Purification and characterization of a salt tolerant metallo-beta-lactamase from Aliivibrio salmonicida

Beta-lactamases are enzymes that inactivate beta-lactam antibiotics by hydrolyzing the amide bond that exists in the beta-lactam ring, disrupting the ring structure and make the antibiotics nonfunctional against bacteria. Metallo-beta-lactamases (MBLs) are a group of beta-lactamases which needs metal ions bound to its active site in order to function and are an important factor in many bacteria in order to be resistant to antibiotics. Resistance to antibiotics is a serious health problem which increases with excessive use of antibiotics. This study is based on the purification and characterization of MBL from the psychrophilic organism Aliivibrio salmonicida which is the causative agent for the Hitra disease, or cold-water vibrosis, in seawater-farmed Atlantic salmon (Salmo salar). Characterization of asMBL has revealed interesting properties in regards of temperature-, salt-, pH optimum and enzyme kinetics. asMBL properties have been compared to VIM-7, a MBL isolated from hospital which thrives in mesophilic conditions. Compared to VIM-7, asMBL proves to be adapted to the colder and saltier environment of the seawater. asMBL enzyme kinetics have also been compared to MBLs from human pathogenic bacteria and asMBL showed a general lower enzymatic efficiency in terms of lower kcat and higher Km at the same experimental conditions as the other MBLs. Nitrocefin, the substrate used for the characterization of asMBL, had its stability in temperature, pH and Tris investigated. From these investigations it seems plausible that nitrocefin is auto-hydrolyzed in a synergetic matter when present at high pH and in Tris buffer. An attempt to crystallize the protein and retrieve a structure was unsuccessful as it turns out that the protein seems to be aggregating at high concentrations.