CN103331035A - Method for decolorizing nucleotide enzymatic hydrolysate - Google Patents
Method for decolorizing nucleotide enzymatic hydrolysate Download PDFInfo
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- CN103331035A CN103331035A CN2013102694366A CN201310269436A CN103331035A CN 103331035 A CN103331035 A CN 103331035A CN 2013102694366 A CN2013102694366 A CN 2013102694366A CN 201310269436 A CN201310269436 A CN 201310269436A CN 103331035 A CN103331035 A CN 103331035A
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- enzymolysis liquid
- decoloured
- enzymolysis
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- 239000002773 nucleotide Substances 0.000 title claims abstract description 47
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000002255 enzymatic effect Effects 0.000 title abstract description 6
- 239000000413 hydrolysate Substances 0.000 title abstract 4
- 229920005989 resin Polymers 0.000 claims abstract description 65
- 239000011347 resin Substances 0.000 claims abstract description 65
- 239000000049 pigment Substances 0.000 claims abstract description 16
- 230000001172 regenerating effect Effects 0.000 claims abstract description 13
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- 239000011148 porous material Substances 0.000 claims abstract description 5
- 238000001179 sorption measurement Methods 0.000 claims abstract description 4
- 239000004793 Polystyrene Substances 0.000 claims abstract description 3
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 40
- 238000004132 cross linking Methods 0.000 claims description 20
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- 238000010521 absorption reaction Methods 0.000 claims description 9
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Abstract
The invention relates to a method for decolorizing nucleotide enzymatic hydrolysate. The method comprises the following steps of: performing pigment adsorption on the nucleotide enzymatic hydrolysate through hyper-crosslinked resin SX-01; and after adsorption is saturated, and regenerating the hyper-crosslinked resin SX-01 by using a regenerant, wherein the hyper-crosslinked resin SX-01 has the following structure units: the skeleton of the resin is polystyrene, the average grain size is 0.3 to 1 mm, the water content is 32 to 49 percent, the aperture is 1.5 to 4 nm, the most probable aperture is 1.8 nm, the average specific surface area is 950 to 1,800 m<2>/g, and the average pore volume is 0.54 to 0.82 cm<3>/g. By adoption of the method, the decolorization ratio of the enzymatic hydrolysate reaches over 80 percent, the treatment quantity reaches 11 times of bed volume (BV), the loss ratio of four kinds of nucleotides is lower than 5 percent, and the consumption quantity of the regenerant is 5 BV.
Description
Technical field
The invention belongs to the separation and purification field of 5 '-nucleotides, be specifically related to the application of superhigh cross-linking resin SX-01 in the decolouring of nucleotides enzymolysis liquid.
Background technology
Nucleosides, nucleotides are the bases that constitutes biological heredity informational DNA and RNA, play an important role in the metabolism of biological cell and power conversion.5 '-mononucleotide and derivative thereof have very important purposes at genetic engineering, medicine, food, agricultural production and scientific research field.In health products, infant food, add nucleotides and be conducive to the additional nucleotides of human body, make nucleotide metabolism be in vigorous state, can reach the purpose of opposing aging, prolonged human body life time, raising immune function of human body and treatment disease; Agriculturally, nucleotides utilizes potentiality very big as plant growth stimulator, experiment showed, that nucleotides can improve the quality of the conversion capability of seed inclusion, emergence rate and seedling, promotes the production of root system of plant, and can deepen the leaf look, improves chlorophyll content; Aspect cosmetics, because nucleotides has the effect that promotes that protein is synthetic, the nucleotides goods are also very extensive in the application of cosmetic industry, can make an addition in washing agent, emulsifying agent, toilet cream, emulsion, the drama cosmetics, can promote skin metabolism, have wrinkle resistant, myogenic is preserved moisture, control sebum secretion, stop ultraviolet radiation absorption, make the effect of skin softness, can both bring into play extremely strong penetration to various skin diseases such as freckle, buckwheat skin, whelk athlete's foots, result for the treatment of is remarkable.
