Molecular cloning and heterologous expression of an acid stable xylanase gene from Alternaria sp HB186

作者：Mao Liangwei; Meng Po; Zhou Cheng; Ma Lixin; Zhang Guimin^*; Ma Yanhe

来源：World Journal of Microbiology and Biotechnology, 2012, 28(3): 777-784.

DOI：10.1007/s11274-011-0924-y

• Alternaria sp HB186
• Heterologous expression
• Pichia pastoris
• Acid stable xylanase
• Gene copy number
• Real-time PCR

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 50A degrees 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.

• 出版日期2012-3
• 单位中国科学院微生物研究所; 湖北大学