Cloning and extracellular expression in Escherichia coli of xylanases from an alkaliphilic thermophilic Bacillus sp. NCIM 59.
A genomic DNA library of an alkaliphilic thermophilic Bacillus was constructed in Escherichia coli with pUC 8 vector and was screened using a Congo red xylan plate clearance assay. Six xylanase positive transformants having identical inserts showed immunological reactivity towards polyclonal antibodies raised against purified xylanase (M(r) 15,800) from the Bacillus. A 4.5-kb HindIII-EcoRI subfragment was found to code for two xylanases of M(r) 14,500 and 35,000. Equivalent amounts of xylanase activity were detected from IPTG induced and noninduced recombinants irrespective of the orientation of the 4.5-kb insert with respect to the lac promoter, indicating that xylanase gene expression was under the control of its own promoter. 95% of the xylanase activity (2 U/ml) was found in the extracellular culture filtrate. The hydrolysis of xylan by the recombinant xylanases yielded mainly xylobiose.
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
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Shendye
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Rao
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014990:Xylans; D006026:Glycoside Hydrolases; D043325:Xylan Endo-1,3-beta-Xylosidase