Namboodiri V M, Chattopadhyaya R
Department of Biochemistry, Bose Institute, Calcutta, India.
Lipids. 2000 May;35(5):495-502. doi: 10.1007/s11745-000-549-3.
An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55 degrees C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.
一种来自黑曲霉的细胞外1,3-特异性脂肪酶，分子量为35.5 kDa，等电点为4.4，通过pH沉淀法纯化了50倍，随后经过一系列色谱步骤，总产率为10%。通过变性聚丙烯酰胺凝胶电泳和尺寸排阻快速高效液相色谱判断，该酶是纯一的。它含有2.8%的糖，经内切糖苷酶F处理后糖被完全去除，去糖基化的酶保留了全部活性。天然脂肪酶在35至55摄氏度以及pH 5.0至6.0之间表现出最佳活性。发现该酶的氨基酸组成和N端序列与先前从黑曲霉中纯化的脂肪酶不同。该酶在天然条件下对胰蛋白酶、胰凝乳蛋白酶、内切蛋白酶Glu-C、凝血酶和木瓜蛋白酶具有抗性，但热变性后易被相同的蛋白酶切割。
