[Purification and characterization of a lipase from Aspergillus niger F044].
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 degrees C , respectively. It was extremely stable at 60 degrees C and retained 98.70% of its original activity for 30min. The stability declined rapidly as soon as the temperature rose over 65 degrees C . 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 Km and Vmax calculated from the Lineweaver-Burk plot using pNPP as hydrolysis substrate were 7.37mmol/L and 25.91 micromol/(min x 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.
Shu
Zheng-Yu
ZY
College of Life Science & Technology, Huazhong University of Science & Technology, Wuhan 430074, China.
Yang
Jiang-Ke
JK
Yan
Yun-Jun
YJ
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D013696:Temperature
D005656:Fungal Proteins; D008049:Lipase