Larva dönemindeki Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae)’dan bir esterazın saflaştırılması ve karakterizasyonu

Öz

Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae) Rosaceae türlerinde zararlara yol açan en önemli böcek türlerinden birisidir. Biyokimyasal veri elde etmek için Cumhuriyet Üniversitesi, Fen Fakültesi, Biyokimya Anabilim Dalı laboratuvarında 2018 yılında başlatılan bu çalışma ile D. fructuum’un larvasından bir esteraz (EC 3.1.1.X) Q Sefaroz anyon değişim, fenil Sefaroz CL-4B ve Sefakril S 100 HR jel filtrasyon kromatografisini kullanarak saflaştırılmıştır. Enzim 6.94 U/mg protein spesifik aktivite, yaklaşık 29 kat saflık ve %8.80 verime sahipti. Nativ-PAGE çalışmalarında sadece bir aktivite bandı gözlenmiştir. Nativ-PAGE ve SDS-PAGE tekniklerini kullanarak, esterazın molekül kütlesi yaklaşık olarak 60 kDa olarak tahmin edilmiştir. Kinetik datadan, enzimin optimum sıcaklık ve pH’ı sırasıyla 40ºC ve 9.0 olarak belirlenmiştir. Enzim, 40ºC ve pH 8.0’da 4 saat kararlıydı. 4 nitrofenil butirat (p-NPB) substrat olarak kullanılarak, K_m ve V_max değerlerinin 0.035 mM and 1.41 µmol/mL.dk olduğu bulunmuştur. Enzim en yüksek aktivitesinip-NPB (%100) ve 4-nitrofenil asetat (%52) üzerinde sergilemiştir. Tüm bu veriler, enzimin diğer böcek türlerinden bildirilen esterolitik enzimlerden farklı kinetik özellik ve molekül kütlesi ile klasik bir esteraz olabileceğini göstermektedir.

Anahtar Kelimeler

• Kolon kromatografisi,Diplolepis fructuum,esteraz,larva,saflaştırma

Purification and characterization of an esterase from larval Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae)

Abstract

Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae) is one of the important insect species that causes damages on Rosaceae species. With this study commenced in 2018 at the laboratory of Department of Biochemistry, Faculty of Science, Cumhuriyet University to get a biochemical data, an esterase (EC 3.1.1.X) from the larvae of D. fructuum was purified using Q Sepharose anion exchange, phenyl Sepharose CL-4B and Sephacryl S100 HR gel filtration chromatography, respectively. The enzyme had 6.94 U/mg protein specific activity, about 29-fold purity, and 8.8% yield. Only one activity band was observed in native-PAGE studies. The molecular weight of the esterase was estimated as 60 kDa using native-PAGE and SDS-PAGE techniques. By the kinetic data, optimum temperature and pH for the enzyme was determined as 40ºC and 9.0, respectively. The enzyme was stable for 4 h at 40ºC and pH 8.0. K_m and V_max values were found to be 0.035 mM and 1.41 µmol/mL.min., using 4-nitrophenyl butyrate (p-NPB) as substrate. The enzyme exhibited its highest activities on p-NPB (100%) and 4-nitrophenyl acetate (52%). All of these data indicate that the enzyme might be a typical esterase with different kinetic properties and molecular weight than esterolytic enzymes reported from other insect species.

