Studies On Synthesis And Purification Process Of Adenosine Triphosphate

With the development of the enzyme engineering and biochemical technology, production of pharmaceutical products by enzymatic method with Adenosine triphosphate (ATP) has reached the industrialization scale,and the demand of ATP on market is increasing.At present,the provision of ATP in domestic market is mainly depending on the technology of synthesis of ATP with adenosine.It will generate by‐products adenosine diphosphate (ADP) on the process of biosynthesis with ATP.ADP is mainly abandoned because there is no feasible route of ADP recovery technology at home and abroad,and it makes not only the pollution and waste of resources.So,it has very important significance on the academic significance、environmental protection、reducing the production cost and realizing the circulation economy to look for the process route of synthesis of ATP with ADP. We Studied the synthesis process of ATP beginning with the recycling by‐products ADP by beer yeast mud enzyme system and recycled ADP as raw materials, groping for recovery method of ADP and carrying on the research of The best substrate concentration, reaction temperature, fermentation medium pH value, fermentation time and so on on the fermentation reaction with recycled ADP. Meanwhile doing a system stud on decolorization、 desalination and purification of the ATPto determining the feasible process route in this paper.Recycling of ADP:adsorbing with205x7benzene sulfonic acid anion exchange resin,eluting with formic acid, ammonium formate buffer solution system, the eluent is desalinated by nanofiltration and then concentrated at low temperature stress until the concentration is qualified,and it can be used as fermentation reaction raw material of ATP. In order to reduce the cost of produce of fermentation reaction,we inspected the affect on the different concentrations of yeast mud, glucose、NaH2PO4、K2HP04、(NH4)3S04、MgS04in fermented liquid with the third generation of beer yeast mud as enzyme system,and the optimum formula is determined: m(glucose)=3.5%,m(yeast mud)=6.5％, m(K2HP04)=3.4%,m(NaH2P04)=7.6%, m((NH4)3S04)=0.2%, m(MgSO4)=1.44%。It need to be purified because the composition of fermented liquid is complex,and contains large amounts of salt substrate and sugar, low concentration of active ingredients.By the research we decide to use frame filter press for yeast mud solid‐liquid separation,No.7filter material,0.2MPa pressure,the filtering medium being desalted with activated carbon769‐1(Shanghai xing long), flow velocity:0.25BV/h, monitoring with ultraviolet spectrophotometer and A260≤0.1。The sample amount is1.5to2times column volume, washing by2b column volume of Purified water, the flow velocity is0.25BV/h, monitoring with ultraviolet spectrophotometer, collecting the fluid flow with A260≥0.1.Eluting with50%ethanol(PH=9with ammonia to adjust), the flow velocity is0.25BV/h,eluent is concentrated at low stress, temperature <70℃, Vacuum‐0.09MPa,recoveryingthe ethanol with athirdvolume, then decolorizatingwith macroporous resin. After the screening,the macroporous resinHZ‐818provided by Shanghai HuaZhen is suitable to decolorize, the flow velocity is0.25BV/h, effluent can be purificated directly with anion exchange resin column.The effluent of macroporous resin is addedin the anion exchange resin column（HZ‐201）, eluting with double column volume by1%NaOH solution to remove the nucleotide and purified water to eutral,eluting AMP、 ADP with0.05M NaCl solution(pH=1.5), then eluting with0.75M NaCl solution(pH=3),to obtainATP. After the inspection, the ATP made with this technology is conform to the pharmacopoeia standard, and the production process is stable withgood reproducibility,ADP is transformed successfully...