Abstract
A xylanase producer, Bacillus pumilus SB-M13, was isolated from soil and identified using various tests based on carbohydrate fermentation preferences and fatty acid analysis. Xylanase gene, isolated using PCR amplification, was partially sequenced and it showed 89–94% sequence similarity to the xylanase genes of other B. pumilus strains. Xylanase with very low level of cellulase was produced on agricultural byproducts. The enzyme has been purified 186-fold by hydrophobic interaction chromatography and biochemically characterized. It has a molecular weight of 24.8 kDa and pI of 9.2. Xylanolytic activity is stable at alkaline pH and highest activity is observed at 60 °C and pH 7.5. Enzyme K m and k cat values were determined as 1.9 mg/mL and 42,600 U/mg, respectively. In aqueous-two-phase system, xylanase always partitioned to the top phase. Basic pH, low PEG concentration, salt addition, and presence of microbial cells enhanced xylanase partitioning. A maximum sevenfold purification, 10-fold concentration and 100% xylanase recovery were obtained, separately, by adjusting system parameters. A fourfold concentrated xylanase was obtained with 70% enzyme recovery only in one step ATPS process without cell harvesting.
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Acknowledgments
This project was supported by TÜBİTAK (MISAG-177, and MISAG-104T473), Middle East Technical University (AFP-99-06-02-10) and BAP-0811-DPT2002K120510-BTEK08. The authors offer sincere thanks to TUBITAK, METU and DPT. We would like to express our gratitude to staff of METU Molecular Biology-Biotechnology R&D Center, who has always offered instant and high quality assistance.
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Ersayin Yasinok, A., Biran, S., Kocabas, A. et al. Xylanase from a soil isolate, Bacillus pumilus: gene isolation, enzyme production, purification, characterization and one-step separation by aqueous-two-phase system. World J Microbiol Biotechnol 26, 1641–1652 (2010). https://doi.org/10.1007/s11274-010-0340-8
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DOI: https://doi.org/10.1007/s11274-010-0340-8