Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina.
A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25 degrees C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml(-1). The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40 degrees C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The Km and Vmax values for birchwood xylan are 2.06 mg ml(-1) and 0.49 mmol min(-1)mg(-1), respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.
Zhang
Gui Min
GM
College of Life Science, Hubei University, Wuhan 430062, People's Republic of China.
Huang
Jun
J
Huang
Guang Rui
GR
Ma
Li Xin
LX
Zhang
Xian En
XE
D001709:Biotechnology; D016386:Chromosome Walking; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020081:Phyllachorales / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D005656:Fungal Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases