Molecular cloning and heterologous expression of an acid stable xylanase gene from Alternaria sp. HB186
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A new xylanase gene, named xyn186, was cloned by the genome-walking PCR method from the Alternaria sp. HB186. The sequence of xyn186 contains a 748 bp open reading frame separated by one intron with the size of 52 bp. The cDNA was obtained by DpnI-mediated intron deletion. The cDNA was cloned into pHBM905A and transformed into Pichia pastoris GS115 to screen xylanase-secreting transformants on RBB-xylan plates. The molecular mass of the enzyme was estimated to be 23 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 50°C, respectively. The K m and V max valued for birchwood xylan are 1.404 mg ml−1 and 0.2748 mmol min−1 mg−1, respectively. The inhibitory effects of various metal ions were investigated, Cu2+ and Hg2+ ions inhibited most of the enzyme activity. The gene copy number of xyn186 in the genome of P. pastoris was estimated as two by the Real-time PCR. To date, xyn186 gene is the first xylanase gene cloned from the genus Alternaria.
KeywordsAlternaria sp. HB186 Heterologous expression Pichia pastoris Acid stable xylanase Gene copy number Real-time PCR
This study was supported by the National Nature Science Foundation (30600014), Hubei Province Nature Science Foundation Key Project, Knowledge Innovative Program of the Chinese Academy of Sciences (KSCX2-EW-G-8) and the Ministry of Sciences and Technology of China (973 programs 2012CB721000).
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