The production method of nucleotides mainly contains 4 kinds: chemical synthesis, RNA enzymatic isolation method, microbe fermentation method and biological catalysis, wherein enzymatic isolation method is that domestic and international nucleotides is produced used major technique.But in the RNA enzymolysis liquid, contain a large amount of pigments, if directly use resin isolation without preliminary treatment, although resin can filter out a part of impurity, pigment residues in the eluent of nucleotides but have greatly, back separation and Crystallization Procedure to nucleotides are all brought very big pressure, influence purity and the colourity of oligonucleotide product, and be adsorbed on pigment on the resin and can cause resin poisoning in various degree, thereby reduce the recycling rate of waterused of resin.Therefore be necessary to remove the pigment in the enzymolysis liquid, thereby alleviate the pressure of downstream separation.
More to nucleotides enzymolysis liquid Study on Decolorization at present, wherein to disclose application number be 200510094493.0 patent " technology of producing nucleotide by enzyme method " to Zhang Jianqiu, uses acid decolorizing resin that nucleotides enzymolysis liquid is decoloured; It is 200810228964.6 patent " production new technique of high-purity 5 '-nucleotides " that Dalian Zhen Ao Bioisystech Co., Ltd discloses application number, uses the nucleotides behind the activated carbon powder foot couple enzymolysis to decolour; Use milipore filter ultrafiltration enzymolysis liquid in the paper of Xiao Linping " research of 5 '-uridylic acid separation purifying technique " and then use activated carbon powder foot couple enzymolysis liquid to decolour.But, to consume a large amount of acid in the process of acid decolorizing resin regeneration, active carbon reclaims difficult, and acid decolorizing resin and active carbon have a spot of absorption to nucleotides, can lose a part of nucleotides, and superhigh cross-linking degree resin SX-01 used in the present invention is less to four kinds of nucleotides absorption, and decolorizing effect is better, percent of decolourization is higher, and regeneration technology is simple, and the regenerative agent consumption is less.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that nucleotides enzymolysis liquid is decoloured.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method that nucleotides enzymolysis liquid is decoloured is carried out pigment absorption with nucleotides enzymolysis liquid by superhigh cross-linking resin SX-01, adsorb saturated after, superhigh cross-linking resin SX-01 utilizes regenerative agent to regenerate;
Wherein, described superhigh cross-linking resin SX-01 has following construction unit:
The skeleton of this resin is polystyrene, and average grain diameter is 0.3～1mm, and water content is 32%～49%, and the aperture is 1.5～4nm, and the most probable aperture is 1.8nm, and average specific surface area is 950～1800m
2/ g, average pore volume is 0.54～0.82cm
3/ g.This construction unit is that the infrared analysis spectrum analysis by Fig. 1 draws: 3100-3200cm
-1The place is the stretching vibration absworption peak of N-H, 2900-3000cm
-1The place is the stretching vibration absworption peak of C-H; 1500-1670cm
-1The place is the skeletal vibration peak of phenyl ring; 1395cm
-1The place is the stretching vibration absworption peak of C-N.
The preparation method of described superhigh cross-linking resin SX-01 is: with chloromethylated polystyrene resin abundant swelling in the o-nitroethylbenzene of 3～8 times of weight, stir the amination reagent that adds 2～5 times of chlorine ball weights after the swelling, the ferric trichloride that adds 10～30% chlorine ball weights is again made the Fu Shi catalyst, 100～150 ℃ carry out Friedel-Crafts after cross-linking reaction, when being 1～3%, resin residual chlorine content stops reaction, reaction finishes the back cooling and leaches the resin spheroid, with ethanol, distilled water washing, make superhigh cross-linking resin SX-01 after the vacuum drying.
Wherein, described nucleotides enzymolysis liquid is to obtain with nuclease p1 enzymolysis RNA powder.Preparation process is as follows:
(1) the 6%RNA solution 600ml of preparation pH=5.5: the NaOH solution with 1mol/l transfers to 600ml deionized water pH about 13 earlier, adding RNA powder (36g) then dissolves, NaOH with 1mol/l transfers pH to 5.5 again, places 70 ℃ of water-baths to be preheated to 70 ℃.
