L-氨基酸脱氨酶的分子改造及其用于全细胞催化法生产α-酮戊二酸条件的优化  

Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis

作　　者：王越 李江华[1] 堵国成[1] 刘龙[1] WANG Yue;LI Jiang-hua;DU Guo-cheng;LIU Long(Key laboratory of Carbohydrate Chemistry and Biotechnology Ministry of Education Jiangnan University, Wuxi 214122, China)

机构地区：[1]江南大学生物工程学院,无锡214122

出　　处：《中国生物工程杂志》2019年第3期56-64,共9页China Biotechnology

基　　金：国家自然科学基金优秀青年基金(31622001);江苏省重点研发计划-社会发展(BE2016638)资助项目

摘　　要：酮戊二酸(α-ketoglutaric acid,α-KG)是谷氨酸脱氨基的酮酸产物,作为一种重要的有机酸广泛用于食品、医药、精细化工等领域。为提高L-氨基酸脱氨酶全细胞催化法合成α-KG的效率及产量,首先通过优化全细胞催化剂制备条件及全细胞转化反应条件,包括发酵过程中的温度、诱导剂浓度、诱导剂添加时刻、诱导时间等;全细胞转化过程中的温度、pH、细胞量、转化时间。各个条件优化后以200g/L谷氨酸钠为底物时,产量最终提高了54. 9%,摩尔转化率为39. 6%。其次,通过定点饱和突变对L-氨基酸脱氨酶进行定向进化以提高其催化能力。经过多次突变、筛选,最优突变体E. coli BL21-pET-20b(+)-pm1152催化200g/L谷氨酸钠生成α-KG最高产量为100. 9g/L,摩尔转化率为64. 7%,较最初对照菌株提高了66. 3%。结果表明,条件优化和饱和突变可有效提高重组大肠杆菌全细胞转化合成α-KG的能力。Alpha-ketoglutaric acid(α-KG),which is a keto acid product deaminated by glutamic acid,is widely used in food,medicine,fine chemicals and other fields as an important organic acid. To improve the yeild and the efficiency of biotransformation for the synthesis of α-ketoglutaric acid. First,by optimizing the conditions of whole-cell biocatalyst preparation and whole-cell biocatalysis conditions. Optimization conditions include the temperature,pH,inducer concentration,induction time in the whole-cell biocatalyst preparation process and the temperature,pH,biocatalyst concentration,biocatalytic time in the whole-cell biocatalysis process. Determine the optimal conditions of each item by detecting the amount of product α-KG. After the conditions were optimized,the maximum yield was increased by 54. 9 % and the molar conversion was 39. 6 %. Secondly,the directed evolution of L-amino acid deaminase by site-directed mutagenesis increased its catalytic ability. Through multiple mutations,screening,the yield of α-ketoglutaric acid biocatalytic synthesized by monosodium glutamate with optimal mutant E. coli BL21-pET-20 b(+)-pm1152 was 100. 9 g/L,and the molar conversion rate was 64. 7 %,an increase of 66. 3% compared to the control strain. The maximal yield and molar conversion of L-glutamic acid to α-KG was reached under the following optimal conditions: 20 g/L whole-cell biocatalyst,30 ℃,pH 6. 0,and 60-h biocatalysis,strain: E. coli BL21-pET-20 b(+)-pm1152. The results showed that the conditional optimization and saturation mutation could effectively increase the whole-cell biocatalyst of recombinant E. coli to synthesize α-ketoglutaric acid.

关 键 词：L-氨基酸脱氨酶 条件优化 定点饱和突变 全细胞催化 

分 类 号：Q819[生物学—生物工程]