Molecular cloning, sequence analysis and expression of a GHF 43 xylanase from Aspergillus niger in Escherichia coli.
A new xylanase gene (xyn43A) from Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The coding region of the gene was separated by only one intron 86 bp in length. It encoded 318 amino acid residues of a protein with a calculated molecular weight (MW) of 33.47 kDa plus a signal peptide of 19 amino acids. The amino acid sequence of the xyn43A gene showed 77.56% amino acid identity to A. nidulans xylanase, and the phylogenetic tree analysis revealed that xyn43A had close relationships with those of family 43 of glycosyl hydrolases reported from other microorganisms. Three-dimensional structure modeling showed that Xyn43A had a typical five-blade β-propeller fold. The mature peptide encoding cDNA was subcloned into pET-28a (+) expression vector. The resultant recombinant plasmid pET-28a-xyn43A was transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. A maximum activity of 61.43 U/mg was obtained from the cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn43A. The recombinant xylanase had optimal activity at pH5.0 and 45°C. Fe(3+), Cu(2+) and EDTA had an obvious active effect on the enzyme.
School of Life Science and Technology, Xinxiang Medical University.
Chen-Yan
CY
Zhou
Yong-Tao
YT
Wang
Tie-Chui
TC
Zhu
Guan-Hua
GH
Fu
Dan-Dan
DD
Wang
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D020536:Enzyme Activators; D011994:Recombinant Proteins; D014995:Xylosidases