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

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. Purification methods by combination of ammonium sulfate [(NH4)2SO4] 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 Ca2+, 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 Vmax and 301.95 mM Km. 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.


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].
To our knowledge, no intracellular lipase has been purified and 126 characterized in wax moth. In our preliminary study [18] we have carried out 127 purification and characterization of midgut lipase from G. mellonella larva. 128 Therefore, the aim of the current study is to purify and characterize fat body lipase 129 of G. mellonella larvae.
lipase assay was carried out by using continuous spectrophotometric rate 158 determination method as described by Tsujita et al. [22] with some modifications.  Samples were first subjected to ammonium sulfate precipitation by using 40 177 and 80% of ammonium sulfate solution and the ammonium sulfate fraction was 178 then collected and centrifuged at 10,000 rpm for 20 min. All the precipitation steps 179 were carried out at 4°C, and in each step, the enzyme activity and protein content 180 were determined.  The molecular mass of the enzyme was estimated using BL Uelf Prestained 204 Protein Ladder (from Genedirex) as molecular mass standards (marker proteins).

205
The gel was scanned with gel documentation system by using a scanner and then, 206 the bands were analyzed by using software: Gel-Pro Analyzer ver. 6.0. The effect of pH on the purified lipase activity was measured by using lipase 220 diluted in 15 μl universal buffer [25]. Buffers were prepared for the pH range from 221 2 to 13. After incubation for 1 h at each pH value, lipase activity was assayed as 222 described above. 223 2. Effect of temperature on lipase activity: 224 Lipase was diluted in 15 μl buffer (50mM Tris-HCl at pH 7 -7.5) then 225 incubated for 1 h at temperatures ranging from 20 to 70°C. Immediately after 226 incubation, lipase activity was measured by using pNPB as the substrate, as   (pNPB, 50 mM). and then the activity was measured as mentioned above.   between crude sample and sample after ammunium sulphate percipitation steps but 288 a large significant difference was observed between latter samples and sample that 289 undergo final step of purification (Fig 2). That means the sephadex G-100 step was 290 more effective in purification process.  The effect of the temperature on the purified lipase activity was assayed over 322 a range from 20 to 70°C (Fig 6). The purified lipase showed a steady increase in its  of the purified lipase is shown in (Fig 7).    24.973 mM (Fig 9). The statistical analysis showed p-value of 0.02. properties by performing purification and biochemical approaches, which is an 378 advantage for studies related to physiology and pest control.

379
The extracted lipase from larval and fat body was purified by using two 380 steps. Non-protein components were separated from proteins using ammonium 381 sulfate in a salting out process.

477
In the current study, the tested synthetic inhibitors had a significant 478 reduction on the lipase activity purified from the fat body of G. mellonella larvae.

479
The previous results indicated that fat body lipase requires metal ions specially 480 calcium, and have a serine residue at their active site. The effect of PMSF was 481 also shown in TAG-lipase activity from the reproductive accessory glands of the sand fly Phlebotomus papatasi [46]