Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for β-lactam synthesis reactions. Rational design of ‘whole variants’ was explored to rapidly improve AEH thermostability by mutating between 3–15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.

氨基酯水解酶（AEH）能够快速合成头孢氨苄，但即使在低温下也会快速失活。之前对 AEH 进行工程设计的努力已经产生了几种改进的变体，但其范围受到限制，部分原因是 β-内酰胺合成反应的活性测定通量有限。探索了“整体变体”的合理设计，通过突变 3-15% 的残基来快速提高 AEH 的热稳定性。发现大多数变体由于钙结合位点突变而失去活性，而其功能之前尚未被描述过。四种活性变体均具有改进的熔化温度，并在合成和水解活性、熔化温度和 25°C 失活方面进行了表征。与最初的 AEH 变体相比，发现两个变体的总营业额有所提高；然而，每个变体的稳定性提高和总体活性之间存在明显的权衡。