一株谷氨酰胺合成酶高产菌株的酶学分析及其基因的克隆分析

Analysis of a high glutamine synthetase producing Arthrobacter globiformis strain

对谷氨酰胺合成酶菌球形节杆菌(Arthrobacter globiformis)突变菌株Y3进行分析.其谷氨酰胺合成酶摇瓶发酵单位是原始菌株的2.81倍;最适反应温度为40℃;最适pH为6.5.以突变菌株和原始菌株基因组为模板,通过同源克隆策略,成功地获得了球形节杆菌谷氨酰胺合成酶基因gln片段.通过序列比对,突变菌株gln基因在第409 bp处产生T→C的碱基的突变,该处突变使酪氨酸(Tyr409)被组氨酸(His409)取代,该突变解决了谷氨酰胺合成酶腺苷酰基化问题,保证了其在高浓度铵盐条件下保持酶活性.

In this study,a mutant strain Y3 with high glutamine synthetase productivity was investigated,its activity was 2.81-fold of the original one.Examined the nature of glutamine synthetase,the results showed that the optimal reaction temperature and pH was 40 ℃ and 6.5,respectively.According to the homology,we designed a pair of PCR primers to clone the gln gene from UV mutant and original strain of Arthrobacter globiformis,respectively.Aligment of the glns revealed that a transversion of T→C at the position 409 bp,and this made Tyr409 was replaced by His409.The mutant of T→C can solve deactivation of glutamine synthetase（GS） resulting from adenylylation in high NH＋4 concentrations.