Enhancing enzyme activity and thermostability of cephalosporin C acylase based on B factor analysis

Abstract:

[Objective] The aim of this study was to obtain a more stable cephalosporin C (CPC) acylase by screening site-directed saturation mutation libraries of Pseudomonas sp. SE83 acyII and to characterize the structure and function of mutant enzyme. [Methods] We computated B factor based on the structure of Pseudomonas diminuta N176, a homologue of Pseudomonas sp. SE83 acyII, and constructed site-directed saturation mutation libraries of SE83 acyII. Combined with pH indicator assay, we used a Biomek FXP automation workstation high-throughput screening method to screen more stable CPC acylase. The enzyme properties were further defined by comparison with the wild type enzyme. The relationship between structure and function of the positive mutants was studied by homology modeling, using SWISS-MODEL software. [Results] We found 9 targeting sites by B factor homologous structure alignment and computation. After 3 rounds of screening, we found that the mutations occurring at residues R218 and K226 could enhance the thermostability of acylase, and the most stable mutants were identified as R218Q and K226V. Their half-lives at 40 °C were approximately 3.77- and 2.77-fold of the wild type enzyme, respectively. The catalytic efficiency kcat/Km was also approximately 1.8- and 3.1-fold of the wild type, respectively. The possible mechanism for the enhanced stability might be the increments of hydrogen bonds and hydrophobic interaction, which were analyzed by homology modeling. [Conclusion] It proved to be an efficient and reliable strategy for improving enzymatic stability based on B factor, and the positive mutant R218Q and K226V could enhance the stability and catalytic efficiency of CPC acylase. Thus, this study may serve as a useful reference for further improving enzyme properties.