Studies On The Biosynthesis And Separation Of Adenosine Triphosphate

The production of adenosine triphosphate(ATP) from adenosine monophosphate (AMP) catalyzed by beer yeast is regarded as one of the most promising techniques in ATP industry. However, there are still some problems existing in this technology such as how to stabilize yeast cell quality, optimize the production conditions and enlarge the production scale. In order to solve these problems, experiments on the reacting condition, generations of beer yeast and permeability of yeast cell were carried out in this paper, with the beer yeast fetched from the beer plant as the enzyme source. At the same time, the pre-separation and further purification of fermentation broth were systematically studied through active carbon and anion-exchange resin. Under the selected conditions, ATP was separated from the fermentation broth. In the end, the environmental influence in ATP production process was estimated.The influences of the factors such as the glucose concentration, phosphate buffer, AMP usage, pH value of the broth, reaction time and temperature were firstly investigated on the fermentation by orthogonal tests. In addition, the effects of yeast amount and stirring rate were also studied. The optimized reaction conditions were obtained as follows, glucose 150 mmol ?L"1, phosphatebuffer 200 m mol-L , AMP 20 gL , pH 8.0 , reaction time 4h, reaction temperature 35癈, yeast 50gL"', 75 rpm, sulphuric acid magnesium 15 mmol -L"1 respectively.The reaction activity of yeast was compared among yeasts of different generations and different conservation time and the yeasts fetched from the first and second pre-fermentation period were found to have a relatively high activity. Several permeabilization methods were employed to treat the yeast. For the ice-frozen yeast, the higher activity was achieved when the frozen time was five to ten days. The yeast treated by acetone behaved the highest production activity with the conversion rate of 89%.In the pre-separation of fermentation broth with active carbon, the selection of active carbon was carried out since different kinds of active carbons have different physical performances. The influence of broth pH, concentration and loading velocity as well as washing velocity and composition proportion was investigated on active carbon adsorption and separation efficiency, respectively. The suitable condition for adsorption is pH 3,concentration 12gL"' at 0.09BVmin"', while the desorption process is favored to operate with ammonia/95% ethanol/distilled water equal to 2 ?5 ' 3(v/v), at 0.23BVmin~'.The anion-exchange resin was selected to recover ATP from the broth. After the best one among different types of resins was determined as the separation resin, the influence of loading solution concentration, pH and velocity was carried out on the adsorption behavior. The best condition foradsorption is pH8.0,concentration ISgL"1 at O.OTBV-min"1. For the desorption process, the suitable conditions were as follows: 0.01 mol -L"1 HAc/0.07 mol -L"' NaAc for eluting AMP; 0.16 mol -L"1 HAc/0.36 mol -L'1 NaAc for ADP, and 0.25 mol-L'1 HC1/0.75 mol-I/1 NaCl for ATP, at O.ieBVmirf1. The UV-absorption spectra, chromatogram of HPLC and Infrared spectra of product are consistent with that of normal sample.Finally, the environmental influence of ATP production process was evaluated and some useful suggestions were put forward to solve the pollution problem engendered.