Sequencing and expression of the xylanase gene 2 from Trichoderma reesei Rut C-30 and characterization of the recombinant enzyme and its activity on xylan.
The Xyn2 gene, which encodes endo-beta-1,4-xylanase2, in Trichoderma reesei Rut C-30 was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to encode 190-amino-acid residues of a protein with a calculated molecular mass of 21 kDa. The cDNA was cloned into pET30alpha expression vector and subsequently expressed in Escherichia coli under the control of strong bacteriophage T7 transcription and translation signals. The enzyme activity assay verified the recombinant protein as xylanase. The isoelectric point and highest activity were 7.5 and 1,600 U/mg, respectively. Like with the T. reesei Xyn2, the highest activity of the recombinant Xyn2 was at 50 degrees C. However, the recombinant enzyme had an improved thermostability and more than 65% of its activity retained after 30 min incubation at 60 degrees C. In addition, the recombinant Xyn2 was active over the range of pH 3.5-7.5 with maximum activity at pH 5.0. Using birchwood xylan, the determined apparent K(m) and k(cat) values were 0.15 mg/ml and 119.7 s(-1), respectively. The enzyme was highly specific towards xylans and exhibited very low activity towards cellulosic substrates. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylose as the main degradation products. These properties should make the enzyme a suitable applicant in various industrial applications.
Jun
He
H
Institute of Animal Nutrition, Sichuan Agricultural University, and Key Laborotary of Animal Disease-Resistance Nutrition, Ministry of Education, Yaan, Sichuan, PR China.
Bing
Yu
Y
Keying
Zhang
Z
Xuemei
Ding
D
Daiwen
Chen
C
D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D014990:Xylans / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014990:Xylans; D043364:Endo-1,4-beta Xylanases