Farklı rekombinant ekspresyon sistemlerinde peynir altı suyunun değerlendirilmesi
Küçük Resim Yok
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tezde süt ürünleri endüstrisinden elde edilen ve katma değeri yüksek bir yan ürün olan peynir altı suyunun Escherichia coli ve Lactococcus lactis rekombinant ekspresyon sistemlerinde değerlendirilmesi incelenmiştir. Peynir altı suyu, laktoz, protein, lipit, vitamin ve mineraller bakımından zengin olan çok değerli bir yan üründür. Bebek mamaları, unlu mamüller, atıştırmalık ürünler ve şekerleme ürünlerinde kullanımlarının bulunmasının yanı sıra farklı mikroorganizmaların büyümesi için substrat olarak da kullanılmaktadır. Rekombinant protein üretiminde peynir altı suyunun kullanımı sayesinde daha düşük maliyetli protein üretimi ve toksik indükleyicilerin kullanımlarının azaltılması amaçlanmıştır. Yapılan bu tez çalışmasında farklı rekombinant ekspresyon sistemlerinde peynir altı suyunun kullanımı değerlendirilmiştir. Tez çalışmasında ilk olarak Escherichia coli Rosetta 2 hücresine klonlanmış model bir protein olan yeşil floresan proteini (YFP) üretiminde indükleyici olarak toksik İzopropil-?-D-tiyogalaktopiranozit (IPTG) yerine demineralize tatlı peynir altı suyu tozu (PAS), proteinlerinden uzaklaştırılmış tatlı peynir altı suyu tozu ve laktoz kullanılarak indükleyici etkileri araştırılmıştır. En düşük 2 mM laktoz içeren proteinlerinden ve minerallerinden uzaklaştırılmış peynir altı suyu tozu kullanımında dahi hücre miktarı ve protein miktarının 0.5 mM IPTG kullanımına oranla daha yüksek olduğu gösterilmiştir. En iyi sonucun 20 mM laktoz içeren PAS kullanımında olduğu saptanmıştır. Tez çalışmasının ikinci bölümünde gıda güvenlikli konakçı bir sistem olan Lactococcus lactis nisin ile indüklenebilir ekspresyon sisteminde peynir altı suyunun kültür ortamı olarak kullanımı sonucunda hücre üremesi ve rekombinant protein üretimine etkisi değerlendirilmiştir. Rekombinant protein olarak bitkisel tatlı proteinler arasından bu çalışma kapsamında literatürde ilk defa biyoinformatik aramalar ile tespit edilen, Tarenaya hassleriana genomunda kodlanan mabinlin II benzeri protein seçilmiştir. Mabinlin benzeri protein kodlayan gen bölgesi nisin ile indüklenebilir PnisA promotörü altında pLEB825 plazmidine klonlanmıştır. Mabinlin benzeri proteini A ve B olmak üzere iki polipeptit zincirinden oluşmaktadır ve bu tez çalışmasında AB ve B olmak üzere iki ayrı formda klonlanmıştır. Elde edilen rekombinant plazmitler L. lactis NZ9000 hücrelerine aktarılmıştır ve PAS varlığında mikroorganizmanın gelişimi incelenmiştir
In this thesis, utilization of whey, a high value-added by-product obtained from the dairy industry, in Escherichia coli and Lactococcus lactis recombinant expression systems was investigated. Whey is a very valuable by-product rich in lactose, protein, lipid, vitamins, and minerals. In addition to its use in infant formulas, bakery products, snack products and confectionery products, it is also used as a substrate for the growth of different microorganisms. With the use of whey in the production of recombinant protein, it is aimed to produce low-cost protein and to reduce the use of toxic inducers. In this thesis, the use of whey in different recombinant expression systems was investigated. In the first part of the study, use of demineralized sweet whey powder, deproteinized whey powder and lactose as inducers were investigated and compared with the classical but toxic inducer Isopropyl-?-D-thiogalactopyranoside (IPTG) in the production of green fluorescent protein (GFP) from Escherichia coli Rosetta 2 cells. Results showed that deproteinized whey powder was an effective inducer at lactose concentrations as low as 2 mM. Whey powder resulted in increased cell concentrations, and this resulted in increased amount of GFP production when compared with results obtained with 0.5 mM IPTG. The best results in terms of protein production was obtained when 20 mM lactose equivalent whey was used. In the second part of the thesis, the effect of using whey as a culture medium in the recombinant protein production from nisin inducible with Lactococcus lactis, which is a food-grade host system, was investigated. The recombinant protein chosen for this purpose was a putative sweet plant protein from Tarenaya hassleriana, which is identified, for the first time, in this study based on its sequence and structure similarity to mabinlin II. The gene region encoding the mabinlin-like protein was cloned into plasmid pLEB825 under the nisin-inducible PnisA promoter. Mabinlin-like protein consists of two polypeptide chains, A and B, and therefore two constructs, AB and B, were cloned in this thesis. L. lactis NZ9000 cells were transformed with the recombinant plasmids and microbial growth using whey-based media were investigated.
In this thesis, utilization of whey, a high value-added by-product obtained from the dairy industry, in Escherichia coli and Lactococcus lactis recombinant expression systems was investigated. Whey is a very valuable by-product rich in lactose, protein, lipid, vitamins, and minerals. In addition to its use in infant formulas, bakery products, snack products and confectionery products, it is also used as a substrate for the growth of different microorganisms. With the use of whey in the production of recombinant protein, it is aimed to produce low-cost protein and to reduce the use of toxic inducers. In this thesis, the use of whey in different recombinant expression systems was investigated. In the first part of the study, use of demineralized sweet whey powder, deproteinized whey powder and lactose as inducers were investigated and compared with the classical but toxic inducer Isopropyl-?-D-thiogalactopyranoside (IPTG) in the production of green fluorescent protein (GFP) from Escherichia coli Rosetta 2 cells. Results showed that deproteinized whey powder was an effective inducer at lactose concentrations as low as 2 mM. Whey powder resulted in increased cell concentrations, and this resulted in increased amount of GFP production when compared with results obtained with 0.5 mM IPTG. The best results in terms of protein production was obtained when 20 mM lactose equivalent whey was used. In the second part of the thesis, the effect of using whey as a culture medium in the recombinant protein production from nisin inducible with Lactococcus lactis, which is a food-grade host system, was investigated. The recombinant protein chosen for this purpose was a putative sweet plant protein from Tarenaya hassleriana, which is identified, for the first time, in this study based on its sequence and structure similarity to mabinlin II. The gene region encoding the mabinlin-like protein was cloned into plasmid pLEB825 under the nisin-inducible PnisA promoter. Mabinlin-like protein consists of two polypeptide chains, A and B, and therefore two constructs, AB and B, were cloned in this thesis. L. lactis NZ9000 cells were transformed with the recombinant plasmids and microbial growth using whey-based media were investigated.
Açıklama
Anahtar Kelimeler
Biyomühendislik, Bioengineering, Biyoteknoloji, Biotechnology
