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

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.

Anahtar Kelimeler:

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

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

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 Km 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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