Abstract: To avoid deactivation of glutamine synthetase (GS) resulting from adenylylation, site-directed mutagenesis was used to replace Tyr405, the putative adenylylation site of GS in Corynebacterium glutamicum, by Phe405. The mutant gene was expressed in E. coli to obtain GS which can enhance the enzymatic conversion from glutamate to glutamine. The wild gene with Tyr405 and the mutant gene with Phe405 were constructed into the recombinant E. coli pET-3a/GSI and pET-3a/GSIM, respectively. GS activity expressed in pET-3a/GSIM is 150 U/L which is 5.0 times as 30 U/L in pET-3a/GSI. Glutamine productivity in pET-3a/GSIM is 17.5 g/L which is 5.1 times as 3.4 g/L in pET-3a/GSI.

Key words: glutamine, E. coli, glutamine synthetase, putative adenylylation site

摘要： 为解决谷氨酰胺合成酶腺苷酰化修饰失活的问题，利用基因定点突变的方法将谷氨酸棒杆菌的谷氨酰胺合成酶(Glutamine Synthetase, GS)腺苷酰化位点由Tyr405突变为Phe405，并在大肠杆菌中获得突变后GS的表达. 对比腺苷酰化位点突变前后的重组大肠杆菌pET-3a/GSI和pET-3a/GSIM在高氨环境下的GS活性和谷氨酰胺产量，发现重组菌pET-3a/GSIM在高氨环境下的最大酶活是150 U/L，产谷氨酰胺浓度为17.5 g/L，分别是pET-3a/GSI酶活(30 U/L)的5.0倍和产谷氨酰胺水平(3.4 g/L)的5.1倍，GS定点突变使谷氨酸转化为谷氨酰胺的途径得到强化.

关键词: 谷氨酰胺, 大肠杆菌, 谷氨酰胺合成酶, 腺苷化位点