Gene Cloning And Characterization Of A Bifunctional Xylanase From Paenibacillus Sp.E18

Xylanases are enzymes to catalyze the hydrolysis of xylan into xylose and oligosaccharide. They are crucial hemicellulases and produced mainly by microorganisms, plants and lower animals. Xylanases have been widely used in many industry processes, such as pulp and paper, foodstuff, animal feed, pharmacy, energy and environmental protection. Microbial xylanases are more promising due to high production, easy to control, low cost and so on.In this study, a strain, E18, showing xylan-degrading activity was isolated by simulation of the natural environment and using selective screening plates, and was identified to be Paenibacillus sp. based on 16S rDNA sequence.By using bioinformatical methods, the amino acid sequences of microbial xylanases from glycoside hydrolase (GH) family 10 were aligned, and a pair of degenerate primers was designed based on the conserved regions. A xyalanse gene fragment was cloned by a Touchdown-PCR, and the flanking regions were obtained by using two step TAIL-PCR. Based on sequence comparison and open reading frame (ORF) analysis, the fragment assembly contained a gene cluster including three genes, xynB-E18, abf43A-E18 and axeE18. The highest homologies of these three genes to known sequences were 85%, 56% and 70%, respectively. xynB-E18 was a xylanase-coding gene containing two highly conserved glutamate, Glu129 and Glu236, of GH 10 members. The deduced amino acid sequence of abf43A-E18 encoded a protein belonging to GH 43, which recombinant protein had both xylanase and arabinofuranohydrolase activities. The sequence of axeE18 exhibited high identity with a putative esterase. The encoded proteins of abf43A-E18 and axeE18 might play a role in xylan degradation as coenzymes. No promoter has been identified yet, suggesting that the xylanolytic gene cluster had more than three members.The gene xynB-E18 was cloned into vector pET22b(+) and then expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The optimum temperature and pH for activity of the recombinant protein XynB-E18 was 50oC and pH 7.0, respectively. The enzyme was pH stable, retaining more than 80% of the initial activity at pH 6.0...