Recombinant production and characterization of new bifidobacterial glycosidases
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Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Çanakkale Onsekiz Mart Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Protein glikozilasyonu, protein katlanması, stabilitesi ve enzimatik koruma dahil olmak üzere protein fonksiyonu gibi önemli rollerle ilişkilendirilmiş yaygın bir translasyon sonrası modifikasyondur. Glikoproteinler (laktoferrin, immünoglobulinler gibi), protein ve glikan ikilisinden oluşmaktadır. Bakteriler, glikoproteinleri sindirebilecek gerekli enzimleri içermediğinden onları karbon kaynağı olarak kullanamaz. Bundan dolayı bakteriler üzerinde gözlemlenen gelişimin karbon kaynağı glikanlardır. Glikanların glikoproteinlerden ayrılıp hücre içine alınabilmesi için ise önce bakterilerin glikosidazlarının bu glikanları kesmeleri gerekmektedir. Glikoproteinlere bağlanan N-glikanlar, konakçı tarafından sindirilemez ve bazı yararlı bakteriler tarafından tüketildikleri kalın bağırsağa ulaşır. Bu nedenle, bağırsak mikrobiyomundaki yararlı mikroorganizmaları seçici bir şekilde uyarabilen yeni nesil prebiyotik bileşikler olarak kabul edilirler. Bifidobakteriler, N-glikan salınımı için benzersiz bir genomik yeteneğe sahiptir. Bu tez kapsamında, glikoproteinlerden N-glikan salınımı için farklı organizmalardan (bebekler, tavşanlar, tavuklar ve yaban arıları) izole edilen Bifidobakteri suşlarından yeni glikosidazların rekombinant üretimi amaçlanmıştır. İlk olarak, Bifidobacterium longum subsp. infantis ATCC 15697'den izole edilen endo-?-N-asetilglukozaminidaz enziminin amino asit dizisine benzerlikleri açısından farklı Bifidobakteri suşlarından yeni glikosidazlar seçilmiştir. Bu enzimlere genetiksel olarak farklı füzyon etiketler (N-His, SUMO, C-His) eklenmiştir ve enzimleri kodlayan genlerin klonlaması için E. cloni 10G hücrelerinde in vivo klonlama tekniği kullanılmıştır. Hedef glikanları değiştirilen enzimler batch methodu ile yüksek verimde üretilmiş ve saflaştırılmıştır. Ardından saflaştırılan enzimlerin model protein RNase B üzerindeki aktiviteleri SDS-PAGE analiziyle test edilmiştir ve aktivite gösteren beş enzimin optimum reaksiyon koşulları ve kinetik parametreleri belirlenmiştir.
Protein glycosylation is a common post-translational modification. It has been associated with important roles such as protein function including protein folding, stability, and enzymatic protection. Glycoproteins (lactoferrin, immunoglobulins, etc.) are composed of protein and glycan structures. Bacteria cannot use them as a carbon source because they do not contain the necessary enzymes that can digest proteins. Therefore, the carbon source of growth observed in bacteria is glycans. For glycans to be released from glycoproteins and taken into the cell, glycosidases of bacteria must first cleave these glycans. N-glycans attached to glycoproteins are indigestible by the host and reach the large intestine, where they are consumed by certain beneficial bacteria. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate beneficial bacteria in the gut microbiome. Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Within the scope of this thesis, the recombinant production of novel glycosidases from Bifidobacterial strains isolated from different organisms (infants, rabbit, chicken, bumblebee) for N-glycan release from glycoproteins was aimed. Firstly, novel glycosidases from different Bifidobacterium strains were selected in terms of their similarity to the amino acid sequence of a reference enzyme, endo-?-N-acetylglucosaminidase. This enzyme was isolated from a microbe in the infant gut, Bifidobacterium longum subsp. infantis ATCC 15697. Different fusion tags (N-His, SUMO, C-His) were genetically added to these enzymes, and in vivo cloning technique was used E. cloni 10G cells to clone the genes encoding the enzymes. All enzymes were produced and purified in high efficiency by the batch method. The activities of the purified enzymes on RNase B protein were then tested by SDS-PAGE analysis. Finally, the optimum reaction conditions and kinetic parameters of five enzymes active on the protein were determined.
Protein glycosylation is a common post-translational modification. It has been associated with important roles such as protein function including protein folding, stability, and enzymatic protection. Glycoproteins (lactoferrin, immunoglobulins, etc.) are composed of protein and glycan structures. Bacteria cannot use them as a carbon source because they do not contain the necessary enzymes that can digest proteins. Therefore, the carbon source of growth observed in bacteria is glycans. For glycans to be released from glycoproteins and taken into the cell, glycosidases of bacteria must first cleave these glycans. N-glycans attached to glycoproteins are indigestible by the host and reach the large intestine, where they are consumed by certain beneficial bacteria. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate beneficial bacteria in the gut microbiome. Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Within the scope of this thesis, the recombinant production of novel glycosidases from Bifidobacterial strains isolated from different organisms (infants, rabbit, chicken, bumblebee) for N-glycan release from glycoproteins was aimed. Firstly, novel glycosidases from different Bifidobacterium strains were selected in terms of their similarity to the amino acid sequence of a reference enzyme, endo-?-N-acetylglucosaminidase. This enzyme was isolated from a microbe in the infant gut, Bifidobacterium longum subsp. infantis ATCC 15697. Different fusion tags (N-His, SUMO, C-His) were genetically added to these enzymes, and in vivo cloning technique was used E. cloni 10G cells to clone the genes encoding the enzymes. All enzymes were produced and purified in high efficiency by the batch method. The activities of the purified enzymes on RNase B protein were then tested by SDS-PAGE analysis. Finally, the optimum reaction conditions and kinetic parameters of five enzymes active on the protein were determined.
Açıklama
Lisansüstü Eğitim Enstitüsü, Biyomoleküler Bilimler Ana Bilim Dalı
Anahtar Kelimeler
Biyoloji, Biology, Biyoteknoloji
