CN101418327A - The new process of production of high purity 5 ' Nucleotide - Google Patents
The new process of production of high purity 5 ' Nucleotide Download PDFInfo
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Abstract
The present invention relates to the production technique of 5 ' Nucleotide.A strain Penicillium citrinum bacterial strain M-71 is cultivated in submerged fermentation, produces the outer nuclease P of born of the same parents
1Rna solution degraded with 2.5～5%, degradation rate reaches more than 80%, use high frequency shale shaker to remove foreigh protein removing and remaining Yeast Nucleic Acid then, pure clear 5 ' Nucleotide solution after the removal of impurity is through ceramic membrane ultrafitration, remove the macromole residuals, again with four posts that strongly basic anion exchange resin is housed connect disposable absorption and stepwise elution, collection, four kinds of mononucleotide solution collecting, selecting molecular weight cut off is 300 daltonian nanofiltration membrane desalinations and concentrated, activated carbon decolorizing makes product after crystallization and the drying.The present invention has separated four kinds of Nucleotide admirably, and wash-out is removed inorganic phosphorus, guarantees to make highly purified 5 ' oligonucleotide product, can reach 98-102% through HPLC testing product purity.
Description
One, technical field:
The present invention relates to the production technique of 5 ' Nucleotide.
Two, background technology:
Producing four kinds of 5 ' Nucleotide at present simultaneously can only be with narrow spectrum nuclease P
1Degraded Yeast Nucleic Acid, through resin absorption, resolve separation and purification and obtain, prepare specificity, high vigor, cheap enzyme is one of key point of this technology for this reason, and the immobilization of use and immobilization nuclease P are arranged at present both at home and abroad
1Method, but technical requirements is very high, preparation cost is also high, is not suitable for being used in China the technology of scale operation 5 ' Nucleotide.Domestic have report to use immersion Fructus Hordei Germinatus to extract nuclease P
1But Fructus Hordei Germinatus is grain resource, preserves also difficulty.
Through nuclease P
1After degraded Yeast Nucleic Acid becomes 5 ' Nucleotide solution, in degradation solution, also stay enzyme, other foreign protein of sex change and fail all nucleic acid of degraded, this part nucleic acid is not 3 '-5 usually ' position syndeton or can not affact this part nucleic acid because of the main body configuration makes enzyme, they produce viscosity in solution, influence 5 ' Nucleotide and on resin, adsorb and resolve effect, the flocculation agent of use is arranged them cohesion and centrifugation method.But all waste time and energy, reduce yield, the gained treatment solution is also pure inadequately clear.Though can solve impurity such as part foreign protein, remaining nucleic acid with ceramic membrane ultrafitration, but the film surface is adhered to by these materials easily, causes the fenestra obstruction and penetrating amount is significantly reduced.
Gained Nucleotide be four kinds of 5 ' mixed nucleotides promptly 5 ' adenylic acid (AMP) (5 ' AMP), 5 ' guanylic acid (5 ' GMP), 5 ' cytidylic acid (5 ' CMP), 5 ' uridylic acid (5 ' UMP), the separation of present four kinds of mixed nucleotides all is to use Zeo-karb and anionite-exchange resin simultaneously or use a kind of resin isolation to open after two kinds of Nucleotide, separates two kinds of Nucleotide in addition with other method again.Its major cause is on Zeo-karb, the acidic conditions upper prop, uridylic acid can not be adsorbed and stream is worn resin, but guanylic acid and cytidylic acid under the condition that with water is eluent very major part resolve simultaneously and can not separate, must with an anionite-exchange resin they be separated again, adenylic acid (AMP) in the end can be by independent wash-out separately, therefore the 1970s and 1980s in last century in order to obtain precursor 5 ' adenylic acid (AMP) of ATP, also once used such technology in industrial production.If use an anionite-exchange resin to separate, then 5 ' cytidylic acid can finely separate with 5 ' guanylic acid.But divide too busy to get awayly with other two kinds of Nucleotide, rely on a positive resin that they are separated again again.
