The emergence of antibiotic-resistant bacteria has become a major threat worldwide [1]. Microorganisms employ several strategies to acquire resistance, of which the most potent and rapid approach is to produce β-lactam hydrolysing enzymes called β-lactamases [1, 2]. β-lactamases are grouped into four molecular classes, from A to D [3]. Class B or metallo-β-lactamases (MBLs) are the only group of enzymes that require 1 or 2 metals for hydrolysis [4]. The MBL family is further subdivided into subclass B1, B2 and B3 [4, 5] based on their sequence homology and hydrolysis mechanisms [6]. In this study, the characteristics, substrate and inhibition profiles of uncharacterised B1 enzymes selected from a combination of 3 public databases containing 325 amino acid sequences have been elucidated to understand their substrate profiles and metal preferences. Protein structure is also solved to structurally compare to the closely-related enzyme from the same cluster. This study shows the effectiveness of the use of available bioinformatics resources and databases to investigate the similarity of protein structure and enzyme characteristics between environmental and clinical-derived enzymes.