Европейский журнал экспериментальной биологии Открытый доступ

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Screening, production, purification and characterization of beta-lactamase from uropathogenic E.coli

Yogini A. Ranade, Smita M. Dharmadhikari and Prasad A. Wadegaonkar

Beta-Lactamase activity was detected in 15 uropathogenic isolates belonging to family Enterobacteriaceae by iodometric method. E. coli Strain Y3 showing multiple antibiotic resistance (MDR) was used to optimize the conditions for production and assay of the enzyme. The major location for the enzyme amongst all uropathogens was found to be extracellular / Periplasmic (72%) including the selected isolate Y3. The enzyme is mainly cell associated and was released from the cell surface by sonication. During periodic monitoring 8 hours of incubation produces maximum cell mass with optimal enzyme activity at pH 7 and temperature of 35°C. The beta lactamase was partialy purified by ammonium sulphate precipitation followed by desalting and dialysis. The pooled ammonium sulphate activity was found to be 5.2 EU (1.5 fold purification). Further dialysed extract gave 2.2 fold purification followed by 4 fold purification in case of desalted concentrated enzyme. The enzyme was found to be active and stable at pH 8 for 48 hrs. and at temperature 35°C for 48 hrs. The enzymes have penicillanase activity, get induced by inducers like penicillin, ampicillin and amoxicillin and are inhibited by Clavulunic acid.