Improvement of the Substrate Specificity and thermostability of Porcine Aminoacylase I by Directed Evolution

Directed evolution was used to improve the specificity and thermostability of porcine aminoacylase I(pACY1) hydrolyzing N-acetyl-L-Arg and N-acetyl-L-Phe.A mutant libarary was constructed by error-prone PCR and DNA shuffling.The positive clones were screened by nutrition selective-culture plates and 96-well plates thermoresistance.After 5 cycle shuffling and screening,One mutant named 7E15 was selected.It exhibited 23-fold increased activity and 7℃ increased Tm by hydrolyzing N-acetyl-L-Arg and 9-fold increased activity by hydrolyzing N-acetyl-L-Phe comparing its wild type parent.Sequence analysis revealed that 7E15 has 7 amino acid substitutions-namely L176A,R196T,A283G,V304G,K306Q,V369A and L370T.L176A,V369A and L370T are near to the activity center in the model of pACY1,and they were suggested to bind with the substrates;while the other 4 were distant,may play key roles in the change of the thermostability of pACY1.