(2) in RNA solution, add the halfhour enzyme liquid of preheating 50mL under 70 ℃ of conditions, shake up enzymolysis 3h50min under 70 ℃ of conditions.
(3) add 0.2%(1.2g again) active carbon, continue enzymolysis 50min.
(4) be cooled to 45 ℃, centrifugal 10min under the 4000rpm condition, suction filtration namely obtains the RNA enzymolysis liquid.
Wherein, the pigment suction type is ADSORPTION IN A FIXED BED, and the resin column ratio of height to diameter is 8～12, and the absorption flow velocity is 1～3BV/h, and column temperature is controlled to be 30～50 ℃.
Wherein, described regenerative agent is 1mol/L NaOH and absolute ethyl alcohol.
Wherein, regenerative process is for regenerating with 3BV1mol/L NaOH earlier, regenerates until flowing out liquid ethanol in the usefulness deionized water flush away resin when colourless with absolute ethyl alcohol then, and the flow velocity of NaOH and ethanol is 1BV/h.
Use among the present invention spectrophotometer the 420nm place to enzymolysis liquid in pigment detect the OD of enzymolysis liquid
420Proportional with the concentration of pigment in the enzymolysis liquid, the percent of decolourization of enzymolysis liquid calculates with following formula:
Percent of decolourization (%)=(A
Before-A
After)/A
Before* 100%
A wherein
Before, A
AfterBe respectively the forward and backward enzymolysis liquid of decolouring in the absorbance at 420nm place.
Use HPLC to adopt external standard method that nucleotide concentration in the enzymolysis liquid is quantitatively detected among the present invention, chromatographic condition is:
1. chromatographic column: the Lichrospher C18(250mm of Chinese nation * 4.6mm i.d., 5 μ m);
2. phase: A flows: ultra-pure water; B: methyl alcohol; C:(NH
4) H
2PO
4And methanol in water (20mmol/L (NH
4) H
2PO
4+ 35ml/L ethanol).Gradient elution program: 0～2min, C phase volume fraction (down together) 100%; 2～8min, B increases to 11%, C by 0% linearity and is reduced to 89% by 100% linearity; 9～10min, B is reduced to 0%, C by 11% linearity and increases to 100% mutually; 10～15min, C are 100% mutually;
3. flow velocity: 1.0ml/min;
4. detection wavelength: 254nm;
5. column temperature: room temperature;
6. sampling volume: 20 μ L.
Detection method and step:
1. the balance of pillar: the water for preparing and buffer solution carry out ultrasonic processing 30min with the mixing filtering with microporous membrane of aperture 0.22 μ m, methyl alcohol with the nylon filtering with microporous membrane of aperture 0.45 μ m after the filtration.Flowing after handling placed on the high performance liquid chromatography mutually, open high performance liquid chromatography, the methyl alcohol with 100%, flow velocity are 1ml/min, the about 20min of flushing pillar; After treating pressure stability, change methyl alcohol 3% into, water 97%, flow velocity are 1ml/min, carry out balance, and about 20min after the pressure stability, changes methyl alcohol 3% into again, and buffer solution 97% carries out balance, turns on light simultaneously, begins to gather baseline, and when treating that baseline is tending towards straight line, balance finishes.
2. the detection of sample: write drip washing program and sample introduction sequence according to chromatographic condition, standard items and the sample of crossing after film is handled is placed on according to the sample introduction sequence on the relevant position of automatic sampler, the beginning sample introduction is also collected the figure spectrum information.
Use following formula to calculate the loss late of nucleotides in the enzymolysis liquid of decolouring back:
Loss late (%)=(C
Before-C
After)/C
Before* 100%
C wherein
Before, C
AfterBe respectively in the enzymolysis liquid four kinds of nucleotides before decolouring and the concentration after the decolouring.
BV/h described in the present invention is for per hour flowing through the amount of solution of resin column bed volume (Bed Volume), and BV is the bed volume in the resin column.
