Characterization And Improvement Of Glutamine Synthetase From Bacillus Megaterium N6

Glutamine synthetase(GS)which catalyzes the conversion of glutamate and ammonia into L-glutamine in an energy dependent reaction with ATP is widely found in animals,plants and microbiology and it is very important in biological nitrogen metabolism.In addition,some GS could also produce L-theanine when the substrate ammonia was substituted with ethylamine.Glutamine has a high nutritional and medicinal value,theanine is good for health,so this two production have a huge market.GS has a great potential industrial application.The study work around Bacillus megateriumN6 which has GS activity and and achieved the following results:The target gene of BGS was obtained by PCR through the relevant gene sequence.The results show that BGS have 1335 bp,encoding 444 amino acids,and the molecular weight of the translated protein is 50.4 kDa.The protein sequences encoded by BGS are 88% identical to those of the reported Bacillus subtilis.Through the study of the enzymatic properties of BGS,it is find that the activity is best at 30? and pH 7.0.The thermal stability of BGS is as high as 90% after treatment at 55 ? for 2.5h,and there is still about 50% activity at 65 ? for 1h,so it has good thermal stability.After thin layer chromatography(TLC)and HPLC show that the gene could synthesize theanine.In order to improve the activity of BGS,the gene wes directed to evolution by random mutagenesis and site mutagenesis.Random mutations were screened by random mutant library constructed by error prone PCR.The results showed that 2 beneficial mutations F1-D10,F1-F10 were screened among 8,000 strains.The catalytic efficiency of F1-D10 to ADP was increased 33%,to glutamine was decreased 20%,to hydroxylamine was improved 79%.The catalytic efficiency of F1-F10 to ADP was improved 65.7 times,to glutamine was increased 56%,to hydroxylamine was increased 67%.Re-mutation of F1-F10,I28 T and L159 I were constructed,and the catalytic efficiency of L159 I mutant was improved 93.7-,2.5-and 3.2-fold that of WT for ADP,glutamine and hydroxylamine,respectively.Seven important catalytic sites were selected by multiple sequence alignment and homologous modeling and E65 A was good in seven site mutations.The results revealed that the E65 A mutant was improved 154.7-,1.4-and 2-fold that of WT for ADP,glutamine and hydroxylamine,respectively.The single-point mutations E65 A and F1-D10 were superimposed to study the effect of superposition and the results did not good.Studies of enzymatic properties show that compared with wild type optimum temperature of E65 A was changed to 50 °C and the optimum temperature for the L159 I was changed to 55 °C.At the same time,the thermal stability of the L159 I was significantly improved.About 90% activity was kept after treated with water bath for 2.5 hours in 65?,while the wild type was only 15% under the same conditions.The theanine production of the L159 I was increased 35% compared with wild type.The results of the structural simulation show that when the 159 Leu in the L159 I becomes Ile,the steric hindrance between the two monomers becomes smaller and the hydrogen bond which is near 159 Leu is more closely so the structure become stability.At the same time such changes make 156 tyrosine closer to the catalytic center and easier to catalyze the reaction.Glutamic acid at position 65 becomes alanine,which makes the active cavity open larger and easier to release the key catalytic site so the catalytic activity of the enzyme become better.In short,this study laid the foundation of structure and function about BGS also good for the further research and development.