Avrupa Deniz Levreği (Dicentrarchus labrax) Pilorik Sekasından Tripsin Enziminin Saflaştırılması ve Kısmi Karakterizasyonu

Year 2022, Volume: 18 Issue: 2, 259 - 272, 01.06.2022

Nihat Karasu , Ali Günlü

https://doi.org/10.22392/actaquatr.1036035

Abstract

Avrupa deniz levreği (Dicentrarchus labrax) pilorik sekasından tripsin enzimi, amonyum sülfat çöktürmesi (%30-%60) ve bir boyut dışlama kromotografisi olan Sephacryl S-200 kolonu kullanılarak saflaştırılmış; saflığı, sıcaklık ve pH’ya hassaslığı hesaplanarak karakterize edilmiştir. Avrupa deniz levreğinden tripsin enzimi 52,71 kat saflaştırılmış olup, %8,60 verimlilik oranına sahiptir. Enzimin molekül büyüklüğü SDS-PAGE elektroforez analizi sonucu 24 kDA molekül büyüklüğünde tek bant olarak tespit edilmiştir. Tripsin enzimi substrat olarak BAPNA (Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride) kullanılarak yapılan stabilite analizlerinde pH 8,0 ve 55 ºC’de optimal aktivite göstermiştir. Ancak, aktivitesinin 50 ºC’den sonra %30 ve fazlasını, 70 ºC’de ise tamamını kaybetmiştir. En iyi stabiliteyi pH 7,0-10,0 arasında göstermiştir. Böylece Avrupa deniz levreğinden geniş pH ve sıcaklık aralıklarında aktivite gösteren, farklı sanayi dallarında kullanılabilecek tripsin enzimi elde edilmiştir. Bulgular, sıcaklık ve pH faktörlerinin tripsin enzimi aktivitesini önemli düzeyde etkilediğini göstermiştir (P<0,05).

Keywords

Dicentrarchus labrax, pilorik seka, tripsin, BAPNA, saflaştırma

Supporting Institution

Muğla Sıtkı Koçman Üniversitesi, Bilimsel Araştırma Projeleri Koordinasyon Birimi

Project Number

16/156 proje

Thanks

Bu çalışma, Nihat KARASU’nun Doktora tezinin bir bölümünden özetlenmiştir. Çalışma Muğla Sıtkı Koçman Üniversitesi, Bilimsel Araştırma Projeleri Koordinasyon Birimi tarafından 16/156 proje numarası ile desteklenmiştir.

Purification and Partial Characterisation of Trypsin from Pyloric caeca of the Seabass (Dicentrarchus labrax)

Abstract

Trypsin was isolated from pyloric caeca of European seabass (Dicentrarchus Labrax) by ammonium sulphate fractionation (30-60%) and size exclusion (Sephacryl S 200) gel filtration chromatography. The enzyme was purified at a rate of 52.71 fold with a yield of 8.60%. The molecular weight of the enzyme was estimated using a low molecular weight marker (Sigma Low Range M3193) and wide molecular weight marker (Sigma Wide Range S8445). The molecular weight of the purified trypsin was estimated to be 24 kDA by sodium dodecyl sulphate-polyacrylamide (SDS-Page) gel electrophoresis, which showed only one band in bromophenol blue staining. The optimum temperature and pH for the trypsin activity were 55 °C and pH 8.0, respectively. The enzyme was extremely stable in the pH range of 7.0-10.0 and highly (70%) stable up to 50 °C after 30 minutes incubation. Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) was used as a substrate for all activity and stability analyzes. Data of the study showed that temperature and pH factors that significantly affect trypsin enzyme activity.

Keywords

Dicentrarchus labrax, pyloric caeca, trypsin, BAPNA, purification

Project Number

16/156 proje

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