| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 103 === β-lactams are one of the most widely used antibiotics in the world, and it works by inhibiting cell wall biosynthesis in the bacterial organism. Bacteria develop resistance to β-lactam antibiotics by synthesizing β-lactamase, which could attack the β-lactam ring and lead to the inactivation of β-lactams. 50% of the food-borne diseases in Taiwan were caused by Vibrio parahaemolyticus infection. Recently, more and more evidence pointed out that V. parahaemolyticus may also develop resistance to β-lactam. In this study, we aimed to characterize an putative β-lactamase, VPA0477, from V. parahaemolyticus. We analyzed the protein sequences of VPA0477 in the Uniprot database, and found that PSE-4 shares 71.3% of similarity and 49.1% of identity to VPA0477, suggesting that VPA0477 is a β-lactamase. The PCR-amplified vpa0477 was cloned into pET-26b and transformed into E. coli C43 (DE3) for protein overexpression. The optimal approaches for VPA0477 production is accomplished by using osmotic shock for protein extraction and purification by using ion-exchange chromatography in pH 6.5. Then His-tag of VPA0477 was removed, and it was named VPA0477ΔHis. Size exclusion chromatography (SEC) and analytical ultracentrifuge showed that VPA0477 had an estimated molecular weight of 31.4 and 29.4 kDa, respectively, suggesting that VPA0477 monomeric. Our kinetics data indicated that VPA0477ΔHis had a Km of 1.37 mM and a kcat of 323 s-1 for ampicillin degradation, and a Km of 1.34 mM and a kcat of 1090 s-1 for carbenicillin degradation; in addition, VPA0477 could be competitively inhibited by clavilanic acid and sulbactam with a Ki of 96.9985 and 1.2580 M, respectively. Our data concluded that VPA0477ΔHis should be classified into Ambler class A and Bush-Jacoby group 2c. Furthermore, VPA0477 doesn’t belong to Extended-spectrum β-lactamases (ESBLs) and carbapenemase.
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