| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 102 === Cholera is potentially epidemic and life-threatening caused by Vibrio cholerae serogroup O1 and O139 that could result in acute diarrheal illness. The statistics from CDC (Centers for Diseases Control and Prevention) showed that there are 3 to 5 million cases and over 100,000 deaths each year globally. If left untreated, the mortality could reach up to 50%. Oral rehydration is the main treatment in clinic, and antibiotics can efficiently shorten the duration and diminish the severity of symptoms. It is reported that antibiotic resistance has emergied in V. cholerae to cause limitation on complete recovery.
Analysis of the completed genome sequence of V. cholerae 01 Biovar Eltor strain N16961 has revealed a chromosomally encoded metallo-β-lactamase, which may be able to hydrolyze most existing β-lactams. We aimed to identify and characterize this new putative metallo-β-lactamase VarG from V. cholerae CVD101. We also wanted to understand its drug specificity and its oligomeric state in neutral buffer, so as to categorize it into Ambler molecular classification. First, the PCR-amplified varG was cloned into pET-26b and transformed into E.coli C43 (DE3) for protein overexpression. VarG was purified by using affinity column purification, and further verified by using SDS-PAGE, Western blotting and ESI-Q-TOF MS/MS protein sequencing. In the other side, size exclusion chromatography, deconvolution by multiply high charged protein, and analytical ultracentrifuge showed that VarG was in dimeric form in neutral buffer. Drug degradation test indicated that VarG showed relatively high activity on carbapenems (imipenem, meropenem kcat = 165.4 s-1, 29.43 s-1), marginal activity on penicillins (ampicillin, piperacillin kcat = 2.54 s-1, 0.33 s-1) and cephalosporins (cephalothin, cefuroxime, cefepime, moxalactam kcat = 0.075 s-1, 0.12 s-1, 0.29 s-1, 0.41 s-1), but no activity on monobactams. In conclusion, VarG could be categorized into Ambler class B2.
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