Yadav R P, Saxena R K, Gupta R, Davidson W S
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
Biotechnol Appl Biochem. 1998 Dec;28 ( Pt 3):243-9.
Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q-Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41+/-1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15-90 degreesC) and was highly thermostable, retaining 100% activity at 60 degreesC for 24 h. It showed good pH tolerance (3.0-12.0) and was stable over a pH range of 4.0-10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non-ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide. At a concentration of 100 microM, 3,4-dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p-nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze-drying and had a shelf-life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3-regiospecificity and was stable in various organic solvents.
土曲霉脂肪酶被纯化至同质，产率为18.0%。该酶的比活性从20.80 U/mg蛋白质提高到250 U/mg蛋白质。在纯化过程中，Q-Sepharose上的离子交换非常有效。通过SDS/PAGE测定，纯化酶的分子量为41±1 kDa。纯化的脂肪酶表现出优异的温度耐受性（15-90℃），并且具有高度的热稳定性，在60℃下24小时保留100%的活性。它表现出良好的pH耐受性（3.0-12.0），在pH 4.0-10.0范围内24小时保持稳定。该酶的活性受到离子型去污剂的抑制，而非离子型去污剂则刺激酶活性。Mg2+和Ca2+离子刺激脂肪酶活性，而Co2+、Cu2+、Ni2+和Fe3+离子则导致抑制。该酶不受金属螯合剂EDTA、2-巯基乙醇和亚铁氰化钾的影响。在100 microM的浓度下，3,4-二氯异香豆素引起微弱抑制，活性损失40%，但相同浓度的对硝基苯基磷酸二乙酯强烈抑制酶活性（活性损失98.12%），证实土曲霉脂肪酶是一种丝氨酸水解酶。该脂肪酶对猪脂肪（相对活性151%）和花生油（相对活性103%）具有高活性，对库苏姆油（相对活性18%）活性最低。长达168小时的广泛透析对酶活性没有影响，表明该酶中不存在任何可透析的辅因子。土曲霉脂肪酶在冷冻干燥后仍保留显著活性，在室温下保质期超过6个月。土曲霉脂肪酶表现出1,3-区域特异性，并且在各种有机溶剂中稳定。
