Molecular cloning, characterization and comparison of bile salt hydrolase enzyme from Lactobacillus gasseri
Küçük Resim Yok
Tarih
2016
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Bolu Abant İzzet Baysal Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Geçtiğimiz on yıl içinde, aktif safra tuzu hidrolaz (STH) enzimi taşıyan probiyotikler hiperkolesterolemi temelli hastalıkların tedavisinde kullanılmaktadır. BSH enziminin safra tuzlarını sebest safra asitlerine ve glisin veya taurin amino asitlerine dönüştürme yeteneği, bu STH enziminin probiyotik seçiminde önemli bir kriter oluşturmaktadır. Bu dekonjugasyon sadece bakterilerin gastro intestinal sistemde hayatta kalabilmesi için tolerans sağlamaz, aynı zamanda insan konakçısının kan kolesterol düzeyinin azaltılmasında önemli bir rol oynar. Bu nedenle, tedavilerde kullanılan probiyotiklerin ve safra tuzu hidrolaz enzimlerin etki mekanizmasının bilinmesi ve anlaşılması önemlidir. Lactobacillus gasseri muhtemelen piyasadaki en yararlı probiyotiklerden biri olsa da, bu mikroorganizmanın STH enziminin katalitik aktivitesi ve substrat tercihleri etki mekanizması ile ilgili çok az bilgi bulunmaktadır. Bu çalışmada, Lb.gasseri ATCC 33323 suşundan elde edilen sth geninin Escherichia coli BLR(DE3) suşuna klonlanmış, ifade edilmiş ve tanımlanmış. Bu yaban tip rekombinan STH (yrSTH) enzimin hidroliz aktivitesi 25°C ile 65°C arasında değişen farklı sıcaklıklarda ve 4.0 ile 8.0 pH aralıklarında yapılan altı farklı safra tuzunun kullanıldığı ninhydrin testleri araştırlmıştır. Yrsth geminin ifadesi ise SDS-PAGE ile belirlenmiştir. Ninhidrin test sonuçları, yrSTH enziminin altı temel insan safra tuzunu hidroliz etmekle birlikte taurine ekli tuzlardan ziyade glisin ekli tzları daha fazla tercih ettiklerrini göstermektedir. yrBSH enziminin maksimum aktiviteleri düşük pH ve yüksek sıcaklıklarda kaydedildi. Elde edilen bulgular, STH enzimleri substratlarını kolat ekinden ziyade aminoasit eklerine göre tanımaktadır önerisini desteklemektedir. Bununla birlikte, STH enziminin katalitik aktivitesi, substrat özgüllükleri ve probiyotk bakteri seçi kriterlerinin daha iyi anlaşılması için yönlendirilmiş mutagenez gibi daha genişletilmiş çalışmalara ihtiyaç duyulmaktadır. ANAHTAR KELİMELER: Probiyotik, Lactobacillus gasseri, Safra tuzu hidrolaz (STH), Katalitik aktivite ve Sübstrat özgüllüğü
Over the past decade, bile salt hydrolase (BSH) active probiotic strains are being used in the treatment of hypercholesterolemia related diseases. The aptitude of this BSH enzyme to deconjugate bile salts into free bile acids and glycine or taurine moiety has been a major criteria in the selection of probiotics. Not only does this deconjugation provides bile tolerance to the bacteria for its continued existence in the GI tract, it also plays a critical part in the reduction of the blood cholesterol level of the human host. Therefore, knowing and understanding the mechanism of action of the BSH enzymes of the probiotic being used in those treatments is of utmost importance. Even though Lactobacillus gasseri is probably one of the most beneficial probiotics on the market, there is little information on the mechanism of action of the catalytic activity and substrate preferences of the BSH enzyme of this microorganism. In this study, the bsh gene from Lb. gasseri ATCC 33323 strain was cloned, expressed, characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activity of the wild-type recombinant BSH (wrBSH) enzyme was then examined in different temperatures ranging from 25°C to 65°C and various pHs from 2.5 to 8.0 along with six different bile acids by ninhydrin assay. The expression of bsh gene was detected by SDS-PAGE. The ninhydrin test results indicated that wrBSH enzyme hydrolyzed six major human bile salts with an obvious preference towards glycine-conjugated compared to tauro-conjugated bile salts. Maximun activities of wrBSH enzyme were also recorded at low pH and high temperatures. Our findings support the suggestion that BSH enzymes recognize their substrates predominantly through their amino acid moieties rather than their cholate moieties. However, more extensive studies like site directed mutagenesis are needed to fully understand the catalytic activity as well as the substrate specificities of the BSH enzymes and to set the standards for the selection of probiotic bacteria. KEYWORDS: Probiotics, Lactobacillus gasseri, Bile Salt Hydrolase (BSH), Catalytic activity and Substrate specificity
Over the past decade, bile salt hydrolase (BSH) active probiotic strains are being used in the treatment of hypercholesterolemia related diseases. The aptitude of this BSH enzyme to deconjugate bile salts into free bile acids and glycine or taurine moiety has been a major criteria in the selection of probiotics. Not only does this deconjugation provides bile tolerance to the bacteria for its continued existence in the GI tract, it also plays a critical part in the reduction of the blood cholesterol level of the human host. Therefore, knowing and understanding the mechanism of action of the BSH enzymes of the probiotic being used in those treatments is of utmost importance. Even though Lactobacillus gasseri is probably one of the most beneficial probiotics on the market, there is little information on the mechanism of action of the catalytic activity and substrate preferences of the BSH enzyme of this microorganism. In this study, the bsh gene from Lb. gasseri ATCC 33323 strain was cloned, expressed, characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activity of the wild-type recombinant BSH (wrBSH) enzyme was then examined in different temperatures ranging from 25°C to 65°C and various pHs from 2.5 to 8.0 along with six different bile acids by ninhydrin assay. The expression of bsh gene was detected by SDS-PAGE. The ninhydrin test results indicated that wrBSH enzyme hydrolyzed six major human bile salts with an obvious preference towards glycine-conjugated compared to tauro-conjugated bile salts. Maximun activities of wrBSH enzyme were also recorded at low pH and high temperatures. Our findings support the suggestion that BSH enzymes recognize their substrates predominantly through their amino acid moieties rather than their cholate moieties. However, more extensive studies like site directed mutagenesis are needed to fully understand the catalytic activity as well as the substrate specificities of the BSH enzymes and to set the standards for the selection of probiotic bacteria. KEYWORDS: Probiotics, Lactobacillus gasseri, Bile Salt Hydrolase (BSH), Catalytic activity and Substrate specificity
Açıklama
Fen Bilimleri Enstitüsü, Biyoloji Ana Bilim Dalı
Anahtar Kelimeler
Biyoloji, Biology
