title: Expression and characterization of a novel lipase from Aspergillus fumigatus with high specific activity.
abstract: A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 10(3) U mg(-1) on a standard substrate of p-nitrophenyl acetate. The Michaelis-Menten kinetic parameters V (max) and K (m) of the lipase were 1.37 mM mg(-1) min(-1) and 14.0 mM, respectively. Ca(2+) and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.
authors: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
authors: Jiao-Jiao
authors: JJ
authors: Shangguan
authors: Yu-Qiang
authors: YQ
authors: Liu
authors: Fu-Jun
authors: FJ
authors: Wang
authors: Jian
authors: J
authors: Zhao
authors: Li-Qiang
authors: LQ
authors: Fan
authors: Su-Xia
authors: SX
authors: Li
authors: Jian-He
authors: JH
authors: Xu
mesh_terms: D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D014169:Transformation, Bacterial
chemical_list: D005656:Fungal Proteins; D009596:Nitrophenols; D011994:Recombinant Proteins; C008642:4-nitrophenyl acetate; D008049:Lipase