Prolin-spesifik endopeptidazların saflaştırılması, karakterizasyonu ve proteolitik proseslerde kullanım potansiyelinin araştırılması
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Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Prolil oligopeptidaz (POP, EC 3.4.21.26) prolin içeren küçük biyolojik peptidleri hidrolizleyen sitozolik bir serin proteazdır. Prolil oligopeptidaz ailesinin üyeleri, nörodejeneratif hastalıklar, peptid hormonları ve nöro peptidlerin yapımı ve yıkımı, öğrenme ve hafıza, DNA sentezi, hücre farklılılaşması ve sinyal iletimi gibi bazı fizyolojik proseslerde önemli rol oynarlar. Bu nedenle teröpatik olarak potansiyel kullanımını açıklayabilmek için çeşitli kaynaklardan saflaştırılmış ve karakterize edilmiştir. Bu çalışmada E.coli'den eksprese edilen rekombinant Myxococcus xanthus Prolil oligopeptidaz metal-şelat affinite ve jel filtrasyon kromatografisi kullanılarak 224 kat saflaştırıldı. Saflaştırılan enzimin molekül kütlesi 70 kDa (monomerik) ve izoelektrik noktası 6.3 olarak belirlendi. Optimum pH ve sıcaklık sırasıyla 7.5 ve 37°C olarak bulundu. Z-Pro-Prolinal ve PMSF gibi çeşitli serin proteaz inhibitörlerinin enzim aktivitesini inhibe ettiği görüldü. Ayrıca saflaştırılan enzimin substrat spesifikliği (sentetik/doğal) ve stabilitesi (termal, pH ve depo kararlılığı) araştırıldı. Elde edilen sonuçlardan Myxococcus xanthus'dan saflaştırılan Prolil oligopeptidazın diğer kaynaklardan elde edilen prolil oligopeptidazlar ile benzerlik gösterdiği görülmektedir. Enzimin potansiyel kullanımı buğday gluten ununun hidrolizi ile test edildi. Bu amaçla buğday gluteni, koşullar optimize edildikten sonra Prolil oligopeptidaz enzimi ile enzimatik hidrolize maruz bırakıldı. Açığa çıkan gluten hidrolizatının antioksidan antimikrobiyal, tripsin ve prolil oligopeptidaz inhibisyon aktiviteleri araştırıldı.
Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small biological peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones and neuropeptides, learning and memory, DNA synthesis, cell differentiation and signal transduction. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus Prolyl oligopeptidase expressed in E.coli was purified 224 fold, using metal-chelate affinity and gel permeation chromatography. The purified enzyme exhibited a monomeric molecular weight of 70 kDa, and an isoelectric point of 6.3. The optimum pH and temperature was estimated as 7.5 and 37°C, respectively. Enzyme activity was inhibited by various serin protease inhibitors such as Z-Pro-Prolinal and PMSF. Furthermore the substrate specificity (synthetic/natural) and the stability (thermal, pH and storage stability) of the purified enzyme were also investigated. The data showed that the Myxococcus xanthus Prolyl oligopeptidase is similar to Prolyl oligopeptidase isolated from other sources. The potential use of the enzyme was tested by the hydrolysis of the wheat gluten. For this purpose wheat gluten was subjected to enzymatic hydrolysis with Prolyl oligopeptidase, after optimizing the conditions. The resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and Prolyl oligopeptidase inhibition activities.
Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small biological peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones and neuropeptides, learning and memory, DNA synthesis, cell differentiation and signal transduction. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus Prolyl oligopeptidase expressed in E.coli was purified 224 fold, using metal-chelate affinity and gel permeation chromatography. The purified enzyme exhibited a monomeric molecular weight of 70 kDa, and an isoelectric point of 6.3. The optimum pH and temperature was estimated as 7.5 and 37°C, respectively. Enzyme activity was inhibited by various serin protease inhibitors such as Z-Pro-Prolinal and PMSF. Furthermore the substrate specificity (synthetic/natural) and the stability (thermal, pH and storage stability) of the purified enzyme were also investigated. The data showed that the Myxococcus xanthus Prolyl oligopeptidase is similar to Prolyl oligopeptidase isolated from other sources. The potential use of the enzyme was tested by the hydrolysis of the wheat gluten. For this purpose wheat gluten was subjected to enzymatic hydrolysis with Prolyl oligopeptidase, after optimizing the conditions. The resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and Prolyl oligopeptidase inhibition activities.
Açıklama
Anahtar Kelimeler
Serin Proteaz, Prolil Oligopeptidaz, Bioaktif Peptid, 2,4,6‐Trinitrobenzen Sülfonik Asit, Serine Protease, Prolyl Oligopeptidase, Bioactive Peptides, 2,4,6‐Trinitrobenzene Sulfonic Acid
