Cloning and extracellular expression in Escherichia coli of xylanases from an alkaliphilic thermophilic Bacillus sp. NCIM 59* Free

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.