Molecular cloning and expression of a xylanase gene from Bacillus polymyxa in Escherichia coli
Genomic fragments of Bacillus polymyxa derived form separate and complete digestion by EcoRI, HindIII, and BamHI were ligated into the corresponding sites of pBR322, and the resulting chimeric plasmids were transformed into Escherichia coli. Of 6000 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 BamHI fragment of B. polymyxa, was subsequently subjected to extensive mapping and a series of subclonings into pUC19. A 2.9-kilobase BamHI-EcoRI 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.
- Research Organization:
- National Research Council of Canada, Ottawa
- OSTI ID:
- 6958327
- Journal Information:
- Appl. Environ. Microbiol.; (United States), Vol. 54:4
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
BACILLUS
GENETIC ENGINEERING
ESCHERICHIA COLI
XYLANASE
DNA-CLONING
GENE REGULATION
GENETIC MAPPING
HYBRIDIZATION
RECOMBINANT DNA
BACTERIA
CLONING
DNA
ENZYMES
GLYCOSYL HYDROLASES
HYDROLASES
MAPPING
MICROORGANISMS
NUCLEIC ACIDS
O-GLYCOSYL HYDROLASES
ORGANIC COMPOUNDS
500200* - Environment
Atmospheric- Chemicals Monitoring & Transport- (-1989)