Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli.
Bacillus subtilis strain B10 was isolated for degumming of ramie blast fibers, and a fragment of 642-bp was amplified from chromosomal DNA by using primers directed against the sequence of Bacillus subtilis xylanase gene given in GenBank. The positive clones were screened on the selected LB agar plates supplemented with xylan by Congo-red staining method. The recombinant plasmid from one positive clone was used for further analysis and DNA sequencing. The gene sequence is different from the reported xylanase gene sequence in sites of two base pairs. The recombinant plasmid was expressed in Escherichia coli, and xylanase activity was measured. The xylanase distribution in extracellular, intracellular and periplasmic fractions were about 22.4%, 28.0% and 49.6%, respectively. The xylanase had optimal activity at pH 6.0 and 50 degrees C.
Huang
Junli
J
College of Biological Engineering, Research Center of Applied Biotechnology, Chongqing University, Chongqing 400044, China. huangjunli@263.net
Wang
Guixue
G
Xiao
Li
L
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004275:DNA, Ribosomal; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019897:Periplasm; D017421:Sequence Analysis; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D004275:DNA, Ribosomal; D014990:Xylans; D043364:Endo-1,4-beta Xylanases