Isolation and Purification of Lipase from the Midgut of Fifth Instar Larvae of Antheraea mylitta drury

Lipases obtained from healthy and pebrinised larvae were purified by 45-85% (NH4)2SO4 fractionation followed by Sephadex S-100 gel filtration and CM-Sepharose. In both the samples final enzyme purification reported was 19.02 and 17.7 folds(magnitude) and the recovery of final purified enzyme was 19.32% and 17.14% with a specific activity of 7.87 and 7.52 µmol/min/mg. Results also show that activity of purified lipase was highest at pH 8 in both the samples. Highest lipase activity was recorded between 37°C to 40°C temperature and lipase activity was maximum at 37°C temperature in healthy sample and 38°C temperature in pebrinised sample. The enzyme activity reduced with addition of NaCl, Urea and MgCl2 whereas EDTA and CaCl2 increased the activity. The molecular weight of the purified enzyme was 30 kDa as determined by SDS-polyacrylamide gel electrophoresis.

Aim: The present study was conducted to isolate, purify and characterize lipases from the midgut of both healthy and pebrinised fifth instar larvae of Antheraea mylitta drury.

Study Design: Study involves dissection of midgut from the fifth instar larvae of fourth day of both healthy and pebrinised larvae, Lipases obtained from both the samples were purified by 45-85% (NH4)2SO4 fractionation followed by Sephadex S-100 gel filtration and CM-Sepharose and specific activity was measured at various temperatures and different pH. Molecular weight of lipase was measured by SDS PAGE.

Place and Duration of Study: Healthy and pebrinised fifth instar larvae of fourth day were collected from the forest patches of Jakaram (18.1E and 79.8 N), Warangal in August 2015.

Methodology: Lipase was isolated from the midgut of both healthy and pebrinised larvae and purification of enzyme was done by (NH4)2SO4 fractionation, Sephadex S-100 gel filtration and CM-Sepharose. Temperature, pH suitable for highest lipase activity was measured and specific activity against various chemicals was also measured. Kinetic parameters like Km and Vmax were estimated by Sigmaplot software version 11. SDS Polyacrylamide gel was used to determine the molecular weight of lipase.

Results: In both healthy and pebrinised larvae final enzyme purification reported was 19.02, 17.7 folds and the recovery % of final purified enzyme was 19.32, 17.14 with a specific activity of 7.87, 7.52 µmol/min/mg. Results also show that activity of purified lipase was highest at pH 8 in both the samples. Highest lipase activity was recorded at temperatures between 37 and 40°C with a remarkable activity at 37°C and 38°C in healthy and pebrinised samples. The enzyme activity reduced with addition of NaCl, Urea and MgCl2 whereas EDTA and CaCl2 increased the activity. The molecular weight of the purified enzyme was 30 kDa as determined by SDS-polyacrylamide gel electrophoresis.

Conclusion: Pebrine disease has reduced the recovery percentage and also the specific activity of the enzyme. Maximum activity was recorded at high temperature in both the samples. During pebrine infection the midgut of fifth instar larvae has got influenced highly with a significant variation in many biochemical components including enzymes. Pebrine spores are the indicators of disease incidence.