Zymomonas mobilis levansukraz geninin Lactococcus Lactis konakçısına klonlama çalışmaları
Küçük Resim Yok
Tarih
2023
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Levan ve levan tipi fruktooligosakkaritlerin oluşumunu katalizleyen levansukraz enzimleri (E.C.2.4.1.10) glikozit hidrolazlar (GH68) ailesine ait olan bakteriyel hücre dışı enzimlerdir. Levansukraz enzimi birçok mikroorganizma tarafından üretilebilmektedir. Bu tez çalışmasında Zymomonas mobilis NRRL B-14023 levansukrazının geninin Lactococcus lactis ekspresyon sistemine moleküler klonlanması ve rekombinant üretiminin incelenmesi hedeflenmiştir. Bu amaçla, pLEB825 plazmidine PnisA promotörü altında Z. mobilis levansukraz geni klonlanarak L. lactis hücresine transfer edilmesi için araştırmalar yapılmıştır. Z. mobilis levansukraz geninin pLEB825 plazmidine entegrasyonu için restriksiyon enzim kesimi ve ligasyon reaksiyonları farklı koşullarda denenmiştir. Elde edilen rekombinant plazmidler tez çalışmasında ara konakçı olarak kullanılan transformasyon veriminin daha yüksek olduğu Escherichia coli TG1 hücrelerine aktarılmıştır. Escherichia coli TG1 hücrelerinden izole edilen rekombinant plazmidler ve doğrudan ligasyon karışımları kompeten L. lactis hücrelerine aktarılması için elektroporasyon denemelerine tabi tutulmuştur. Sonuç olarak, bu çalışma Z. mobilis levansukrazının gıdalar ic?in gu?venli konakc?ı sisteminden olan L. lactis ekspresyon sistemine klonlanması için ilk adımları atmayı amaçlamaktadır. Bu araştırma, gelecekte biyoteknoloji ve endüstriyel uygulamalarda levansukraz enzimlerinin üretimi ve kullanımı açısından önemli bir adım olabilir.
Levansucrases (E.C.2.4.1.10), which catalyze the formation of levan and levan-type fructooligosaccharides, are bacterial extracellular enzymes belonging to the family of glycoside hydrolases (GH68). Levansucrase enzyme can be produced by many microorganisms. In this thesis, it was aimed to investigate the molecular cloning and recombinant production of Zymomonas mobilis NRRL B-14023 levansucrase in Lactococcus lactis expression system. In the recombinant production studies of the enzyme, the cloning of the gene region encoding the Z. mobilis NRRL B-14023 levansucrase protein into the plasmid pLEB825 under the PnisA promoter and the resulting recombinant plasmids were transferred to L. lactis NZ9000 cells was investigated. For that purpose, different conditions were investigated in the restriction enzyme and ligation reactions of the Z. mobilis levansucrase gene and the pLEB825 plasmid. The recombinant plasmids obtained were transferred into Escherichia coli TG1 cells with higher transformation efficiency, which were used as intermediate hosts in the thesis study. Electroporation experiments were performed to transfer the obtained recombinant plasmids to competent L.lactis cells. In conclusion, it has been reported the investigation of the cloning and recombinant production of Z.mobilis levansucrase in L. lactis expression system, which has recently emerged as a promising host for the production of recombinant proteins. This research represents an important advancement in the production and utilization of levansucrase enzymes in future biotechnology and industrial applications.
Levansucrases (E.C.2.4.1.10), which catalyze the formation of levan and levan-type fructooligosaccharides, are bacterial extracellular enzymes belonging to the family of glycoside hydrolases (GH68). Levansucrase enzyme can be produced by many microorganisms. In this thesis, it was aimed to investigate the molecular cloning and recombinant production of Zymomonas mobilis NRRL B-14023 levansucrase in Lactococcus lactis expression system. In the recombinant production studies of the enzyme, the cloning of the gene region encoding the Z. mobilis NRRL B-14023 levansucrase protein into the plasmid pLEB825 under the PnisA promoter and the resulting recombinant plasmids were transferred to L. lactis NZ9000 cells was investigated. For that purpose, different conditions were investigated in the restriction enzyme and ligation reactions of the Z. mobilis levansucrase gene and the pLEB825 plasmid. The recombinant plasmids obtained were transferred into Escherichia coli TG1 cells with higher transformation efficiency, which were used as intermediate hosts in the thesis study. Electroporation experiments were performed to transfer the obtained recombinant plasmids to competent L.lactis cells. In conclusion, it has been reported the investigation of the cloning and recombinant production of Z.mobilis levansucrase in L. lactis expression system, which has recently emerged as a promising host for the production of recombinant proteins. This research represents an important advancement in the production and utilization of levansucrase enzymes in future biotechnology and industrial applications.
Açıklama
Anahtar Kelimeler
Gıda Mühendisliği, Food Engineering
