易错PCR法提高L-氨基酸脱氨酶全细胞转化L-异亮氨酸生产α-酮-β-甲基正戊酸效率

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

对奇异变形杆菌Proteus mirabilis中的L-氨基酸脱氨酶进行定向进化,利用易错PCR技术向基因中引入随机突变,建立突变文库来筛选可获得较高α-酮-β-甲基正戊酸产量的突变株。突变株7/23-6最适全细胞转化反应条件是以pH 8.5 Tris-HCl为缓冲液,用900 mmol/L L-异亮氨酸在30℃下进行催化反应21～24 h,其α-酮-β-甲基正戊酸产量可以达到102 g/L,底物转化率达到87%。与对照菌相比,α-酮-β-甲基正戊酸产量和底物转化率均提高了13%,热稳定性提高了36.7%。

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 ofα-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/Lα-keto-β-methylvaleric acid could be got with 87%substrate conversion rate as a result.Compared with the original strain,both theα-keto-β-methylvaleric acid production and substrate conversion rate were improved 13%,and its thermostability was improved 36.7%.