Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa |
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Authors: | Harry J Gilbert Gail Jenkins Debra A Sullivan and Judith Hall |
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Institution: | (1) Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, NE1 7RU Newcastle upon Tyne, UK |
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Abstract: | Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl- -D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC
0.15 M NaCl, 0.015 M sodium citrate
- Smr
resistance to streptomycin
- Kmr
resistance to kanamycin
- Apr
resistance to ampicillin
- Tcr
resistance to tetracycline
- Cmr
resistance to chloramphenicol
- CMCase
carboxymethylcellulase |
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Keywords: | Carboxymethylcellulases Cloning Pseudomonas Multiple genes |
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