Protein Engineering of d‐Succinylase from Cupriavidus sp. for d‐Amino Acid Synthesis and the Structural Implications

Abstract

d‐Amino acids are important chiral building blocks for pharmaceuticals and agrochemicals. Previously, we have used d‐Succinylase (DSA) from Cupriavidus sp. P4‐10‐C and N‐succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503 to produce d‐amino acids via the dynamic kinetic resolution of N‐succinyl‐dl‐amino acids. However, the use of this bioconversion system remains challenging for industrial application due to the insufficient enantioselectivity of DSA toward N‐succinyl‐d‐amino acids. Therefore, we screened DSA mutants for improved enantioselectivity by directed evolution. Several mutants showed improved enantioseletivity compared to wild‐type DSA. L182E mutant had superior enantioselectivity, and the thermal stability was also remarkably improved by this single mutation. We solved the crystal structure of the L182E mutant in complex with succinic acids at a resolution 2.0 Å. The mutated residues in all generated mutants that showed improved enantioselectivity (including the substituted Glu182 in the L182E mutant) are found very close to the active site. The solved crystal structure also provides some rationale to explain the higher thermostability of the L182E mutant compared to wild‐type DSA. d‐phenylalanine and d‐tryptophan were produced in high conversion (approximately 90%) with 98.8% ee and 99.6% ee, respectively, using coupled L182E DSA and NSAR with the one‐pot enzymatic method. These data suggested that L182E DSA may be a useful biocatalyst for industrial d‐amino acids production.