{Reference Type}: Journal Article
{Title}: Identification and structure-based engineering of a dipeptidase CpPepD from Clostridium perfringens for the synthesis of l-carnosine.
{Author}: Zhang X;Liu X;Chen X;Feng J;Zhao Q;Wu Q;Zhu D;
{Journal}: J Biotechnol
{Volume}: 389
{Issue}: 0
{Year}: 2024 Jun 20
{Factor}: 3.595
{DOI}: 10.1016/j.jbiotec.2024.05.001
{Abstract}: l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94 mM (14.46 g L-1) or 67.02 mM (15.16 g L-1) l-Car was produced at the substrate concentrations of 6 M β-Ala and 0.2 M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.