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

Year 2018, , 1508 - 1517, 01.12.2018

Sema Bilgin , Yakup Ulusu , Hülya Kuduğ , İsa Gökçe

https://doi.org/10.16984/saufenbilder.327153

Cited By: 2

Abstract

 Bu çalışmada, Bacillus subtilis akky1 straini Türkiye’nin
Ordu İli’nin Akkuş ilçesindeki kayın ormanından alınan topraktan izole
edilmiştir. akky1 straini 16S rRNA analizi ile tanımlanmıştır. 16S rRNA dizisi
Bacillus subtilis strain B7 (KC310823.1) ile %100’lük bir benzerlik
göstermiştir. GenBank’da verilen Bacillus subtilis ksilanazına ait gen dizisi
esas alınarak tasarlanan primerler kullanılarak genomic DNA’dan 642 bp’lik bir
DNA fragmenti elde edilmiştir. Ksilanazı kodlayan gen, pET28b (+) ekspresyon vektörüne
klonlanmış ve Escherichia coli BL21 (DE3)’de ekpresse edilmiştir. Rekombinant
olarak üretilen protein nikel afinite kromatografisi ile saflaştırılmış ve
ksilanaz aktivitesi belirlenmiştir. Saflaştırılan ksilanazın moleküler ağırlığı
SDS-PAGE ile 26.0 kDa olarak belirlenmiştir. Rekombinant enzimin optimum pH’sı
6.0 ve sıcaklığı 60°C, Km değeri ise 3.33 mg/ml olarak tespit edilmiştir.  

Keywords

Bacillus subtilis, Ksilanaz, Rekombinant Protein, Endüstriyel Enzimler, Escherichia coli

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

Abstract

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

Keywords

Bacillus subtilis, Xylanase, Recombinant Protein, Industrial Enzymes, Escherichia coli

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