Beneficial effect of the present invention:
1. superhigh cross-linking degree resin SX-01 is applied to the decolouring of nucleotides enzymolysis liquid, this resin price is cheap, big to the pigment adsorbance in the enzymolysis liquid, percent of decolourization higher (reaching more than 80%), the enzymolysis liquid treating capacity is big (reaching 11BV), can improve the appearance luster of enzymolysis liquid greatly, can not introduce new impurity in the decolorization, and the loss late of four kinds of mononucleotides lower (being lower than 2%), reduce greatly behind the nucleotides separate, the difficulty of crystallization;
2. the regeneration of the resin SX-01 wash-out behind the adsorpting pigment is easier to, and it is more complete to regenerate, regenerative agent consumption less (the regenerative agent consumption is 5BV), and the resin life cycle is longer.
3. the employed superhigh cross-linking resin SX-01 among the present invention not only can be used for the decolorization of nucleotides enzymolysis liquid, can also be applied to the decolorization of various organic acid fermentation liquid, amino acid zymotic fluid etc.
Description of drawings
Fig. 1 is the infrared spectrogram of superhigh cross-linking resin SX-01 of the present invention;
Fig. 2 is the sem photograph (SEM figure) of superhigh cross-linking resin SX-01 of the present invention;
Fig. 3 is the BET figure of superhigh cross-linking resin SX-01 of the present invention;
Fig. 4 is the graph of pore diameter distribution of superhigh cross-linking resin SX-01 of the present invention;
Fig. 5 is the consumption of different eluant, eluents when resin column is regenerated in the invention process 5.
The specific embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the preparation of nucleotides enzymolysis liquid.
The enzymolysis process of RNA adopts nuclease p1 to act on RNA solution and obtains 4 kinds of 5 '-mononucleotides, is 5 '-adenylate (AMP), 5 '-cytidine monophosphate (CMP), 5 '-guanylic acid (GMP) and 5 '-uridylic acid (UMP).Concrete steps are as follows:
(1) the 6%RNA solution 600ml of preparation pH=5.5: the NaOH solution with 1mol/L transfers to pH about 13 earlier, adds RNA powder (36g) then and dissolves, and transfers pH to 5.5 with the NaOH solution of 1mol/L again, places 70 ℃ of water-baths to be preheated to 70 ℃.
(2) in RNA solution, add the 70 ℃ of halfhour enzyme liquid of preheating 50ml, shake up enzymolysis 3h50min under 70 ℃ of conditions.
(3) add 0.2%(1.2g again) active carbon, continue enzymolysis 50min.
(4) be cooled to 45 ℃, centrifugal 10min under the 4000rpm condition, suction filtration namely obtains the RNA enzymolysis liquid, and the pH of enzymolysis liquid reduces to 4.5 behind the enzymolysis.
Embodiment 2: the acquisition of superhigh cross-linking resin SX-01.
In cleaning, in the dry 2000mL there-necked flask, the chloromethylated polystyrene resin (chlorine ball) that adds the 200g drying, add the 1200g o-nitroethylbenzene, stir and evenly mix, under the room temperature fully more than the swelling 2h, stir the methylamine that adds 4 times of chlorine ball weights after the swelling, add catalyst anhydrous ferric trichloride 20g again, temperature programming to 135 under agitation～150 ℃, carry out cross-linking reaction certain hour behind the Friedel-Crafts again under this temperature, control residual chlorine content is 1～3%, stop reaction, cooling back sucking-off mother liquor, with industrial alcohol repeatedly the extracting resin to extract flow out liquid colourless limpid till, using 4% hydrochloric acid and distilled water washing resin to neutral successively, dry for standby.
Products therefrom is brown rounded grain shape, and average grain diameter is 0.3～1mm, and water content is 32%～49%, and the aperture is 1.5～4nm, and the most probable aperture is 1.8nm, and average specific surface area is 950～1800m
2/ g, average pore volume is 0.54～0.82cm
3/ g.Derive the chemical structural formula of resin by the infrared spectrogram of Fig. 1, calculate the resin specific area by Fig. 2 and Fig. 3, obtain aperture and the most probable aperture of resin by Fig. 4.
