Hatice Aysun MERCİMEK TAKCI, Gökhan SERTBAŞ

Bacillus spp. Suşlarından Ksilanaz Enziminin Kısmi Saflaştırılması ve Karakterizasyonu

β-1,4 bağlı ksilopiranozil rezidülerinden oluşan ksilanın β-1,4 glikozidik bağlarını yıkan ksilanaz, başta kağıt ve kağıt hamuru endüstrileri olmak üzere tekstil, yem, fırın ve biyoyakıt endüstrilerinde de geniş potansiyel uygulamalara sahip endüstriyel bir enzimdir. Bu çalışmada, Kilis 7 Aralık Üniversitesi merkez kampüs toprak örneğinden ksilanaz üretici Bacillus spp suşlarının izolasyonu ve saflaştırılan ksilanazın çeşitli endüstrilerdeki uygulanabilirliği araştırılmıştır. Ksilan içeren sıvı fermantasyon ortamında geliştirilen Bacillus spp suşundan hücre serbest elde edilen ham ksilanaz preparatına amonyum sülfat ve alkol çöktürme uygulanarak saflaştırılmıştır. En yüksek total ve spesifik enzim aktivitesi 723 U/L ve 64.81 U/mg olarak ham enzim preparatında saptanmıştır. Ksilanazın optimum aktivitesi 90°C ve pH 10.6’da belirlenmiştir. Enzimin 90°C sıcaklıkta 15 dakika ön inkübasyonu sonrası aktivitenin %167’e kadar arttığı saptanmıştır. Karakterizasyon çalışmalarındaki en yüksek aktivitesi 2935 U/L olarak hesaplanmıştır. SDS-PAGE ve zimogram analizlerinde, kısmi saflaştırılmış ekstraselüler ksilanaza ait ortalama 38.75 kDa moleküler ağırlığında tek protein bandı gözlenmiştir. Ksilanın ksilanaz enzimi ile hidrolizi sonucunda parçalama ürünleri olarak ksiloz ve ksilo-oligosakkaritler belirlenmiştir.

Anahtar Kelimeler:

Bacillus spp., karakterizasyon, kısmi saflaştırma, ksilan, ksilanaz

Partial Purification and Characterization of Xylanase Enzyme from Bacillus spp. Strains

Xylanase that cleaves β-1,4-glycosidic linkages of xylan composed of β-1,4-linked xylopyranosyl residues, is a industrial enzyme, have widespread potential applications in the textile, feed, beverage and biofuel industries, especially the paper and pulp industry. In this study, we were examined to the isolation of Bacillus spp strains, produced extracellular xylanase, from Kilis 7 Aralık University central campus soil sample and the application in various industries of purificated xylanase. The cell-free crude xylanase preparate obtained from Bacillus spp strain developed in a liquid fermentation medium including xylan was purified by using ammonium sulfate and alcohol precipitation. The highest total and specific enzyme activities were detected as 723 U/L and 64.81 U/mg in cell-free crude xylanase preparate. The optimum activity of xylanase was emerged at 90°C and pH 10.6. After the pre-incubation of enzyme at 90°C for 15 min, its activity was detected to increase up to 167%. In characterization studies, the highest enzyme activity was calculated as 2935 U/L. In SDS-PAGE and zymogram analysis, single protein band in 38.75 kDa molecular weight belonging to the partially purified extracellular xylanase was observed. Xylose and xylo-oligosaccharides were detected as the products by hydrolysis of xylan with xylanase.

Keywords:

Bacillus spp., characterization, partially purification, xylan, xylanase,

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