Modification of L-amino Acid Deaminase Gene by Error-prone PCR to Improve the Production of α-keto-β-methylvaleric Acid from L-isoleucine Through Whole-Cell Biocatalyst

Abstract:

In order to make directed evolution on L-amino acid deaminase coming from Proteus mirabilis，the error-prone PCR was used to introduce random mutagenesis to the gene to construct a mutation library and screen for mutants which could have high production of α-keto-β-methylvaleric acid. The optimal reaction conditions of the mutant named 7/23-6 were as following. The optimal substrate concentration was 900 mmol/L L-Ile. The optimal reaction tempreture was 30 ℃. The optimal reaction pH was 8.5. And the optimal reaction time was about 21~24 h. Under these conditions，102 g/L α-keto-β-methylvaleric acid could be got with 87% substrate conversion rate as a result. Compared with the original strain，both the α-keto-β-methylvaleric acid production and substrate conversion rate were improved 13 %，and its thermostability was improved 36.7 %.