Embodiment 3: the preliminary treatment of superhigh cross-linking resin SX-01.
With the ethanol of 2BV with the flow velocity flushing resin of 2BV/h → wash away with the deionized water HCl solution of ethanol → 2BV0.1mol/L with the flow velocity flushing resin of 1BV/h → be washed to neutrality → with the NaOH solution of 2BV0.1mol/L with the flow velocity flushing resin of 1BV/h → be washed to neutrality → wash resin → be washed to neutrality with the flow velocity of 1BV/h with the HCl solution of 2BV0.1mol/L.
Embodiment 4: the decolouring of fixed-bed resin post.
The enzymolysis liquid resin column of flowing through is carried out pigment absorption, and the ratio of height to diameter of resin column is 10, and the pH of enzymolysis liquid is 4.5, and the enzymolysis liquid flow velocity is 1BV/h, and the resin column column temperature is controlled to be 30 ℃, 40 ℃, 50 ℃ respectively, the OD when the column outlet place
420OD near material liquid
420The time finish upper prop, calculate upper prop percent of decolourization, four kinds of mononucleotide loss lates and enzymolysis liquid treating capacity, percent of decolourization under each temperature conditions, nucleotides loss late and enzymolysis liquid treating capacity such as table 1, temperature is little to percent of decolourization, nucleotides loss late and the influence of enzymolysis liquid treating capacity, but in the time of 30 ℃, enzymolysis liquid agglutination, the phenomenon that pillar in the process of upper prop decolouring, can occur gambling, and consumed energy is many 50 ℃ the time, therefore selects 40 ℃ of best upper prop bleaching temperatures of conduct.
Table 1
Embodiment 5: the decolouring of fixed-bed resin post.
The enzymolysis liquid resin column of flowing through is carried out pigment absorption, and the ratio of height to diameter of resin column is 10, and the pH of enzymolysis liquid is 4.5, and the resin column column temperature is controlled to be 40 ℃, and the enzymolysis liquid flow velocity is respectively 1BV/h, 2BV/h, 3BV/h, the OD when the column outlet place
420OD near material liquid
420The time finish upper prop, calculate upper prop percent of decolourization, four kinds of mononucleotide loss lates and enzymolysis liquid treating capacity, treating capacity such as the table 2 of enzymolysis liquid under the different in flow rate condition.The nucleotides loss late is all very low under three kinds of flow velocitys as can be seen from the table, and when flow velocity was 1BV/h, percent of decolourization was higher and treating capacity is maximum, was respectively 84.8% and 11.6 times of bed volume.
Table 2
Embodiment 6: the fixed-bed resin column regeneration.
The resin column that regenerative agent is flowed through behind the adsorpting pigment carries out regeneration of resin, and regenerant flow rate is 1BV/h, and used regenerative agent kind is respectively following a kind of:
1 1mol/L NaOH solution;
The aqueous solution of 2 10wt%NaCl+0.7wt%NaOH;
The aqueous solution of 3 20wt%NaCl+0.7wt%NaOH;
The aqueous solution of 4 10wt%NaCl+0.5mol/LNaOH;
The aqueous solution of 5 10 (v/v) % ethanol+1mol/LNaOH;
The aqueous solution of 6 25 (v/v) % ethanol+1mol/LNaOH;
The aqueous solution of 7 50 (v/v) % ethanol+1mol/LNaOH;
8 carry out wash-out with 3BV1mol/LNaOH solution earlier, and then carry out wash-out with absolute ethyl alcohol.
Finish wash-out when colourless up to the column outlet drop, calculate the eluant, eluent consumption, the consumption of various eluant, eluents such as Fig. 5.The 8th kind of eluant, eluent wherein namely earlier carries out wash-out with 3BV1mol/LNaOH solution, and then it is lower to carry out wash-out eluant, eluent consumption with absolute ethyl alcohol, is approximately 5BV.