Keywords

• Column chromatography,Diplolepis fructuum,esterase,larvae,purification

Kaynakça

1. Arrese, E. L. & M. A. Wells, 1994. Purification and properties of a phosphorylatable triacylglycerol lipase from the fat body of an insect, Manduca sexta. Journal of Lipid Research, 35: 1652-1660.
2. Fahmy, A. S., S. S. Abdel-Gany, T. M. Mohamed & S. A. Mohamed, 2004. Esterase and lipase in camel tick Hyalomma dromedarii (Acari: Ixodidae) during embryogenesis. Comparative Biochemistry and Physiology Part B, 137: 159-168.
3. Akpınar, M. A., S. Görgün, L. Gençer & A. Aktümsek, 2017. Fatty acid composition of Diplolepis fructuum (Rübsaamen, 1895) (Hymenoptera: Cynipidae) during its developmental stages. Journal of the Entomological Research Society, 19 (3): 109-118.
4. Ma, M., H. Jia, X. Cui, N. Zhai, H. Wang & X. Guo, 2018. Isolation of carboxylesterase (esterase FE4) from Apis cerana cerana and its role in oxidative resistance during adverse environmental stress. Biochimie, 144: 85-97.
5. Nauen, R., 2007. Insecticide resistance in disease vectors of public health importance. Pest Management Science, 63: 628-633.
6. Qerioz, J. A., C. T. Tomaz & J. M. S. Cabral, 2001. Hydrophobic interaction chromatography of proteins. Journal of Biotechnology, 87: 143-159.
7. Raman, A., 2011. Morphogenesis of insect-induced plant galls: facts and questions. Flora, 206: 517-533.
8. Ronquist, F., 1999. Phylogeny, classification and evolution of the Cynipoidea. Zoologica Scripta, 28: 139-164.

