Screening Of Glutamine Synthetase-producing Strain

Glutamine synthetase (GS,glutamine synthetase;EC6.3.1.2)is an enzyme widespread in vivo, it is involved in many organisms, nitrogen and carbon metabolism. And nitrogen metabolism is necessary for organisms to maintain life, therefore, it is one of the most critical enzymes in vivo. It is also very thorough in vivo distribution and physiological functions. In addition, it is an important enzymes that can catalyze the synthesis of theanine in vitro. glutamine is extremely important in industrial production of catalysts, is very valuable material, so it is also increasing for demanding . Microbial fermentation is economic and efficient way to produce glutamine synthetase.Currently glutamine synthetase producing- strain mainly belong to some of the bacteria Arthrobacter. The use of mold fermentation of glutamine synthetase rarely, Aspergillus is not being studied. In this study, first screening a high glutamine synthetase-producing strain,from strains preserved in laboratory. and researching UV mutagenesis , optimized fermentation conditions and the enzymatic properties.First of all, screening a high glutamine synthetase-producing strain- Aspergillus from strains preserved in laboratory. Then, Aspergillus oryzae was chosen for UV mutagenesis. the optimal conditions for UV mutagenesis of Aspergillus oryzae were determined as follows: UV lamp 15W, irradiation distance 20cm, irradiation time 120S. Finally, after rescreening, and genetic stability testing, the enzyme production capacity increased up to 15.6%Secondly, out of the breeding conditions of Aspergillus oryzae fermentation optimization, first the single factor experiments, including:optimal carbon and nitrogen sources, initial pH, medium volume, fermentation temperature, rotation speed and the inducing factor. The results show that the optimum carbon source was sucrose, the optimum nitrogen source was peptone, and the optimum initial pH 6, the optimum liquid volume of 60ml, optimum temperature is 28℃, the optimum rotation speed 160r/min The optimum induced factor was sodium glutamate. Determined by orthogonal experiment of four factors add significantly affect the amount of optimization of medium components and liquid fermentation conditions for the breeding out of glutamine synthetase in Aspergillus oryzae enhance the ability.Again, on the ultrasonic breaker broken the conditions of the cell wall of Aspergillus study, setting different working time and intermittent time, and then optimize the total time, the results show that the ultrasonic power 400W breaker conditions as follows: working time 1S, intermittent time 3S, ultrasonic time 20min, was the optimum condition.Finally, the extracted glutamine synthetase in crude extract for further purification,Aspergillus oryzae by experiments on enzymatic properties of glutamine synthetase in apreliminary study. The results show that the optimal enzymatic properties as follows:reaction time was 30min, the optimum pH was 6.5, the optimum temperature is 60℃, Theresults also shown that both Mg²⁺ and Mn²⁺ were essential to maintain GS activity.