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

Yıl 2018, Cilt: 22 Sayı: 6, 1508 - 1517, 01.12.2018

Sema Bilgin , Yakup Ulusu , Hülya Kuduğ , İsa Gökçe

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

Cited By: 2

Öz

 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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

• References
• [1] Beg Q K, Kapoor M, Mahajan L, Hoonda G S (2001). Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56: 326–338.
• [2] Belancic A, Scarpa J, Peirano A, Diaz R, Steiner J (1995). Eyzayuirre J. Penicillium purpurogenum produces several xylanases:purification and properties of two of the enzymes. J Biotechnol 41: 71–79.
• [3] Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290
• [4] Dey D, Hinge J, Shendye A, Rao M (1992). Purification andproperties of extracellular endo-xylanases from alkalophilic thermophilic Bacillus sp. Can J Microbiol 38: 436–442.
• [5] Dhiman SS, Sharma J, Battan B (2008) Industrial applications and future prospects of microbial xylanases: a review. BioResources 3(4):1377–1402
• [6] Elegir G, Sykes M, Jeffries TW (1995). Differential and synergisticaction of Streptomyces endoxylanases in prebleaching of kraftpulp. Enzyme Microb Technol 17: 954–959.
• [7] Fernandez-No I C, Böhme K, Díaz-Bao M, Cepeda A, Barros-Velázquez J (2013). Characterisation and profiling of Bacillus subtilis, Bacillus cereus and Bacillus licheniformis by maldi-tof mass fingerprinting. Food Microbiol, 33: 235–242.
• [8] Georis J, Giannotta F, de Buyl E, Granier B, Fre`re J M (2000). Purification and properties of three endo-b-1,4-xylanases produced by Streptomyces sp. strain S38 which diver in their ability to enhance the bleaching of kraft pulps. Enzyme Microb Technol, 26:178–186.
• [9] Huang J, Wang G, Xiao L (2006). Clonning, Sequencing and Expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli. Bioresource Technol, 97: 802-808.
• [10] Jalal A, Rashid N, Rasool N, Akhtar, M (2008). Gene Cloning and Characterization of a xylanase from a newly isolated Bacillus subtilis strain R5. J Biosci Bioeng, 107: 360-365.
• [11] Kiddinamoorthy J, Alfredo J, Gulelat DH, Rakshit SKV (2008) Production, purification and characterization of Bacillus sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching. World J Microbiol Biotechnol 24:605–612
• [12] Kulkarni N., Shendye A., Rao M (1999). Molecular and biotechnological aspects of xylanases, Fems Mıcrobıol Rev 23: 411-456.
• [13] Lineweaver H, Burk D (1934). The determination of enzyme dissociation constants. J Am Chem Soc, 56: 658-666.
• [14] Maalej-Achouri I, Guerfali M, Gargouri A, Belghith H (2009) Production of xylo-oligosaccharides from agro-industrial residues using immobilized Talaromyces thermophiles xylanase. J Mol Cata B Enzymatic 59:145–152
• [15] Miller G L (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 31: 426-428.
• [16] Polizeli M L, Rizzatti A C, Monti R, Terenzi H F, Jorge J A, Amorim D S (2005). Xylanases from fungi: properties and industrial applications.Appl Microbiol Biotechnol 67: 577–591.
• [17] Rani S, Nand, K (1996) Development of cellulase-free xylanase-producing anaerobic consortia for the use of lignocellulosic wastes. Enzyme Microb Technol 18:23–28
• [18]Sanghi A, Garg N, Kuhar K, Kuhad RC, Gupta VK (2009) Enhanced production of cellulase-free xylanase by alkalophilic Bacillus subtilis ASH and its application in biobleaching of kraft pulp. BioResources 4(3):1109–1129
• [19] Ulusu Y, Bilgin Ş S, Kuduğ H, Gökçe İ (2016). Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOL system. PREP Biochem Biotech. DOI: 10.1080/ 10826068. 2015.1085399.
• [20] Wood P J, Erfle J D, Teather R M (1998). Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases. Method Enzymol, 160:59-7