%0 Journal Article
%T 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
%A 郭濛檬
%A 刘龙
%A 李江华
%A 堵国成
%A 陈坚
%J 食品与生物技术学报
%D 2018
%R 10.3969/j.issn.1673-1689.2018.11.009
%X 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%。</br>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 %
%K L-氨基酸脱氨酶 易错PCR 全细胞转化 α-酮-β-甲基正戊酸&lt
%K /br&gt
%K L-amino acid deaminase
%K error-prone PCR
%K whole-cell biocatalyst
%K α-keto-β- methylvaleric acid
%U http://spyswjs.cnjournals.com/spyswjs/ch/reader/view_abstract.aspx?file_no=201811009&flag=1