Xylanase from a soil isolate, Bacillus pumilus: gene isolation, enzyme production, purification, characterization and one-step separation by aqueous-two-phase system

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.

Application of cellulase-free xylano-pectinolytic enzymes from the same bacterial isolate in biobleaching of kraft pulp

“…Use of xylanase and pectinase in combination has been reported earlier by Ahlawat et al (2007) and Dhiman et al (2009) but in both reports xylanase and pectinase were produced from the different bacterial species. Production of xylanases from Bacillus pumilus have been reported by several workers Nagar et al, 2010;Yasinok et al, 2010). Similarly, pectinase production from B. pumilus has been reported by Sharma and Satyanarayana (2006) and Basu et al (2008).…”

Enhancing Jatropha oil extraction yield from the kernels assisted by a xylan-degrading bacterium to preserve protein structure

“…In case of B. pumilus , previous investigations have reported the potential application of B. pumilus as xylanase producer (Ahlawat et al 2007; Battan et al 2007; Kapoor and Kuhad 2007; Kapoor et al 2008; Nagar et al 2010; Wang et al 2010; Yasinok et al 2010). In contrast, we found that only a few publications are available on the potential application of B. altitudinis .…”

STATISTICAL APPROACH FOR PECTINASE PRODUCTION BY Bacillus firmus SDB9 AND EVALUATION OF PECTINO-XYLANOLYTIC ENZYMES FOR PRETREATMENT OF KRAFT PULP

“…Production of xylanase from Bacillus species has been reported by several researchers (Ersayin et al, 2010;Nagar et al, 2010). Similarly, pectinase production from Bacillus species has also been reported (Basu et al, 2008;Sharma & Satyanarayana, 2006).…”