Expression of a Trichoderma reesei beta-xylanase gene in Escherichia coli and activity of the enzyme on fiber-bound substrates.
A recombinant gene Xyn2 (570bp) encoding the main Trichoderma reesei Rut C-30 endo-beta-1,4-xylanase was successfully cloned and expressed in Escherichia coli BL21 under the control of strong bacteriophage T7 transcription and translation signals. The molecular weight of the recombinant protein was estimated by SDS-PAGE to be 24 kDa. The expressed protein was purified by Ni(2+)-NTA affinity chromatography and enzyme activity assay verified the protein as a xylanase. Like with the T. reesei Xyn2, the optimal activity of the recombinant Xyn2 was at 50 degrees C. However, the recombinant xylanase had an improved thermostability and more than 70% of its activity remained after 30 min incubation at 60 degrees C. In addition, the recombinant xylanase was active over the range of pH 3.5-7.5 with maximum activity at pH 5.0. The enzyme was highly specific towards xylans but exhibited very low activities towards cellulosic substrates. Using Birchwood xylan, the determined apparent K(m) and k(cat) values were 0.11 mg/ml and 106 s(-1), respectively. These properties should make the enzyme an attractive candidate for various industrial applications.
Jun
He
H
Institute of Animal Nutrition, Sichuan Agricultural University, Ya'an, Sichuan 625014, PR China.
Bing
Yu
Y
Keying
Zhang
Z
Xuemei
Ding
D
Daiwen
Chen
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
D011134:Polysaccharides; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases