黑曲霉F044脂肪酶的分离纯化及酶学性质研究

Purification and Characterization of a Lipase from Aspergillus niger F044

摘要:

黑曲霉F044脂肪酶发酵上清液经硫酸铵沉淀、透析、DEAE Sepharose Fast Flow阴离子交换层析和Sephadex G-75凝胶过滤层析得到电泳纯的脂肪酶，纯化倍数为73.71倍，活性回收率为34%。对纯化脂肪酶性质研究表明：该脂肪酶分子量约为35～40kD，水解橄榄油的最适温度和最适pH分别为45℃和7.0，在60℃以下和pH 2.0～9.0之间有很好的稳定性。该脂肪酶的水解活性对Ca²⁺表现明显的依赖性，而Mn²⁺、Fe²⁺和Zn²⁺对脂肪酶则有显著的抑制作用。在最适条件下水解pNPP的K_m和V_max分别为7.37mmol/L和25.91μmol/(min·mg)。其N-端的15个氨基酸序列为Ser(Glu/His)-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln。

Abstract:

A lipase from Aspergillus niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 gel filtration chromatography. This purification protocol resulted in a 73.71-fold purification of lipase with 33.99% final yield, and the relative molecular weight of the enzyme was determined to be approximately 35～40kD using SDS-PAGE. The optimum pH and temperature for lipolytic activity of the lipase was 7.0 and 45℃, respectively. It was extremely stable at 60℃ and retained 98.70% of its original activity for 30min. The stability declined rapidly as soon as the temperature rose over 65℃. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4h. Ca²⁺ and Mg²⁺ ions stimulated lipolytic activity, whereas Mn²⁺, Fe²⁺ and Zn²⁺ ions caused inhibition. The values of K_m and V_max calculated from the Lineweaver-Burk plot using pNPP as hydrolysis substrate were 7.37mmol/L and 25.91μmol/(min·mg), respectively. The N-terminal sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln, which is highly homogeneity with that of lipase, as reported by Torossian.