In addition in the nucleolysis process, owing to exist a small amount of monoesterase activity, and the nucleic acid end has the phosphate radical of 3 ' end, therefore inorganic phosphorus significantly increases in the degradation process, can produce 10g-15g inorganic phosphorus according to observation degraded per kilogram nucleic acid, need one of research badly and remove inorganic phosphorus and do not influence the isolating method of Nucleotide.
Final nucleotide resin isolation strength of solution is rarer and contain salt, and can not reach crystallization concentration, and routine can be used the decompression thin film concentration, but can not desalination, influences product purity, and using charcoal absorption to resolve can desalination again, but complex process, yield reduce.
Three, summary of the invention:
The present invention seeks to overcome above-mentioned not enough problem, a kind of new process of production of high purity 5 ' Nucleotide is provided, technology is simple to operation, and reactant is stable, product yield height, purity height.
The technical scheme that the present invention is adopted for achieving the above object is: a strain Penicillium citrinum bacterial strain M-71 is cultivated in submerged fermentation, produces the outer nuclease P of born of the same parents
1Rna solution degraded with 2.5～5%, degradation rate reaches more than 80%, use high frequency shale shaker to remove foreigh protein removing and remaining Yeast Nucleic Acid then, pure clear 5 ' Nucleotide solution after the removal of impurity is through ceramic membrane ultrafitration, remove the macromole residuals, adsorb and wash-out Nucleotide with four post series connection that strongly basic anion exchange resin is housed again, be the purifying mononucleotide, selecting molecular weight cut off is 300 daltonian nanofiltration membrane desalinations and concentrated, activated carbon decolorizing makes four kinds of highly purified 5 ' mononucleotide products after crystallization and the drying.
Described cultivation Penicillium citrinum bacterial strain M-71 under a suitable medium and submerged fermentation condition, in 30 hours, produces nuclease P
1Vigor 1200-1400 units per ml.
Described substratum ingredient and per-cent are: glucose 5%, peptone 0.5%, K
2HPO
40.05%, KH
2PO
40.05%, MgSO
40.04%, CaCl
20.04%, ZnSO
40.02%; PH6.0.
The described high frequency shale shaker that removes foreigh protein removing and the use of remaining Yeast Nucleic Acid, the vibration highest frequency is 3000 times/minute, using filter screen is 200-300 orders.
The ceramic membrane filter that described ultrafiltration is adopted is the ceramic membrane filter of 20-50nm.
Described sublimed substantially 5 ' Nucleotide solution is adsorbed on four columnss in series that strongly basic anion exchange resin is housed, and adopts the formate solution of different concns to carry out stepwise elution, is separated to the solution of four kinds of mononucleotides.
Four kinds of mononucleotide solution that described separation obtains respectively through the nanofiltration membrane desalination, concentrate and through the activated carbon decolorizing of routine, add an amount of methyl alcohol or water crystallization becomes highly purified 5 ' Nucleotide in separately iso-electric point.
