Purification and characterization of a lipase from Aspergillus niger F044.
- Author:
Zheng-Yu SHU
1
;
Jiang-Ke YANG
;
Yun-Jun YAN
Author Information
1. College of Life Science & Technology, Huazhong University of Science & Technology, Wuhan 430074, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Aspergillus niger;
enzymology;
Chromatography, Gel;
Chromatography, Ion Exchange;
Electrophoresis, Polyacrylamide Gel;
Enzyme Stability;
Fungal Proteins;
chemistry;
isolation & purification;
metabolism;
Hydrogen-Ion Concentration;
Lipase;
chemistry;
isolation & purification;
metabolism;
Molecular Sequence Data;
Molecular Weight;
Sequence Analysis, Protein;
Temperature
- From:
Chinese Journal of Biotechnology
2007;23(1):96-100
- CountryChina
- Language:Chinese
-
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 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.
