Fetuin O-glikanlarının, Bioaktif N-Glikanların Yeni Endo-B-N-asetilglukozaminidaz Tarafından İzole Edilmesindeki Katkısının Belirlenmesi
İnek fetuini N- ve O- glikanları içerdiğinden dolayı farklı glikosidazların aktivitesini test etmek için kullanılan model bir proteindir. Yeni bir glikosidaz olan endo-B-N-asetilglukozaminidaz enzimi (EndoBI-1) bebek bağırsaklarında bulunan Bifidobakteri infantis’ten tarafımızca daha önceden izole edilmiştir. Bu enzim farklı protein yapılarında bulunan N-glikan merkezlerini kesebilmektedir. Enzimin yüksek aktivite ve geniş substrat aralığından dolayı, peynir altı suyu proteinleri gibi karmaşık yapılarda aktivite gösterebilmektedir. Ayrıca, bu enzim aktivitesini yüksek sıcaklıklarda koruyabildiği için, pastörizasyon gibi ısıl işlem gerektiren süreçlerde de kullanılabilmektedir. İnek peynir altı suyu, yıllık milyonlarca ton üretilen bir glikoprotein kaynağıdır. Fakat, EndobI-1 enziminin bu substrata uygulanması, peynir altı proteinlerinin O-glikanları tarafından bloke edilip aktiviteyi düşürdüğü düşünülmemektedir. O-glikanların, proteinleri bir korucuyu görevi ile koruyarak, yeni bir prebiyotik kaynağı olarak kabul edilen N-glikanların izolasyonunu zorlaştırmakta olduğu sanılmaktadır. Bu çalışmada, O-glikanlar fetuinden ayrılarak, bunun N-glikanların enzimatik olarak EndoBI-1 tarafından ayrılmasına olan katkısı incelenmiştir. Ayrılan glikanlar kütle spektrometresi ile analiz edilmiş ve izomerler dahil 22 farklı yapı gözlemlenmiştir. Sonuçlara göre, O-glikanların fetuinden ayrılması, N-glikan izolasyon etkinliğini (Kcat/Km) 0.52 ‘den 1.54 ml/mg x min-1’a çıkarırken, Km değerini 0.32 ‘den 0.22 mg/ml’ye düşürmüştür. Bu sonuçlara göre, EndobI-1 süt endüstrisinde çok daha etkili bir şekilde kullanabilmesinin yolu açılmıştır.
Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-β-N-acetylglucosaminidase
Bovine fetuin is a model protein to study the activity of variousglycosidases since it contains both N- and O- glycans attached to thepolypeptide chain. We recently showed a novel glycosidase, endo-β-Nacetylglucosaminidaseisolated from an infant gut microbe,Bifidobacterium infantis. This enzyme is capable of cleaving the N-N’-diacetyl chitobiose moiety found in the N-glycan core of a wide varietyof proteins. It is considered a promising approach to release N-glycansfrom complex substrates such as whey proteins due to its high activityand wide substrate specificity. Moreover, it also maintains its activityat high temperatures enabling the use of this enzyme in thermal dairyprocesses such as during the pasteurization. Bovine whey is apotential source of glycans providing million tons of glycoproteinsannually. Application of EndoBI-1 on bovine whey is challenging dueto the complexity of the whey proteins and their O-glycosylationpattern. O-glycans are considered to be a protective agent for Ndeglycosylationthat hinders the isolation of these recently found novelcompounds. In this study, O-glycans were removed from fetuin (bothO- and N- glycosylated model glycoprotein) and the contribution of Oglycansto the accessibility of EndoBI-1 to bovine fetuin N-glycanswere tested. Released glycans were characterized by advanced massspectrometry and 22 different N-glycans (including isomers) weremonitored. According to the results, it was shown that removing Oglycansfrom Fetuin increases the Kcat/Km value 0.52 to 1.54 ml/mgx min-1 and the affinity of EndoBI-1 (Km value from 0.32 to 0.22mg/ml) to target N-glycans enabling more feasible application of thisenzyme in dairy streams.
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