Identification of two distinct Bacillus circulans xylanases by molecular cloning of the genes and expression in Escherichia coli.
Two genes coding for xylanase synthesis in Bacillus circulans were cloned and expressed in Escherichia coli. After digestion of genomic DNA from Bacillus circulans with EcoRI and PstI, the fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Restriction enzyme mapping of the two inserts coding for xylanase activity indicated distinctly different nucleotide sequences. Cross-hybridization assays confirmed the absence of sequence homology between the two genes. In vitro transcription-translation assays indicated that the cloned genes encoded for proteins with molecular weights of 22,000 and 59,000. The gene products displayed different substrate specificities. The 22,000-dalton enzyme readily hybrolyzed aspeen, larchwood, and oat spelt xylans, whereas the second was unable to extensively depolymerize oat spelt xylan and resulted in very limited reducing sugar release from any of the xylan substrates tested. Both of the xylanases had isoelectric points of approximately 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; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D013379:Substrate Specificity; D014158:Transcription, Genetic; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism
D014990:Xylans; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase