Investigation of a plasmid-associated heat stable protease produced by Pseudomonas fluorescens T20

Hatfield, E.A. (1990) Investigation of a plasmid-associated heat stable protease produced by Pseudomonas fluorescens T20. Masters thesis, Memorial University of Newfoundland.

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Abstract

Pseudomonas fluorescens T20, a psychrotroph of milk origin produces an extracellular heat-stable protease which in large quantities causes spoilage of milk and milk products. The protease was purified by affinity column chromatography using carboxy-D-phenylalanine-triethylenetetramine Sepharose 4B. It was shown to have a molecular weight of 44,000. Two mutant strains of P. fluorescens T20 were generated using N-methyl- N'nitro- N•-nitrosoquanidine. The extracellular extracts of both mutants were shown by immunoprecipitation to be negative for the presence of the heat-stable protease. The intracellular extracts in turn were tested by immunoprecipitation and only one, the Type A mutant strain, showed the presence of cross reacting material. Thus two types of mutants were generated, one which could express the sequence encoding the heat-stable protease but which was unable to export the protease out of the cell and another, which could not express or export the heatstable protease. The mutant strains were designated Type A and Type B respectively. The genetic origin of the heat-stable protease was investigated and it was determined that the resident plasmid in P. fluorescens T20 carried the gene encoding the heat-stable protease. The plasmid had a molecular weight of 44.06 Kbp. A 3.3 Kbp Sal 1 fragment was cloned from the plasmid into the vector, pUC12. The cells transformed with the recombinant shov.-ed phenotypic expression of the heat-stable protease. In furtherance of the attempt to find that region of the plasmid encoding the heat-stable protease a restriction endonuclease map of the plasmid was constructed.

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