Claims (5)
1. the method that nucleotides enzymolysis liquid is decoloured is characterized in that, nucleotides enzymolysis liquid is carried out pigment absorption by superhigh cross-linking resin SX-01, adsorb saturated after, superhigh cross-linking resin SX-01 utilizes regenerative agent to carry out resin regeneration;
Wherein, described superhigh cross-linking resin SX-01 has following construction unit:
The skeleton of this resin is polystyrene, and average grain diameter is 0.3～1mm, and water content is 32%～49%, and the aperture is 1.5～4nm, and the most probable aperture is 1.8nm, and average specific surface area is 950～1800m
2/ g, average pore volume is 0.54～0.82cm
3/ g.
2. the method that nucleotides enzymolysis liquid is decoloured according to claim 1 is characterized in that, described nucleotides enzymolysis liquid is to obtain with nuclease p1 enzymolysis RNA powder.
3. the method that nucleotides enzymolysis liquid is decoloured according to claim 1 is characterized in that, the pigment suction type is ADSORPTION IN A FIXED BED, the resin column ratio of height to diameter is 8～12, the pH of enzymolysis liquid is 4.5, and the absorption flow velocity is 1～3BV/h, and column temperature is controlled to be 30～50 ℃.
4. the method that nucleotides enzymolysis liquid is decoloured according to claim 1 is characterized in that, described regenerative agent is 1mol/L NaOH and absolute ethyl alcohol.
5. according to claim 1 or the 4 described methods that nucleotides enzymolysis liquid is decoloured, it is characterized in that, regenerative process is for regenerating with 3BV1mol/L NaOH earlier, regenerating with absolute ethyl alcohol then approaches the ethanol in usefulness deionized water flush away resin when colourless until flowing out liquid, and the flow velocity of NaOH and ethanol is 1BV/h.
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| CN105964146A (en) * | 2016-06-29 | 2016-09-28 | 南京工业大学 | Method of separating nucleotide acid from enzymolytic liquid |
| CN106632519A (en) * | 2016-12-12 | 2017-05-10 | 南京工业大学 | Technology for separating nucleotide by adopting continuous ion exchange chromatography technique |
| CN107854864A (en) * | 2017-10-24 | 2018-03-30 | 陕西蓝深特种树脂有限公司 | A kind of phenylglycine produces mother liquor decolorizing process technique |
| CN107930183A (en) * | 2017-12-20 | 2018-04-20 | 浙江云涛生物技术股份有限公司 | A kind of phenylglycine highly basic mother liquor resin decoloration device and decoloration process |
| CN110330541A (en) * | 2019-05-22 | 2019-10-15 | 南阳师范学院 | A kind of separation method to 5 '-guanylic acids and 5 '-cytidylic acids |
| CN113438967A (en) * | 2019-05-13 | 2021-09-24 | 因都菲尔公司 | Method for removing degraded components from hydrocarbon fluids and porous media for carrying out the method |
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| CN106632519A (en) * | 2016-12-12 | 2017-05-10 | 南京工业大学 | Technology for separating nucleotide by adopting continuous ion exchange chromatography technique |
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| CN107854864A (en) * | 2017-10-24 | 2018-03-30 | 陕西蓝深特种树脂有限公司 | A kind of phenylglycine produces mother liquor decolorizing process technique |
| CN107930183A (en) * | 2017-12-20 | 2018-04-20 | 浙江云涛生物技术股份有限公司 | A kind of phenylglycine highly basic mother liquor resin decoloration device and decoloration process |
| CN113438967A (en) * | 2019-05-13 | 2021-09-24 | 因都菲尔公司 | Method for removing degraded components from hydrocarbon fluids and porous media for carrying out the method |
| CN110330541A (en) * | 2019-05-22 | 2019-10-15 | 南阳师范学院 | A kind of separation method to 5 '-guanylic acids and 5 '-cytidylic acids |
| CN110330541B (en) * | 2019-05-22 | 2023-03-21 | 南阳师范学院 | Method for separating 5 '-guanine nucleotide and 5' -cytosine nucleotide |
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