Year 2018, Volume 21 , Issue 3, Pages 286 - 291 2018-06-15

Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-β-N-acetylglucosaminidase
Fetuin O-glikanlarının, Bioaktif N-Glikanların Yeni Endo-B-N-asetilglukozaminidaz Tarafından İzole Edilmesindeki Katkısının Belirlenmesi

Sercan KARAV [1]


Bovine fetuin is a model protein to study the activity of various glycosidases since it contains both N- and O- glycans attached to the polypeptide chain. We recently showed a novel glycosidase, endo-β-N-acetylglucosaminidase isolated 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 variety of proteins. It is considered a promising approach to release N-glycans from complex substrates such as whey proteins due to its high activity and wide substrate specificity. Moreover, it also maintains its activity at high temperatures enabling the use of this enzyme in thermal dairy processes such as during the pasteurization. Bovine whey is a potential source of glycans providing million tons of glycoproteins annually. Application of EndoBI-1 on bovine whey is challenging due to the complexity of the whey proteins and their O-glycosylation pattern. O-glycans are considered to be a protective agent for N-deglycosylation that hinders the isolation of these recently found novel compounds. In this study, O-glycans were removed from fetuin (both O- and N- glycosylated model glycoprotein) and the contribution of O-glycans to the accessibility of EndoBI-1 to bovine fetuin N-glycans were tested. Released glycans were characterized by advanced mass spectrometry and 22 different N-glycans (including isomers) were monitored. According to the results, it was shown that removing O-glycans from Fetuin increases the Kcat/Km value 0.52 to 1.54 ml/mg x min-1and the affinity of EndoBI-1 (Km value from 0.32 to 0.22 mg/ml) to target N-glycans enabling more feasible application of this enzyme in dairy streams.

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.

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Primary Language en
Subjects Science
Journal Section RESEARCH ARTICLE
Authors

Orcid: 0000-0003-4056-1673
Author: Sercan KARAV (Primary Author)

Dates

Publication Date : June 15, 2018

APA KARAV, S . (2018). Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-β-N-acetylglucosaminidase. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi , 21 (3) , 286-291 . DOI: 10.18016/ksudobil.335396