Purification, production and investigation of commercial use of 1,4-β-endo xylanase in various Bacillus species isolated from thermal resources

In this study; thermophilic bacteria have been isolated from hot water springs in Eastern and Southeastern Anatolia. The morphological characteristics of the isolated samples were identified and total xylanase activities were determined. The 16s rDNA regions from the isolated DNAs were amplified by PCR and then clonning and sequence analysis was performed. Gene sequences coding for xylanase enzyme from Bacillus subtilis strain with high activity were determined by bioinformatics analysis methods. Purification of recombinant proteins with 6X-His tag was performed through nickel affinity chromatography. The purified enzyme was caged by the ANADOLUCA method. Five different isolates (Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bacillus thuringiensis and Geobacillus kaustophilus) were identified and the xylanase from B. subtilis isolate which showed highest activity was produced as recombinant enzyme. The optimal pH of both the recombinant and recombinant nano-xylanase enzyme was 7.0, the optimum temperature was 68 °C for the recombinant enzyme and 75 °C for the nano-enzyme. Optimum enzyme activity was determined to be 1803 U/mg for the recombinant enzyme and 1898 U / mg for the nano-enzyme. The Km and Vmax values of the isolates were determined to be 2,298 (mM) and 5,691 (EU/mL.dk) for the recombinant enzyme, 2,402 (mM) and 6,195 (EU/mL.dk) for the Nano enzyme, respectively. It was found that the recombinant and nano xylanase showed different relative activity when metal ions were included: recombinant xylanase; MgO4 (80%), CuSO4 (57%), CaCl2 (74%), ZnSO4 (5%) and FeSO4 (72%), nano xylanase; MgO4 (85%), CuSO4 (71%), CaCl2 (85%), ZnSO4 (50%) and FeSO4 (94%).