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

Year 2019, Volume: 43 Issue: 4, 367 - 376, 01.12.2019

Mazen Althaljı Salih Görgün

https://doi.org/10.16970/entoted.533752

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

Supporting Institution

This work is MSc study of the first author This study was supported by Sivas Cumhuriyet University, Scientific Research Unit, (CUBAP), Turkey, Grant Project No: F-601.

Project Number

F-601

Thanks

The authors convey their special thanks to Scientific Research Fund of Sivas Cumhuriyet University (CUBAP) for financial support under the project number F-601. Also, the authors thank Prof. Dr. Lütfiye Gençer (Department of Biology, Sivas Cumhuriyet University) for her helps during the rearing and diagnosis of the insect.

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

Abstract

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.

Keywords

Kolon kromatografisi, Diplolepis fructuum, esteraz, larva, saflaştırma

Project Number

F-601

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