||D-amino acid oxidase (DAO, EC126.96.36.199) is a key enzyme for the production of 7-aminocephalosporanic acid, the precursor of semi-synthetic cepham antibiotics. It was found to be inactivated by H2O2 generated from its own catalysis. Methionine oxidation usually plays an important role in protein damage. Therefore, using the SOEing (splicing by overlapped extension) method, the methionine residues in Rhodosporidium toruloides and Candida boidinii DAO were replaced by leucine residues to improve their resistance to H2O2.
Three R. toruloides mutant DAOs M59L, M88L, and M213L as well as two C. boidinii mutant DAOs were produced. The specific activities of R. toruloides mutant DAOs M59L, M88L, and M213L were 101.9, 84.1, and 106.7 U/mg, respectively. The former two were higher than that of wild type DAO (98.6 U/mg). The kinetic analysis also showed that M59L had a kcat/Km of 39.9 s-1 mM-1 which was better than that of wild type DAO (35.7 s-1 mM-1). On the other hand, C. boidinii mutant DAOs didn’t show any detectable activity.
The resistance to H2O2 of M59L and M213L were slightly better than that of wild type DAO. The treatment with 100 mM H2O2 for 20 minutes resulted in a decrease in the specific activities of M59L, M213L, and wild type DAO by 7.88, 12.36 and 0.66%, respectively.