The new process of production of described high purity 5 ' Nucleotide at first prepares nuclease P
1: the deep liquid ventilation is cultivated Penicillium citrinum bacterial strain M-71 and is got nuclease P
1, nanofiltration condensed nucleic acid enzyme P
1, with nuclease P
1Enzyme liquid through concentrated 6-16 times of 300-10000 dalton's nanofiltration membrane nanofiltrations is standby;
Adopt condensed nucleic acid enzyme P then
1Carry out the degraded of Yeast Nucleic Acid: RNA is made into 2.5-5.0% solution, adds concentrated enzyme 1.4-4% (V/V), be degraded into 5 ' Nucleotide mixed solution;
Degradation solution is crossed high frequency shale shaker: the degradation solution that temperature is 30～40 ℃, its maximum frequency of oscillation are 3000 times/minute, use filter screen 200-300 orders, remove a large amount of shape protein, remaining nucleic acid and other impurity admittedly by the degradation solution behind the vibratory screening apparatus;
The ultrafiltration of degradation solution: cross the ceramic membrane ultrafitration of the degradation solution of vibratory screening apparatus, remove soluble proteins through 20-50nm, purifying 5 ' Nucleotide solution, the control import is pressed and is 4kg/cm during ultrafiltration
2, use pure washing film after the ultrafiltration, get ultrafiltrated;
Absorption on strongly basic anion exchange resin, separation, four kinds of 5 ' Nucleotide of wash-out: four post series connection of polymeric adsorbent, degradation solution after the ultrafiltration is squeezed into columns in series, flow velocity is 0.6-1 column volume per hour, is washed till ultraviolet determination OD with pure water again
260＜20, washing is complete uses the formic acid solution stepwise elution, collects OD respectively
26020 elutriant, collect liquid and be respectively 5 ' CMP, 5 ' AMP, 5 ' UMP, 5 ' GMP, 12～15 ℃ of absorb-elute process control temps; Wash-out adopts formic acid-sodium formate solution (formic acid concn 0.04-0.08M, the phosphoric acid salt on sodium formate concentrations 0.03-0.05M) the flush away post more than 60% midway;
Collection liquid desalination behind the wash-out, concentrated: 5 ' CMP collects the nanofiltration employing NF of liquid and the film of two kinds of models of DK, 300 dalton, and it is 1～1.2kg/cm that the control import is pressed
2, must concentrate 7-17 times nanofiltration liquid.
5 ' AMP collects the same CMP of nanofiltration that liquid, 5 ' UMP collection liquid and 5 ' GMP collect liquid.
Nanofiltration liquid rises membrane concentration: vapor pressure 0.03-0.05MP is advanced in control, and 50-70 ℃ of temperature outs concentrate 4.4-9 times with nanofiltration liquid, make concentrated solution concentration reach 10-15%;
Single nucleic acid behind the liter membrane concentration adopts powdered carbon to decolour respectively, and with known isoelectric point crystallizing method, methanol usage is 0-2 times of concentrated solution volume, time 2-10hr, centrifugal after the crystallization, methanol wash, product is put 45-50 ℃ of HWZ-20B type microwave dryers, oven dry.
Novel process of the present invention is compared with the like product production technique has significant technical characterstic: independent research produces nuclease P
1The cultivation of bacterial strain, the unique and acquisition high reactivity nucleic acid enzyme P of method
1, this high reactivity nucleic acid enzyme P
1Degraded Yeast Nucleic Acid produces 5 ' Nucleotide solution, and its degradation rate is usually more than 80%.High frequency shale shaker is adopted in the removal of impurity, and degradation solution is removed a large amount of shape protein, remaining nucleic acid and other impurity admittedly by vibratory screening apparatus, and penetrating flow velocity is fast, and is easy for operation.The separation of Nucleotide adopts four post series systems that strongly basic anion exchange resin is housed to adsorb and the wash-out mononucleotide, owing to be conventional four times blade diameter length ratio, single-column is improved the Nucleotide upper column quantity, selected suitable eluent, separated four kinds of Nucleotide admirably.
At producing a class P contained compound in the Yeast Nucleic Acid degradation process, it can be adsorbed onto on the resin simultaneously, wash with product during wash-out, influence the problem of product purity, the present invention has designed the method for wash-out inorganic phosphorus in the resin elution process, promptly use formic acid-sodium formiate (formic acid concn 0.04-0.08M, sodium formate concentrations 0.03-0.05M) mix reagent can be removed phosphoric acid salt more than 60%, phosphoric acid salt can not be separated out again when the product crystallization, assurance makes highly purified 5 ' oligonucleotide product, can reach 98-102% through HPLC testing product purity.
Four, embodiment:
Embodiment 1
New process of production production high purity 5 ' Nucleotide by following 5 ' Nucleotide, comprise that condensed nucleic acid enzyme P1 degraded Yeast Nucleic Acid (RNA) becomes 5 ' Nucleotide solution, through vibratory screening apparatus, ultrafiltration, resin absorption, stepwise elution, collection, nanofiltration, concentrate, pure Nucleotide 5 ' CMP, 5 ' AMP, 5 ' UMP, the 5 ' GMP of decolouring, crystallization, dry four kinds of content 〉=98%.
At first prepare nuclease P
1: deep liquid ventilate to be cultivated Penicillium citrinum bacterial strain M-71, cultivated through 30 hours nuclease P
1, enzyme activity 1200 units per ml.Nanofiltration condensed nucleic acid enzyme P
1, with nuclease P
1Be condensed into greater than the enzyme liquid of 7500 units per ml standby through 300 dalton's nanofiltration membrane;
Adopt condensed nucleic acid enzyme P then
1Carry out the degraded of Yeast Nucleic Acid: get pure 17.5kg RNA and be made into 2.5% solution, add concentrated enzyme 3.2% (V/V), be degraded into 5 ' Nucleotide mixed solution.
Degradation solution is crossed high frequency shale shaker: the degradation solution that temperature is 30～40 ℃, speed with 1000～1200L/hr is crossed high frequency shale shaker, its maximum frequency of oscillation is 3000 times/minute, uses filter screen 300 orders, removes a large amount of shape protein, remaining nucleic acid and other impurity admittedly by the degradation solution behind the vibratory screening apparatus.
The ultrafiltration of degradation solution: the degradation solution of crossing vibratory screening apparatus is 8M through the 50nm membrane area
2Ceramic membrane ultrafitration, remove soluble proteins, purifying 5 ' Nucleotide solution, the control import is pressed and to be 4kg/cm during ultrafiltration
2, use the pure washing film of 400L, average flux 137.5L/hrcm after the ultrafiltration
2, altogether ultrafiltrated 1100L, 60 minutes times spent, ultrafiltration loss 0.56%.
Absorption on strongly basic anion exchange resin, separation, four kinds of 5 ' Nucleotide of wash-out:
Polymeric adsorbent: four post series connection, adorn resin 190L altogether, degradation solution after the ultrafiltration is squeezed into columns in series, flow velocity 120L/hr, upper prop rate 96.7%.
Washing: be washed till ultraviolet determination OD with 380～770L pure water
260＜20 finish, washing loss 4.2%.
Wash-out: washing is complete uses the formic acid solution stepwise elution, collects OD respectively
26020 elutriant, to collect liquid and be respectively 5 ' CMP 1680L, 5 ' AMP 2100L, 5 ' UMP 840L, 5 ' GMP1530L, 12～15 ℃ of absorb-elute process control temps are collected liquid total losses 13.52%.
Owing in the RNA degradation process, can produce a class P contained compound, be adsorbed on the resin simultaneously, wash with product again, influenced product purity, therefore adopt formic acid-sodium formate solution (formic acid concn 0.04-0.08M during wash-out, phosphoric acid salt on sodium formate concentrations 0.03-0.05M) the flush away post more than 60%, product phosphoric acid salt when crystallization can not separated out again like this, and four kinds of product purities are all 〉=98%.
The nanofiltration of elutriant: make and collect the liquid desalination, concentrate.
5 ' CMP collects the nanofiltration of liquid and adopts NF film, 300 dalton, membrane area 30M
2, the nanofiltration of 1680LCMP solution is moisturizing 400L midway, average flux 21.73L/min, and 110 minutes consuming time, it was 1～1.2kg/cm that the control import is pressed
2, get nanofiltration liquid 120L, lose 2.74% in the penetrating fluid, film internal loss 6.04%, total losses 8.78%.
2,5 ' AMP collects the nanofiltration of liquid:
The same CMP of nanofiltration equipment and control pressure.
2100L 5 ' AMP collects liquid, and nanofiltration is moisturizing 500L midway, average flux 27.47L/min, and 95 minutes consuming time, lose 2.26% in the penetrating fluid, film internal loss 8.6%, total losses 10.86% gets nanofiltration liquid 125L.
3,5 ' UMP collects the nanofiltration of liquid
The same CMP of nanofiltration equipment and control pressure.
840L 5 ' UMP collects liquid, and nanofiltration is moisturizing 500L midway, average flux 17.33L/min, and 75 minutes consuming time, lose 2.12% in the penetrating fluid, film internal loss 15.94%, total losses 18.06% gets nanofiltration liquid 120L.
4,5 ' GMP collects the nanofiltration of liquid
The same CMP of nanofiltration equipment and control pressure.
1530L 5 ' GMP collects liquid, and nanofiltration is moisturizing 630L midway, average flux 34L/min, and 65 minutes consuming time, penetrating fluid loss 1.77%, film internal loss 1.07%, total losses 2.84% gets nanofiltration liquid 125L.
Rise membrane concentration
Vapor pressure 0.03-0.05MP is advanced in control, and 50-70 ℃ of temperature outs, concentrated solution concentration reach 10-15%, 5 ' CMP and be concentrated into 13.5L from 120L, and 5 ' AMP is concentrated into 16.72L from 125L, and 5 ' UMP is concentrated into 27L from 120L, and 5 ' GMP is concentrated into 20L from 125L.
Single nucleic acid behind the liter film adopts powdered carbon to decolour respectively, with known isoelectric point crystallizing method.Methanol usage is 0-2 times of concentrated solution volume, time 2-10hr.Centrifugal after the crystallization, methanol wash.Product is put 45-50 ℃ of HWZ-20B type microwave dryers, oven dry.
Products obtained therefrom is analyzed and the yield measuring and calculating, and product is analyzed through HPLC, the results are shown in following table:
Claims (9)
1, the new process of production of high purity 5 ' Nucleotide is characterized in that: a strain Penicillium citrinum bacterial strain M-71 is cultivated in submerged fermentation, produces the outer nuclease P of born of the same parents
1Rna solution degraded with 2.5～5%, degradation rate reaches more than 80%, use high frequency shale shaker to remove foreigh protein removing and remaining Yeast Nucleic Acid then, pure clear 5 ' Nucleotide solution after the removal of impurity is through ceramic membrane ultrafitration, remove the macromole residuals, again with four posts that strongly basic anion exchange resin is housed connect disposable absorption and stepwise elution, collect, four kinds of mononucleotide solution collecting, selecting molecular weight cut off is 300 daltonian nanofiltration membrane desalinations and concentrated, activated carbon decolorizing makes four kinds of highly purified 5 ' mononucleotide products after crystallization and the drying.
2, the new process of production of high purity 5 ' Nucleotide according to claim 1 is characterized in that: cultivate Penicillium citrinum bacterial strain M-71, under substratum and submerged fermentation condition, in 30 hours, produce nuclease P
1Vigor 1200-1400 units per ml.
3, the new process of production of high purity 5 ' Nucleotide according to claim 2 is characterized in that: substratum ingredient and per-cent are: glucose 5%, peptone 0.5%, K
2HPO
40.05%, KH
2PO
40.05%, MgSO
40.04%, CaCl
20.04%, ZnSO
40.02%; PH6.0.
4, the new process of production of high purity 5 ' Nucleotide according to claim 1 is characterized in that: remove the high frequency shale shaker that foreigh protein removing and remaining Yeast Nucleic Acid use, maximum frequency of oscillation is 3000 times/minute, and using filter screen is 200-300 orders.
5, the new process of production of high purity 5 ' Nucleotide according to claim 1 is characterized in that: the ceramic membrane filter that ultrafiltration is adopted is the ceramic membrane filter of 20-50nm.
