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

DOI：10.3969/j.issn.1673-1689.2018.11.009

中文关键词: L-氨基酸脱氨酶 易错PCR 全细胞转化 α-酮-β-甲基正戊酸

英文关键词: L-amino acid deaminase,error-prone PCR,whole-cell biocatalyst,α-keto-β- methylvaleric acid

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中文摘要:

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%。

英文摘要:

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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