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首页> 外文期刊>British Biotechnology Journal >Isolation and Purification of Lipase from the Midgut of Fifth Instar Larvae of Antheraea mylitta drury
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Isolation and Purification of Lipase from the Midgut of Fifth Instar Larvae of Antheraea mylitta drury

机译：my蚕五龄幼虫中肠脂肪酶的分离与纯化

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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.
机译：通过45％至85％（NH4） 2 SO 4 分级分离纯化健康和去蛋白幼虫的脂肪酶，然后进行Sephadex S-100凝胶过滤和CM-Sepharose。在两个样品中，报告的最终酶纯化度均为19.02和17.7倍（大小），最终纯化酶的回收率为19.32％和17.14％，比活性为7.87和7.52μmol/ min / mg。结果还表明，在两个样品中，纯化的脂肪酶的活性在pH 8时最高。在37°C至40°C的温度之间记录到最高的脂肪酶活性，健康样品中的脂肪酶活性最高，在37°C的温度下，在纤维化样品中则为38°C。加入NaCl，尿素和MgCl 2 会降低酶的活性，而EDTA和CaCl 2 会增加酶的活性。通过SDS-聚丙烯酰胺凝胶电泳测定，纯化的酶的分子量为30kDa。目的：进行本研究以分离，纯化和鉴定来自健康和卵白化的五色花An幼虫中肠的脂肪酶。研究设计：研究涉及从健康和卵白幼虫的第四天第五龄幼虫中肠解剖，从两个样品中获得的脂肪酶均通过45-85％（NH4） 2 SO 4 分级分离，然后进行Sephadex S-100凝胶过滤和CM-Sepharose，在不同温度和不同pH下测量比活性。脂肪酶的分子量通过SDS PAGE测定。研究的地点和时间：2015年8月，从瓦兰加尔的贾卡拉姆邦（18.1E和79.8 N）的森林中收集了健康的第四天幼化的五龄幼虫。 （NH4） 2 SO 4 分级分离，Sephadex S-100凝胶过滤和CM-Sepharose酶纯化。测量适合最高脂肪酶活性的温度，pH，并测量针对各种化学物质的比活性。动力学参数（例如Km和Vmax）通过Sigmaplot软件版本11进行估算。使用SDS聚丙烯酰胺凝胶测定脂肪酶的分子量。结果：在健康幼虫和去皮幼虫中，最终酶的纯化率分别为19.02、17.7倍和最终纯化酶的回收率分别为19.32、17.14和7.87、7.52μmol/ min / mg的比活。结果还表明，在两个样品中，纯化的脂肪酶的活性在pH 8时最高。在37至40°C的温度下记录到最高的脂肪酶活性，在健康和白蛋白化的样品中，在37°C至38°C的温度下具有显着的活性。加入NaCl，尿素和MgCl 2 会降低酶的活性，而EDTA和CaCl 2 会增加酶的活性。通过SDS-聚丙烯酰胺凝胶电泳测定，纯化的酶的分子量为30kDa。结论：小儿麻痹症降低了该酶的回收率并降低了其比活性。在两个样品中均记录了在高温下的最大活性。在小白菜感染期间，五龄幼虫的中肠受到很大的影响，包括酶在内的许多生化成分发生了显着变化。小儿孢子是疾病发病率的指标。

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