Barallon, Valerie Rita; (1990) Study of Streptomyces β-lactamase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

β-lactamases play an important part in the resistance of many bacteria to β-lactam antibiotics. This degradative enzyme hydrolyses the amide bond in the β-lactam ring of susceptible penicillins and cephalosporins. 70% of antibiotics including the β-lactam compounds are produced by Streptomyces. In addition, Streptomyces produce a number of extracellular enzymes, including β-lactamases. In this study, the production of beta-lactamase by Streptomyces was examined. Out of 15 Actinomycete strains screened 3, Streptomyces alhofaciens, Streptomyces rimosus and Streptomyces violaceonlger, were found to produce significant amounts of β-lactamase. From tine course studies, it was round that all 3 organisms produced the enzyme early in the life cycle and that S. alhofaciens had the greatest yield of β-lactamase. Analysis of affinity chromatography purified β-lactamase from S. alboiaciens, indicated that the enzyme was a serine type β-lactamase with a molecular weight of aproximately 52kDa. Semi-purified β-lactamase from S. alhofaciens was used to raise anti-sera in rabbit. In Western-blot analysis of crude extra cellular enzyme irom S. alhofaciens, S. rimosus, S. violaceoniger, and the semi-purified β-lactamase, there appeared to be some degree of homology between the β-lactamases of these 3 microorganisms. The anti-sera was also used to screen a gene bank of S. albofaciens in xL47. 12 recombinants were isolated and DMA prepared from them was mapped and sub-cloned into an E.coli-Streptomyces shuttle vector pQR1, In addition attempts were made to subclone the bla gene into pQR1 and transformed into the Streptomyces host Streptomyces lividans. β-lactainase activity from the Streptomyces lividans transformants displayed β-lactamase activity on agar plates.

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