Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

Abstract

Background: Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results: Purification methods by combination of ammonium sulfate [(NH 4) 2 SO 4 ] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7-10. The partially purified enzyme showed significant stimulation by Ca 2+ , K + , and Na + metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg − 1 V max and 301.95 mM K m. Conclusion: Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

Study of the dual biological impacts of aqueous extracts of normal and gamma-irradiated Galleria mellonella larvae

“…Certain enzymes have been reported to be secreted by G. mellonella larvae, such as acid phosphatase, 47 protease, 48 and lipase. 49 Furthermore, it has been reported that gamma irradiation doses can regulate enzyme cascades in insects, leading to biological control, 50 consistent with the present study, which shows that a 6 Gy irradiation dose leads to a beneficial secretion of enzymes that enhance the antibacterial and antitumour effects of samples.…”