Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product.
A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.
Yang
R C
RC
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
MacKenzie
C R
CR
Bilous
D
D
Narang
S A
SA
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D015139:Blotting, Southern; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D043325:Xylan Endo-1,3-beta-Xylosidase
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase