Cloning and expression of a Paecilomyces thermophila xylanase gene in E. coli and characterization of the recombinant xylanase.
A cDNA library of Paecilomyces thermophila was constructed, and the gene encoding xylanase (designated Pt xynA) was isolated from the library. Pt xynA consisted of 681 bp, and the translated protein encoded 226 amino acids. This is the first functional gene cloned from P. thermophila. The gene was successfully expressed in Escherichia coli BL21 and the recombinant xylanase (XynA) was purified to homogeneity by Ni-NTA and Sephadex G50. XynA showed an optimum activity at 75 degrees C and pH 7.0. Its residual activity was more than 60% after being treated at 85 degrees C for 30 min. K(m) values of XynA for birchwood xylan, beechwood xylan and oat-spelt xylan were 4.4, 3.6 and 9.7 mg ml(-1), respectively. The enzyme has an endohydrolytic mode of action and can hydrolyse xylotriose to xylobiose through transglycosylation. These results indicate the XynA is a thermostable enzyme and has great potential in various industries.
Zhang
Min
M
Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Jiang
Zhengqiang
Z
Yang
Shaoqing
S
Hua
Chengwei
C
Li
Lite
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010143:Paecilomyces / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013696:Temperature
D017931:DNA Primers; D004271:DNA, Fungal; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases