Study On Separating And Purifying Technique Of Mixture Of 5'-deoxymononucleotides

DNA is an essential substance with genetic characters, which is also a main part of organism. It plays an important role during the periods of growth, reproduction, inheritance, mutation for organism. DNA can promote cell's growth, increase cell's energy and improve the metabolism of organism. 5'-deoxymononucleotides is the production of hydrolyzed DNA with nuclease P1 as its activator. And it is comprised of deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxycytidine monophosphate (dCMP) and thymidine monophosphate (TMP). Mixture of 5'-deoxymononucleotides can provide necessary nutritive material for the recovery of cells and tissues. It also takes part in the course of metabolism of nucleic acid in vivo. Clinically, 5'-deoxymononucleotides can cure many diseases, including leucopenia, thrombocytopenia, anaemia, aplastic anemia and diversified inflammation.Nuclease P1 (E.C.3.1.30.1) is a glucoprotein with three zinc atoms. Its molecular weight is 36000 Da. The proportion of carbohydrate is 17%. The zymoprotein is comprised of 270 amino acids and is very rich in hydrophobe amino acid. Nuclease P1 can hydrolyze single-strand DNA and RNA. Thus, 5'-mononucleotide comes into being. Nuclease P1 is one of the few proleases which are widely demanded and can be produced in large scale.The commonly-used industrial production process of 5'-deoxymononucleotides is described as follows: DNA is hydrolyzed by Nuclease P1. Then, the digested solution is separated and purified. In the production of 5'-deoxymononucleotides, the courses of separation and purification are the key steps, which can greatly influence the quality andthe costs of these production courses. Nowadays, there are some methods for purifying 5'-deoxymononucleotides, including 201x7 anion-exchange resin chromatography, 201x8 anion-exchange resin chromatography, D293 anion exchange resin chromatography and ultrafiltration technique. These methods have been used industrially for decades, but they have the same disadvantages: bad repeatability, changeful quality, low purity, and high dependence on experience in production.To solve those problems, the research program is designed as follows: first, optimize culture medium and culture conditions;second, use 717 anion exchange resin, 001x7 cation exchange resin and 769 activated charcoal to produce qualified mixture of 5'-deoxymononucleotides on the basis of good repeatability, high recovery, convenience and low cost;third, use DEAE Sepharose Fast Flow to separate and purify 5'-deoxymononucleotides. Thus, we build up a method which can produces high-purity 5'-deoxymononucleotides stably with good repeatability, and provides scientific basis for industrial production.1. Acquiring Nuclease PI:1.1 Incubating penicillium citrinum and producing Nuclease PI by fermentation:1.1.1 Strain:The strain is bought from CICC, number 4011. It can be cultured in solid medium or liquid medium.1.1.2 Culture medium:Solid medium: 2%agar, 20%potato, 5%glucose, 0.05%KH2PO4, 0.05%KH2PO4, 0.04%MgSO4,0.04%CaCl2,natural pH, autoclaving 20min at 121 °C.Liquid medium: 20%potato, 5%glucose, 0.05%KH2PO4, 0.05%KH2PO4, 0.04% MgSO4,0.04%CaCl2,natural pH, autoclaving 20min at 121°C.Fermentation medium: 5%glucose, 0.5%peptone, 0.5%groundnut flour, 0.05 % KH2PO4, 0.05 % KH2PO4, 0.04 % MgSO4, 0.04 % CaCl2, 0.02 % ZnSO4 , pH6.0, autoclaving 20min at 121 °C.1.1.3 Culture method:Plate culture: inoculate penicillium citrinum on solid medium, temperature 30°C,incubated for four or five days.Seed culture: put 10 mL liquid medium in 50 mL conical flask, then inoculate penicillium citrinum, and incubated for 24 hours at 200r/min, temperature 28 °C.Fermentation culture: put 50 mL Fermentation medium in 500 mL conical flask, the inoculation quantity of penicillium citrinum is 10%, and incubated for 72 hours at 200r/min, temperature 28°C. 1.1.4 Result of fermentation:The activity of nuclease PI is improved from 48IU/mL to 168.63IU/mL at this condition. 1.2 Precipitating nuclease PI with ammonium sulfate(80% saturation):Filtrate nuclease PI which is acquired by fermentation, precipitate it with ammonium sulfate (80% saturation), then get the precipitation by centrifuging. At last, dialyze nuclease PI in dialysis bag (5000 Mr).2. Hydrolyzing DNA:Dissolve DNA in H2O, denaturalize it by heating the solution up to 90°C, adjust pH to 5.4 with HC1, then put nuclease PI in and keep hydrolyzing for at least 1.5h. When the hydrolyzation is finished, heat the solution up to 90°C again to make nuclease PI denaturalized, put the solution in refrigerator to make denaturalized nuclease PI precipitated.3. Study on Separating and purifying method of Mixture of 5'-deoxymononucleotides:3.1 Measuring purity and recovery rate of 5'-deoxymononucleotides:Measure the purity of 5'-deoxymononucleotides in eluate as well as the concentration of 5'-deoxymononucleotides in sample solution and eluate. Then calculate the content of 5'-deoxymononucleotides in sample solution and eluate to get the recovery rate.3.2 Separating and purifying 5'-deoxymononucleotides by ultrafiltration:denaturali zat i onammoni a , pHT. 0protein Jiydrolyzate------? filtrate----------------?? filtrateSOXZ. 10MIM, filter769 activated charcoalpII3. 0 concentration----------------------------?■ eluatc------------■?■ concentrateclutinn, nthnnnl and nmmnni afilter ultrafiltering ultrafiltering------?'filtrate ----------------m* filtrate------<---------?■0. 45^? 10000MW 1000 Iff1yophi1i zat i on filtrate--------------------^ g* -deoxy?osaonucleotidesThis method has high speed and high recovery rate (80%-85%), but the purity of 5'-deoxymononucleotides is very low (only 50%). The purity can't meet the standard of national pharmacopoeia and it needs to be improved.33 Separating and purifying 5'-deoxymononucIeotides by 717 anion exchange resin:denat ur ali zat i on, pH7. 0protein hydrolyzate-------? filtrate------------------*■ fil trale90C,10IIN> filteranion exchangeC?1T anion resin] precipitate protein. 5 10mol/L NaOH.pHT. 0*■ eluatcelation,0.Zmol/L HC1 filterT$9 activated Ghajrcoal 769 activated charcoalpH3. 0 pHS. 0-........... ? eluote - ...........ipluti on, Kthnrird mid nmmnni n elul inn, fithannl and nnunnni aconcentration filter 1yophi11zation------------?■ concentrate------------?. filtrate_<sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub><sub>0. 22 MmThis method can make qualified 5'-deoxymononucleotides (70%), but the recovery rate is very low (only 70%).3.4 Separating and purifying 5'-deoxymononucleotides by 717 anion exchange resin and 001x7 cation exchange resin:denat ur ali z at i on ammoni a , pH7. Oprotein hydrolyzatc-------? filtrate —---■■—■-------——? filtrate90C, 1 OHM. filteranion exchange[717 anion TBsin] precipitate proteinpH8. 5 lOmol/L NaOH. pH7. 0---------------------------?■ eluate -------------------infiltratefilterelution, 0. 2md1/L HC1cation "change 769 activated charcoal001X7 resin pH3. 0—...........----—.........——-—*■ effluent ....................—_---.........—---_<sub>...............—......([>H1. b clution. cthanol and ammoniaconcentration filter lyophilix?tion--------■--------?■ concentrate -----------------*■ filtrate-----------------------?■0, 22mjb&* -deoxymononucleotidesThis method can make qualified 5'-deoxymononucleotides (70%), at the same time, the recovery rate is high enough (almost 40%). This method has good value for industrial production.3.5 Separating and purifying 5'-deoxymononucleotides by DEAE Sepharose Fast Flow:denat uralix at ionamiBon i a , pHY. Oprotein hydruiyzate—----? Filtrate ^—————-— ? filtrntr?90C10IIN, filter769 activated charcoalj!3 q cuticen I ration----------------------------------------.—?- eluate------------------*- cunceiitraleclution, cthanol and ammoniaDEAE Sepharose fact flow ?Filttvr binding buffcr(20mmol/L Tris, pH9. 0)"*? filtrate0. 45*?m elution buf f er (20niinol Tria+0. 025mol/I. NnCl,pH9. 0)5' -dRoiymfinnnus] noti dnsThis method use DEAE Sepharose Fast Flow to separate 5'-deoxymononucleotidesdirectly. The chromatographic grade purity of the eluate is above 98% by HPLC, the recovery rate of dCMP,dAMP and TMP is above 95%, the recovery rate of dGMP is above 90%.This method has ideal unanimity, high recovery rate. The gel has high chemical and physical stabilities that enable them to withstand the rigorous conditions. Most importantly, they are easy to scaled up and they are specially supported for use in the industrial production.0020Altogether, conclusions as following:1. After optimized culture medium and cultural conditions, the activity of nuclease PI is improved from 48IU/mLto 168.63IU/m.2. After studying on separating and purifying 5'-deoxymononucleotides by ultrafiltration, 717 anion exchange resin, 717 anion exchange resin and 001><7 cation exchange resin, we get a technique which is supported for use in the industrial production.3. At the same time, we have used DEAE Sepharose Fast Flow to separate and purify 5'-deoxymononucleotides. The result shows that the method can get high-purity 5'-deoxymononucleotides. The method has ideal unanimity, high recovery rate, and it is easy to scaled up and is supported for use in the industrial production. This research provides a good foundation for industrial production in the future.