saujs

Sakarya University Journal of Science

2147-835X

Sakarya University

10.16984/saufenbilder.327153

Structural Biology Chemical Engineering

Yapısal Biyoloji Kimya Mühendisliği

Bacillus subtilis’e Ait Ksilanaz (xyn-akky1)’in Escherichia coli’de Klonlanması, Ekspresyonu ve Karakterizasyonu

Cloning, Expression and Characterization of Xylanase (xyn-akky1) from Bacillus subtilis in Escherichia coli

Bilgin

Sema

GAZİOSMANPAŞA ÜNİVERSİTESİ, FEN-EDEBİYAT FAKÜLTESİ, KİMYA BÖLÜMÜ Ulusu

Yakup

KARAMANOĞLU MEHMETBEY ÜNİVERSİTESİ, MÜHENDİSLİK FAKÜLTESİ, BİYOMÜHENDİSLİK BÖLÜMÜ Kuduğ

Hülya

GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ Gökçe

İsa

GAZİOSMANPAŞA ÜNİVERSİTESİ, MÜHENDİSLİK VE DOĞA BİLİMLERİ FAKÜLTESİ

12 01 2018

22 6 1508 1517 07 07 2017 11 28 2017

1997

Sakarya University Journal of Science

Bu çalışmada, Bacillus subtilis akky1 straini Türkiye’ninOrdu İli’nin Akkuş ilçesindeki kayın ormanından alınan topraktan izoleedilmiştir. akky1 straini 16S rRNA analizi ile tanımlanmıştır. 16S rRNA dizisiBacillus subtilis strain B7 (KC310823.1) ile %100’lük bir benzerlikgöstermiştir. GenBank’da verilen Bacillus subtilis ksilanazına ait gen dizisiesas alınarak tasarlanan primerler kullanılarak genomic DNA’dan 642 bp’lik birDNA fragmenti elde edilmiştir. Ksilanazı kodlayan gen, pET28b (+) ekspresyon vektörüneklonlanmış ve Escherichia coli BL21 (DE3)’de ekpresse edilmiştir. Rekombinantolarak üretilen protein nikel afinite kromatografisi ile saflaştırılmış veksilanaz aktivitesi belirlenmiştir. Saflaştırılan ksilanazın moleküler ağırlığıSDS-PAGE ile 26.0 kDa olarak belirlenmiştir. Rekombinant enzimin optimum pH’sı6.0 ve sıcaklığı 60°C, Km değeri ise 3.33 mg/ml olarak tespit edilmiştir.

In thisstudy Bacillus subtilis akky1 strainwas isolated from the soil of beech forest in Akkuş City, Ordu Province,Turkey. The identification of the strain akky1 done by PCR amplification 16SrRNA. The full-length 16S rRNA sequence showed the highest nucleotidesimilarity (100%) with Bacillus subtilisstrain B7 (KC310823.1). Spesific oligonucleotides targeting the sequence of Bacillus subtilis xylanase gene given inGenBank were used to amplify a 642-bp fragment from genomic DNA. The geneencoding xylanase was cloned into pET28b (+) plasmid vector, sequenced andexpressed in Escherichia coli BL21(DE3). The hexahistidine (6xHis) tagged fusion protein was purified usingnickel affinity chromatography and the xylanase activity was measured. Themolecular mass of the purified xylanase was approximately 26 kDa as estimatedby SDS-PAGE. The xylanase had optimal activity at pH 6.0 and 60°C.The Km values of the recombinant enzyme towards beechwood was 3,33mg/ml.

Bacillus subtilis Xylanase Recombinant Protein Industrial Enzymes Escherichia coli

Bacillus subtilis Ksilanaz Rekombinant Protein Endüstriyel Enzimler Escherichia coli

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