Engineering of the critical residues at the stereochemistry-gate loops of Brevibacillus agri dihydropyrimidinase for the production of l-homophenylalanine

Brevibacillus agri dihydropyrimidinase (BaDHP) exhibits a substrate preference for d-homophenylalanylhydantoin (d-HPAH). Site-directed mutagenesis of BaDHP was performed specifically to the residues proposed to be important in the enzyme activity. M63A, F65A, L94A, L159A and L159V variants exhibited the increased activity (54–469%) toward l-HPAH. L159V variant was used to convert HPAH to l-homophenylalanine (l-HPA) in the hydantoinase process. As compared with the wild-type enzyme, the conversion yield of l-HPA was increased from 39 to 61% by L159V variant. The conversion yield for l-HPA production was further increased up to 90% by coupling L159V variant with Bacillus kaustophilus l-N-carbamoylase and Deinococcus radiodurans N-acylamino acid racemase in the biocatalysis process.