6, the new process of production of high purity 5 ' Nucleotide according to claim 1, it is characterized in that: sublimed substantially 5 ' Nucleotide solution is adsorbed on four columnss in series that strongly basic anion exchange resin is housed, and adopt the formate solution of different concns to carry out stepwise elution, be separated to the solution of four kinds of mononucleotides.
7, the new process of production of high purity 5 ' Nucleotide according to claim 1, it is characterized in that: separate four kinds of mononucleotide solution obtaining respectively through the nanofiltration membrane desalination, concentrate and through the activated carbon decolorizing of routine, add an amount of methyl alcohol or water crystallization becomes highly purified 5 ' Nucleotide in separately iso-electric point.
8, according to the new process of production of the arbitrary described high purity 5 ' Nucleotide of claim 1-7, it is characterized in that: at first prepare nuclease P
1: the deep liquid ventilation is cultivated Penicillium citrinum bacterial strain M-71 and is got nuclease P
1, nanofiltration condensed nucleic acid enzyme P
1, with nuclease P
1The enzyme liquid that is concentrated into 6-16 times through 300-10000 dalton's nanofiltration membrane is standby;
Adopt condensed nucleic acid enzyme P then
1Carry out the degraded of Yeast Nucleic Acid: RNA is made into 2.5-5% solution, adds and concentrate enzyme 1.4-4% (V/V), be degraded into 5 ' Nucleotide mixed solution;
Degradation solution is crossed high frequency shale shaker: the degradation solution that temperature is 30～40 ℃, its maximum frequency of oscillation are 3000 times/minute, use filter screen 200-300 orders, remove a large amount of shape protein, remaining nucleic acid and other impurity admittedly by the degradation solution behind the vibratory screening apparatus;
The ultrafiltration of degradation solution: cross the ceramic membrane ultrafitration of the degradation solution of vibratory screening apparatus, remove soluble proteins through 20-50nm, purifying 5 ' Nucleotide solution, the control import is pressed and is 4kg/cm during ultrafiltration
2, use pure washing film after the ultrafiltration, get ultrafiltrated;
Absorption on strongly basic anion exchange resin, separation, four kinds of 5 ' Nucleotide of wash-out: four post series connection of polymeric adsorbent, degradation solution after the ultrafiltration is squeezed into columns in series, the column volume that flow velocity is 0.6-1 times is washed till ultraviolet determination OD with pure water
260＜20 finish, and washing is complete uses the formic acid solution stepwise elution, collects OD respectively
26020 elutriant, and collect liquid and be respectively 5 ' CMP, 5 ' AMP, 5 ' UMP, 5 ' GMP, 12～15 ℃ of absorb-elute process control temps; Adopt the phosphoric acid salt more than 60% on formic acid-sodium formate solution flush away post during wash-out;
Collection liquid desalination behind the wash-out, concentrated: 5 ' CMP collects the nanofiltration of liquid and adopts the NF film, and molecular weight cut off is 300 dalton, and it is 1～1.2kg/cm that the control import is pressed
2, get nanofiltration liquid.
5 ' AMP collects the same CMP of nanofiltration that liquid, 5 ' UMP collection liquid and 5 ' GMP collect liquid;
Nanofiltration liquid rises membrane concentration: vapor pressure 0.03-0.05MP is advanced in control, 50-70 ℃ of temperature outs, and concentrated solution concentration reaches 10-15%.
Single nucleic acid after concentrating decolours with powdered carbon respectively, and with known isoelectric point crystallizing method, methanol usage is 0-2 times of concentrated solution volume, and time 2-10hr is centrifugal after the crystallization, methanol wash, and product is put 45-50 ℃ of HWZ-20B type microwave dryers, oven dry.
9, the new process of production of high purity 5 ' Nucleotide according to claim 8 is characterized in that: adopting formic acid-sodium formate solution during wash-out is formic acid concn 0.04-0.08M, the solution of sodium formate concentrations 0.03-0.05M.
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