Molecular cloning and heterologous expression of an acid stable xylanase gene from <Emphasis Type="Italic">Alternaria</Emphasis> sp. HB186

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.