9. Rossiter, L. C., C. M. Conyers, A. D. MacNicoll & H. A. Rose, 2001. Two qualitatively different B-esterases from two organophosphate-resistant strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) and their roles in fenitrothion and chlorpyrifos-methyl resistance. Pesticide Biochemistry and Physiology, 69: 118-130.
10. Arrese, E. L., R. T. Patel & J. L. Soulages, 2006. The main triglyceride-lipase from the insect fat body is an active phospholipase A1: identification and characterization. Journal of Lipid Research, 47: 2656-2667.
11. Yaicharoen, R., R. Kiatfuengfoo, T. Chareonviriyaphap & P. Rongnoparut, 2005. Characterization of deltamethrin resistance in field populations of Aedes aegypti in Thailand. Journal of Vector Ecology, 30 (1): 144-150.
12. Bhardwaj, K., A. Raju & R. Rajasekharan, 2001. Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family. Plant Physiology, 127 (4): 1728-1738.
13. Bollag, D. M., M. D. Rozycki & S. J. Edelstein, 1996. Protein Methods. A John Wiley and Sons Inc. Publication, 432pp.
14. Bompensieri, S., R. Gonzalez, R. Kok, M. V. Miranda, I. Nutgeren-Roodzant, K. J. Hellingwerf, O. Cascone & B. C. Nudel, 1996. Purification of lipase from Acinetobacter calcoaceticus AAC323-1 by hydrophobic interaction methods. Biotechnology and Applied Biochemistry, 23 (1): 77-81.
15. Bülow, L. & K. Mosbach, 1987. The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters. Federation of European Biochemical Societies, 210 (2): 147-152.
16. Canavaso, L.E., Z. E. Jouni, K. J. Karnas, J. E. Pennington & M. A. Wells, 2001. Fat metabolism in insects. Annual Review of Nutrition, 21: 23-46.
17. Dahan-Moss, Y. L. & L. L. Koekemoer, 2016. Analysis of esterase enzyme activity in adults of the major malaria vector Anopheles funestus. Parasites & Vectors, 9: 110.
18. Fojan, P., P. H. Jonson, M. T. N. Petersen & S. B. Petersen, 2000. What distinguishes an esterase from a lipase: A novel structural approach. Biochimie, 82 (11): 1033-1041.
19. Gong, Y. H., G. M. Ai, M. Li, X. Y. Shi, Q. Y. Diao & X. W. Gao, 2017. Functional characterization of carboxylesterase gene mutations involved in Aphis gossypii resistance to organophosphate insecticides. Insect Molecular Biology, 26 (6): 702-714.
20. Görgün, S. & M. A. Akpınar, 2012. Purification and characterization of lipase from the liver of carp, Cyprinus carpio L. (1758), living in Lake Tödürge (Sivas, Türkiye). Turkish Journal of Fisheries and Aquatic Sciences, 12 (2): 207-215.
21. Görgün, S. & G. Zengin, 2015. Determination of fatty acid profiles and esterase activities in the gills and gonads of Vimba vimba (L., 1758). Journal of the American Oil Chemist’s Society, 92 (3): 353-360.
22. Katılmış, Y. & S. Kıyak, 2009. The oak gall wasp Aphelonyx persica: a new record from Turkey, with some new host records. Phytoparasitica, 37: 95-97.
23. Li, X., M. A. Schuler & M. R. Berenbaum, 2007. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Annual Review of Entomology, 52: 231-253.
24. Lotfalizadeh, H., R. Ezzati-Tabrizi & A. Masnadi-Yazdinejad, 2009. Diplolepis fructuum (Rübsaamen) (Hym.: Cynipidae) a new host for Exeristes roborator (Fabricius) (Hym.: Ichneumonidae) in Iran. Biharean Biologist, 3: 171-173.
25. Montella, I. R., R. Schama & D. Valle, 2012. The classification of esterases: an important gene family involved in insecticide resistance-A review. Memorias do Instituto Oswaldo Cruz, Rio de Janeiro, 107 (4): 437-449.
26. Ødegaard, F., 2000. How many species of arthropods? Erwin’s estimate revised. Biological Journal of the Linnean Society, 71 (4): 583-597.
27. Ranjbar, M., A. Zibaee & J. J. Sendi, 2015. Purification and characterization of a digestive lipase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae). Frontiers in Life Science, 8 (1): 64-70.
28. Santana, C. C., L. A. Barbosa, I. D. B. Junior, T. G. do Nascimento, C. B. Dornelas & L. A. M. Grillo, 2017. Lipase activity in the larval midgut of Rhynchophorus palmarum: Biochemical characterization and the effects of reducing agents. Insects, 8 (100): 1-7.
29. Satoh, T., 2005. Toxicological implications of esterases-From molecular structures to functions. Toxicology and Applied Pharmacology, 207: 11-18.
30. Satoh, T., P. Taylor, W. F. Bosron, S. P. Sanghanı, M. Hosokawa & B. N. La Du, 2002. Current progress on esterases: From molecular structure to function. Drug Metabolism and Disposition, 30 (5): 488-493.
31. Shin, D. & C. T. Smartt, 2016. Assessment of esterase gene expression as a risk marker for insecticide resistance in Florida Culex nigripalpus (Diptera: Culicidae). Journal of Vector Ecology, 41 (1): 63-71.
32. Tabasian, H., S. Ravan, A. R. Bandani & B. A. Siahsar, 2010. The effect of esterase activity in resistance of Aphis gossypii to selective insecticides. Journal of Food, Agriculture & Environment, 8 (3&4): 1108-1112.
33. Zibaee, A., 2012. A digestive lipase of Pieris brassicae L. (Lepidoptera: Pieridae): purification, characterization, and host plants effects. Archieves of Insect Biochemistry and Physiology, 81 (1): 1-19.
34. Zibaee, A., A. R. Bandani & S. Ramzi, 2008. Lipase and invertase activities in midgut and salivary glands of Chilo suppressalis (Walker) (Lepidoptera, Pyralidae), rice striped stem borer. Invertebrate Survival Journal, 5: 180-189.
35. Zibaee, A. & M. Fazeli-Dinan, 2012. Purification and characterization of a digestive lipase in rice green semi-looper, Naranga aenescens Moore (Lepidoptera: Noctuidae). Signpost Open Access Journal of Entomological Studies, 1: 38-54.
36. Zou, C.-S., C.-W. Cao, G.-C. Zhang & Z.-Y. Wang, 2014. Purification, characterization, and sensitivity to pesticides of carboxylesterase from Dendrolimus superans (Lepidoptera: Lasiocampidae). Journal of Insect Science, 14 (260): 1-6.