Molecular Cloning and Expression of a Xylanase Gene from Bacillus polymyxa in Escherichia coli

Abstract

Genomic fragments of Bacillus polymyxa derived from separate and complete digestion by Eco RI, Hin dIII, and Bam HI were ligated into the corresponding sites of pBR322, and the resulting chimeric plasmids were transformed into Escherichia coli. Of 6,000 transformants screened, 1 (pBPX-277) produced a clear halo on Remazol brilliant blue xylan plates. The insert in the pBPX-277 recombinant, identified as an 8.0-kilobase Bam HI fragment of B. polymyxa , was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 2.9-kilobase Bam HI- Eco RI subfragment was found to code for xylanase activity. Xylanase activity expressed by E. coli harboring the cloned gene was located primarily in the periplasm and corresponded to one of two distinct xylanases produced by B. polymyxa. Xylanase expression by the cloned gene occurred in the absence of xylan and was reduced by glucose and xylose. Southern blot hybridization with the cloned fragment as a probe against complete genomic digests of the bacilli B. polymyxa, B. circulans , and B. subtilis revealed that the cloned xylanase gene was unique to B. polymyxa. The xylanase expressed by the cloned gene had a molecular weight of approximately 48,000 and an isoelectric point of 4.9.