Cloning and expression of putative beta-xylosidase B from Phanerochaete chrysosporium
Nội dung tóm tắt: White-rot fungus P. chrysosporium has been reported to produce complex hemicelluloses degradation enzymes including β-xylosidases. Genes encode for these enzymes have been predicted, namely β-xylosidase A and β-xylosidase B, however, the functional of these genes have not been characterized. This study aimed to clone a putative cDNA encoding for β-xylosidase B (PcXylB) from P. chrysosporium BKM-F-1767. Sequence analysis indicated the gene consisted of 981 nucleotides, coding for 326 amino acids. N-terminal containing 18 amino acids was identified as secretion signal peptide. The predicted secondary structure of PcXylB was composed of 27 beta strands, while three catalytic size residues Asp37, Asp152, and Glu221 were found to be general base, pKa module, and general acid, respectively. PcXylB was constructed with expression vectors pPICZA and pPICZαA and integrated into P. pastoris genome using electroporation method. The rPcXylB secretion of recombinant pPICZA-PcXylB P. pastoris was driven by intrinsic secretion signal while pPICZαA- PcXylBα P. pastoris was promoted of α-secretion signal from Saccharomyces cerevisiae. The rPcXylB secrection was carried out by adding methanol to final concentration of 1 % for every 12 h. The recombinant pPICZA-PcXylB constructed P. pastoris did not secret rPcXylB. Western dot-blot analysis showed the difference on recombinant protein released among of twenty pPICZαA-PcXylBα transformants. Free-cell medium culture of recombinant P. pastoris on the fourth day cultivation was harvested to purify enzyme using anti his-tag column. Purified rPcXylB exhibited a single band of approximately 33 kDa on SDS-PAGE. The present study was first report on the cloning and expression of putative P. chrysosporium β-xylosidase B.
Thể loại: Trong nước
Tác giả: Nguyễn Đức Huy, Seung-Moon Park
Đơn vị: Ban lãnh đạo viện
Đăng tại: Tạp chí Công nghệ sinh học
Tập, (số), trang: 10, 4A, 785-791
Số ISSN/ISBN: 1859 - 2201
Chỉ số Impact factor:
Điểm số theo QĐ của HĐ chức danh GS Nhà nước:
Năm công bố: 2012
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Số lần xem: 23
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