Purification and characterization of fat body lipase from the Greater Wax MothGalleria mellonella(Lepidoptera: Pyralidae)

Abstract

43Lipid mobilization and transport in insects is under investigation, especially 44 lipases and lipophorin because of their roles in energy production and transport of 45 lipids at flying activity. The present study has been conducted to purify 46 intracellular fat body lipase for the first time, from last larval instar of Galleria 47 mellonella. Purification methods by combination of ammonium sulfate 48 precipitation and gel filtration using Sephadex G-100 demonstrated that the 49 amount of protein and the specific activity of fat body lipase were 50 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, 51 with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step 52 was more effective in purification process. SDS-PAGE and zymogram revealed 53 that fat body lipase showed two monomers with molecular weights of 178.8 and 54 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried 55 out through testing its activities against several factors such as; different 56 temperatures, pH ranges, metal ions and inhibitors ending by determination of their 57 kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. 58The highest activities of enzyme were determined at the temperature ranges of 35-59 37°C and 37-40°C and pH ranges of 7-9 and 7-10. The partially purified enzyme 60 showed significant stimulation by Ca 2+ , K + and Na + metal ions indicating that fat 61 body lipase is metalloproteinase. Additionally, lipase activity was strongly 4 62 inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-63 diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) 64 providing an evidence of presence of serine residue and activation of enzymes by 65 metal ions. Kinetic parameters were 301.95mM K m and 0.316 Umg -1 V max . By 66 considering the purification of fat body lipase from larvae and using some 67 inhibitors especially ion chelating agents, it is suggested to develop this study by 68 using lipase inhibitors to reach a successful control of Galleria mellonella in the 69 near future. 70 Introduction 73 Lipids are utilized efficiently by insects as substrate for reproduction 74 embryogenesis, metamorphosis and flight. The importance of lipids to insects had 75 been recognized by several authors [1,2]. 76 Lipids are large and heterogeneous group of substances which are insoluble 77 in water but soluble in nonpolar solvents. Some lipids contain fatty acids (e.g., fats 78 and waxes) while others lack them (e.g., terpenes). Lipids containing fatty acids 79 involve several groups; sphinogolipids, glycerolipids, glycerophospholipids, sterol 80 lipids, saccahrolipids, prenol lipids and polykiteds [3]. Others that contain 5 81 metabolites play an important role in biological reactions [4]. Lipids containing 82 fatty acids present as storage lipids and membrane lipids. Glycolipids and 83 phospholipids are included in membrane lipids, while the storage lipids represented 84 by fats in adipose tissue of animals,...

Ontogenic development of the digestive enzymes and chemical composition of Hermetia illucens larvae of different ages

“…Lipase has a key role in insect lipid acquisition, storage, and mobilisation. Also, it is fundamental to many physiological processes (Santana et al, 2017) and lipid transmission in insects (Mahdy et al, 2020), including the reproductive system function and energy supplement during starvation (Ziegler, 1991). In the current study, we found the lipase activity occurring in alkaline conditions (pH 11).…”