Molecular cloning and heterologous expression of an acid stable xylanase gene from Alternaria 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, Cu(2+) and Hg(2+) 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.
College of Life Sciences, Hubei University, Wuhan 430062, China.
Liangwei
L
Mao
Po
P
Meng
Cheng
C
Zhou
Lixin
L
Ma
Guimin
G
Zhang
Yanhe
Y
Ma
D000143:Acids; D000528:Alternaria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D018628:Gene Dosage; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010843:Pichia / Q000235:genetics; D017422:Sequence Analysis, DNA; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000143:Acids; D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D008670:Metals; D014995:Xylosidases