Cloning, characterization, and expression of xylanase A gene from Paenibacillus sp. DG-22 in Escherichia coli.
The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a 182-residues mature protein of a calculated molecular weight of 20,000 Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and 60 degrees C, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.
Lee
Tae Hyeong
TH
Department of Biotechnology, Dongguk University, Kyongbuk 780-714, Korea.
Lim
Pyung Ok
PO
Lee
Yong-Eok
YE
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases