Directed evolution of N-carbamyl-d-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability

摘要

Directed evolution of n-carbamyl-d-amino acid amidohydrolase for simultaneous improvement of oxidative and thermal stability.A mutant library was generated by DNA shuffling,and positive clones with imporved oxidative and thermal stability were screened on the basis of the activity staning method on a solid agar plate containing pH indicator (phenol red) and substrate 9N-carbamyl-D-p-hydroxyphenylglycine).Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly imporved stability.Oxidative stability of theevolved enzyme 2S3 was about 18-fold higher than that of the wild type,and it alsoshowed an 8-fold increased thermostability.The K_m value of 2S3 was comparable to that of wild-type enzyme,but k_(cat) was slightly decreased.DNA sequence analysis revealed that six aminoa acid residues (Q23L,V40A,H58Y,G75S,M184L,and T262A) were substituted in 2S3.From the mutational analysis,four mutations (Q23L,H58Y,M184L,and T262A) were found to lead to an improvement of bot oxidative and thermal stability.Of them,T262A had the most significant effect,and V40A and G75S only increased the oxidative stability.