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Title: The plasmid-mediated OXA-2 beta-lactamase yield improvement, purification and characterisation
Author: Holland, Susan
ISNI:       0000 0001 3580 455X
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1983
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The OXA-2 bla gene as specified by the R factor R46 was cloned into the plasmid vector pMB9 to produce the chimaeric plasmid pSU3 in order to increase the gene copy number and concurrently the level of beta-lactamase production. The pSU3 plasmid was transformed into a high cell yielding Porton strain (E. coli CA265) which was then utilised for a 400 litre batch culture fermentation. The total yield of the OXA-2 beta-lactamase was approximately 78-fold greater than that from an E. coli strain carrying the R46 plasmid, as reported by Dale and Smith (1971). The conditions necessary for the large scale purification of the OXA-2 beta-lactamase were investigated. The large scale purification protocol was capable of processing 2.25 litres of crude cell extract (22.5 litres of original 400 litre culture) containing 116 g of cell protein. The yield of purified enzyme from each 2.25 litres was 90mg and the recovery of enzyme activity was 15%. Sufficient purified enzyme was therefore available to fulfill the needs of this project and those of future research. Various techniques were performed in order to characterise the OXA-2 enzyme. A short NH2-terminal sequence of the OXA-2 enzyme was determined. The occurrence of a leucine-glutamate sequence at positions 5 and 6 in the sequence may indicate some homology with the class A beta-lactamases. The interaction of the OXA-2 enzyme with a range of beta-lactamase inhibitors was investigated with particular emphasis on the mechanism of clavulanate-mediated inhibition. The features of this interaction were correlated into a minimal scheme for the interaction of clavulanic acid with the OXA-2 beta lactamase. The OXA-2 and OXA-3 beta lactamases were shown to be related via immunological cross-neutralisation. The OXA-2 enzyme showed no relationship to any of the other plasmid-mediated beta-lactamases tested. Presumably if the OXA-2 enzyme is a class A beta-lactamase; then in view of the relationship between the OXA-2 and OXA-3 enzymes, the OXA beta-lactamase may also be assigned to this class.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry