Department of Food, Life and Environmental Sciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan.
Appl Biochem Biotechnol. 2020 Nov;192(3):910-922. doi: 10.1007/s12010-020-03360-4. Epub 2020 Jul 2.
A novel lipolytic enzyme-encoding gene, lipO745, from Aspergillus oryzae RIB40 was cloned and expressed in Pichia pastoris. Purified recombinant LipO745 (rLipO745) had a molecular mass of approximately 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. rLipO745 exhibited maximum activity at 40 °C and pH 7.0 and was stable at temperatures ≤ 40 °C. The substrate specificity of purified rLipO745 was analyzed using α-naphthyl esters as artificial substrates and various triacylglycerol and sterol esters as natural substrates. From among a panel of α-naphthyl esters (C2-C16), α-naphthyl butyrate (C4), with an activity of 269 ± 3.3 units/mg protein, was the optimal substrate for hydrolysis by the purified recombinant protein. The K and k values of rLiO745 for the C4 substrate were 0.073 ± 0.0012 mM and 608 ± 108 s, respectively. The purified recombinant enzyme had considerable hydrolytic activity toward tributyrin, tripalmitin, and triolein, indicating lipase activity, and toward cholesteryl acetate, butyrate, palmitate, and oleate, indicating sterol esterase activity. Transesterification activities between tributyrin and cholesterol or between tributyrin and campesterol were also determined.
从米曲霉 RIB40 中克隆并在毕赤酵母中表达了一种新型脂肪酶编码基因 lipO745。纯化的重组 LipO745（rLipO745）在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的分子量约为 60 kDa。rLipO745 在 40°C 和 pH 7.0 下表现出最大活性，在温度≤40°C 下稳定。使用α-萘基酯作为人工底物和各种三酰基甘油和固醇酯作为天然底物分析了纯化的 rLipO745 的底物特异性。在一系列α-萘基酯（C2-C16）中，α-萘丁酸（C4）是水解的最佳底物，其活性为 269±3.3 单位/mg 蛋白。rLiO745 对 C4 底物的 K 和 k 值分别为 0.073±0.0012 mM 和 608±108 s。纯化的重组酶对三丁酸甘油酯、三棕榈酸甘油酯和三油酸甘油酯具有相当大的水解活性，表明具有脂肪酶活性，对乙酸胆固醇酯、丁酸、棕榈酸和油酸具有固醇酯酶活性。还测定了三丁酸甘油酯与胆固醇或三丁酸甘油酯与菜油甾醇之间的转酯化活性。
