m of 50 °C. Differential scanning calorimetry analysis showed melting temperature and calorimetric enthalpy of 55.5 °C and 444 kJ/mol respectively. Dynamic light scattering analysis indicated that the lipase was prone to aggregation upon unfolding at high temperature. It can be concluded that RoL-ZAC3 possesses promising potential for numerous biotechnological applications.

摘要

研究了从米根霉ZAC3（Ro L-ZAC3）中纯化的脂肪酶的特性。Ro L-ZAC3 是一种 15.8 kDa 的蛋白质，在 pH 8 和 55 °C 下具有最佳活性，在 60 °C 下的半衰期为 126 分钟。使用对硝基苯基丁酸作为底物的动力学参数对于K _m、V _max和k _cat分别为 0.19 ± 0.02 mM、126 ± 5.6 U/ml 和 122 s ^{-1 。}Ro L-ZAC3 在甲醇和异丙醇中表现出稳定性，Na ⁺增强了活性。p对硝基苯油酸酯和蓖麻油分别是测试的对硝基苯酯和植物油中最好的底物。在 75°C 孵育 30 分钟后观察到约 43% 的残留活性。圆二色性热扫描显示脂肪酶即使在高温下也表现出强烈的负椭圆率。如色氨酸荧光所示，随着温度升高，三级结构的扰动导致疏水侧链暴露于水环境中，t - T _m50°C。差示扫描量热分析显示熔解温度和量热焓分别为 55.5 °C 和 444 kJ/mol。动态光散射分析表明，脂肪酶在高温下展开时容易聚集。可以得出结论，Ro L-ZAC3 具有广阔的生物技术应用潜力。