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.

Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

“…The recombinant A. fumigatus lipase, whose optimal temperature was 65°C, constitutes an exception. However, this enzyme was not stable at this temperature, losing almost 100% of its activity after 1 h of incubation [29]. The thermal stability profile of A. japonicus LAB01 lipase indicated that this enzyme was stable at 45°C for at least 3 h, retaining 70% of its original activity.…”

“…Most of the described microbial lipases are more active in the cleavage of long or medium chain fatty acid substrates, as noted for lipases mentioned before as well as lipases from A. carneus, Staphylococcus aureus, and Penicillium aurantiogriseum , which showed a preference for pNPD substrate (C10) [13, 30, 50]; A. niger MYA lipase prefers pNPO (p-nitro substrate) (C18) [27]; P. crustosum lipase prefers pNPL (C12) [48]. However, a new type of lipase gene, which was cloned from the genomic DNA of A. fumigatus (CGMCC 2873), showed extremely high hydrolytic activity toward p-short-chain nitrophenyl esters, such as pNPA (C2) [29]. …”

Cloning, Expression, and Biochemical Characterization of an Enantioselective Lipase, YLIP9, from Yarrowia lipolytica MSR80

“…b Effect of temperature on ZZ-YLIP9 activity. Lipase assay was performed at temperatures ranging from 30 to 80°C to determine the optimum temperature where optimum activity has been reported in the range of 35-45°C, but similar to AFL1-1 of A. fumigatus that has an optimum at 65°C and 60 % activity at temperatures 30-50°C [6,9,16]. Various inhibitors were used to study their effect on ZZ-YLIP9 (Fig.…”

Effect of Metal Ions on the Hydrolytic and Transesterification Activities of Candida rugosa Lipase

“…The specific activity of the pure lipase was observed as 10.61 IU 100 activity and same activity was used as base while calculating the relative activity of CRL in presence of metal ions as given in 14,15 regarding the effect of metal ions on lipase hydrolytic activity. The effect varies from metal to metal and even same metal found to effect the lipase activity in a different manner if lipase source has been varied 16 .…”