الفهرس | Only 14 pages are availabe for public view |
Abstract The present study has been conducted to purify intracellular fat body lipase and midgut lipase, for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100, yielded a 98.9 and 49.15- fold purity and recovery of 50.81% and 18.3% for fat body lipase and midgut lipase, respectively. SDS-PAGE and zymogram revealed that the molecular weight of midgut lipase was 104.23 kDa, while fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of, fat body lipase and midgut lipase, was carried out through testing their activities against several factors such as; different temperatures, pHs, 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 for midgut and fat body lipase, respectively. The partially purified enzymes showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that both enzymes are metalloproteinases. Also, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride(PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions.Kinetic parameters were 301.95mM Km and 0.361Umg-1 Vmax for fat body lipase and 381.46 mM Km and 0.7893Umg-1 Vmax for midgut lipase. Key words: Galleria mellonella, purification, characterization, digestive lipase, midgut lipase, intracellular lipase and fat body lipase. |