Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli.
The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.
Zhou
Chenyan
C
School of Biotechnology, Southern Yangtze University, 170 Huihe Road, Wuxi 214036, PR China.
Bai
Jianyu
J
Deng
Shanshan
S
Wang
Jin
J
Zhu
Jie
J
Wu
Minchen
M
Wang
Wu
W
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases