Improvement of oxidative and thermostability of N-carbamyl-d-amino Acid amidohydrolase by directed evolution.
N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermostability. We attempted the simultaneous improvement of the oxidative and thermostability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, the best mutant 2S3 with improved oxidative and thermostability was selected, purified and characterized. The temperature at which 50% of the initial activity remains after incubation for 30 min was 73 degrees C for 2S3, whereas it was 61 degrees C for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at 25 degrees C resulted in a complete loss of activity, but 2S3 retained about 79% of the initial activity under the same conditions. The K(m) value of 2S3 was estimated to be similar to that of wild-type enzyme; however k(cat) was decreased, leading to a slightly reduced value of k(cat)/K(m), compared with wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3 and substitutions included Q23L, V40A, H58Y, G75S, M184L and T262A. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L and T262A were found to enhance both oxidative and thermostability of the enzyme and of them, T262A showed the most significant effect. V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of the stabilizing effects of each amino acid substitution was also carried out.
D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019020:Directed Molecular Evolution; D015723:Gene Library; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007218:Industrial Microbiology; D008958:Models, Molecular; D018384:Oxidative Stress; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D006861:Hydrogen Peroxide; D000581:Amidohydrolases; C112401:N-carbamyl-D-amino acid amidohydrolase