Production of thermotolerant xylanase in Paenibacillus macerans
Date Issued
2014
Date
2014
Author(s)
Chang, Hui-Fen
Abstract
Our reports showed that when cultured in 500 mL Hinton’s flask, the optimum temperature for Paenibacillus macerans is 30℃, the optimum pH of induction medium is pH 7.0, and the optimum induction temperature is 45℃. The xylanase of P. macerans was optimally active at 55℃ and pH 4.5. Compared with glucose, maltose, and xylose, the most suitable carbon source in growth stage was glycerol. Compared with bagasse, soya powder, white poplar powder, and beechwood xylan, the best carbon source we investigated to induce xylanase activity was wheat bran. While grown for 20 hours at 37℃, we added wheat bran to start induction phase, and rising temperature to 45℃ at the same time, continuing to culture for 34 hours, having the xylanase with the concentration of 15 IU/mL. To scale up, we cultured P. macerans in the 5 L bioreactor. While grown for 9.5 hours at 37℃, we added wheat bran, and rising temperature to 45℃, continuing to culture for 28 hours, having the xylanase activity with 15 IU/mL. Compared with flask culture, we could obtain the same amount of xylanase with shortened incubation time of 16.5 hours. Coordinated with laccase, we carried out functional assay─the bio-bleaching of pulp. The xylanase significantly reduced kappa number by 3.7 units, and increased the ISO brightness by 2%. We used 4 conserved sequences of glycoside hydrolase family 11 (GH11) and genome-walking technique to acquire xylanase nucleotide sequence in moderately thermophilic bacterium P. macerans. A xylanase gene of 633 bp was cloned that encodes a protein containing 211 amino acid residues. When the xylanase gene was cloned and expressed in Escherichia coli M15/pREP4, we used anti-His taq antibody to detect the recombinant protein, figuring out the recombinant strain produced almost all the xylanase in the inclusion body, even after long-time induction at low temperature. The soluble protein in the supernatant after sonication showed low xylanase activity, even concentrated. The xylanase of P. macerans is one of the rare xylanases that exhibits thermo- and acid stability, and thus, it is a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides from agro-residues for use as prebiotics.
Subjects
類芽孢桿菌
山毛櫸木聚醣
麩皮
木聚醣酶
染色體步移技術
Type
thesis
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