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-  2018 

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

DOI: 10.3969/j.issn.1673-1689.2018.11.009

Keywords: L-氨基酸脱氨酶 易错PCR 全细胞转化 α-酮-β-甲基正戊酸
L-amino acid deaminase
,error-prone PCR,whole-cell biocatalyst,α-keto-β- methylvaleric acid

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Abstract:

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 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 %

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