Evolutionary robustness requires that the number of highly conserved amino acid residues in proteins is minimized. In enzymes, such conservation is observed for catalytic residues but also for some residues in the second shell or even further from the active site. β-lactamases evolve in response to changing antibiotic selection pressures and are thus expected to be evolutionarily robust, with a limited number of highly conserved amino acid residues. As part of the effort to understand the roles of conserved residues in class A β-lactamases, we investigate the reasons leading to the conservation of two amino acid residues in the β-lactamase BlaC, Glu37 and Trp229. Using site-directed mutagenesis, we have generated point mutations of these residues and observed a drastic decrease in the levels of soluble protein produced in Escherichia coli, thus abolishing completely the resistance of bacteria against β-lactam antibiotics. However, the purified proteins are structurally and kinetically very similar to the wild type enzyme, only differing by exhibiting a slightly lower melting temperature. We conclude that conservation of Glu37 and Trp229 is solely caused by an essential role in the folding process and we propose that during folding Glu37 primes the formation of the central β-sheet and Trp229 contributes to the hydrophobic collapse into a molten globule.