CN103331035B - Method for decolorizing nucleotide enzymatic hydrolysate - Google Patents
Method for decolorizing nucleotide enzymatic hydrolysate Download PDFInfo
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- CN103331035B CN103331035B CN201310269436.6A CN201310269436A CN103331035B CN 103331035 B CN103331035 B CN 103331035B CN 201310269436 A CN201310269436 A CN 201310269436A CN 103331035 B CN103331035 B CN 103331035B
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- enzymolysis solution
- enzymolysis
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- 239000002773 nucleotide Substances 0.000 title claims abstract description 46
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 29
- 230000002255 enzymatic effect Effects 0.000 title abstract 4
- 239000000413 hydrolysate Substances 0.000 title abstract 4
- 229920005989 resin Polymers 0.000 claims abstract description 49
- 239000011347 resin Substances 0.000 claims abstract description 49
- 239000000049 pigment Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000011148 porous material Substances 0.000 claims abstract description 9
- 238000001179 sorption measurement Methods 0.000 claims abstract description 6
- 239000004793 Polystyrene Substances 0.000 claims abstract description 3
- 229920002223 polystyrene Polymers 0.000 claims abstract description 3
- 230000001172 regenerating effect Effects 0.000 claims abstract description 3
- 229920006395 saturated elastomer Polymers 0.000 claims abstract description 3
- 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 27
- 239000002952 polymeric resin Substances 0.000 claims description 16
- 229920003002 synthetic resin Polymers 0.000 claims description 16
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- 238000010521 absorption reaction Methods 0.000 claims description 10
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- 101710163270 Nuclease Proteins 0.000 claims description 3
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- PXWYZLWEKCMTEZ-UHFFFAOYSA-N 1-ethyl-2-nitrobenzene Chemical compound CCC1=CC=CC=C1[N+]([O-])=O PXWYZLWEKCMTEZ-UHFFFAOYSA-N 0.000 description 2
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- 239000012535 impurity Substances 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
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Landscapes
- Saccharide Compounds (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 '-Nucleotide, be specifically related to the application of hypercrosslinked polymeric resin SX-01 in the decolouring of Nucleotide enzymolysis solution.
Background technology
Nucleosides, Nucleotide are the bases forming biological heredity informational DNA and RNA, play an important role in the substance metabolism and energy transformation of biomass cells.5 '-mononucleotide and derivative thereof have very important purposes at genetic engineering, medicine, food, agriculture production and scientific research field.In healthcare products, infant or baby food, add Nucleotide be conducive to human body and supplement Nucleotide, make nucleotide metabolism be in healthy state, the object of opposing aging, prolonged human body life time, raising immune function of human body and disease therapy can be reached; Agriculturally, Nucleotide is very large as plant growth stimulator Exploitative potential, and experiment proves, Nucleotide can improve the quality of the conversion capability of seed inclusion, seedling rate and seedling, promotes the production of root system of plant, and can deepen leaf look, improve chlorophyll content; In makeup, because Nucleotide has the effect promoting protein synthesis, Nucleotide goods are also very extensive in the application of cosmetic industry, can make an addition in washing composition, emulsifying agent, vanishing cream, emulsion, theatrical makeup, the effect that can promote the metabolism of skin, there is wrinkle resistant, myogenic moisturizing, control sebum secretion, stop ultraviolet radiation absorption, make skin emolliency, can play extremely strong seepage force to various tetter such as freckle, buckwheat skin, comedo tinea pediss, result for the treatment of is remarkable.
The production method of Nucleotide mainly contains 4 kinds: chemical synthesis, RNA enzymolysis process, microbe fermentation method and biological catalysis, and wherein enzymolysis process is that domestic and international Nucleotide produces major technique used.But containing a large amount of pigment in RNA enzymolysis solution, if directly use resin isolation without pre-treatment, although resin can filter out a part of impurity, but have a big chunk pigment residue in the elutriant of Nucleotide, very large pressure is all brought to the rear separation of Nucleotide and Crystallization Procedure, affect purity and the colourity of oligonucleotide product, and the pigment be adsorbed on resin can cause resin poisoning in various degree, thus reduce the repeating utilization factor of resin.Therefore be necessary to remove the pigment in enzymolysis solution, thus alleviate the pressure of downstream separation.
More to the research of Nucleotide enzymolysis solution decolouring at present, wherein Zhang Jianqiu discloses the patent " technique of producing nucleotide by enzyme method " that application number is 200510094493.0, uses acid decolorizing resin to decolour to Nucleotide enzymolysis solution; Dalian Zhen Ao Bioisystech Co., Ltd discloses the patent " new process of production of high-purity 5 '-Nucleotide " that application number is 200810228964.6, uses the Nucleotide after activity charcoal powder foot couple enzymolysis to decolour; Use ultra-filtration membrane ultrafiltration enzymolysis solution in the paper " research of 5'-UMP separation purifying technique " of Xiao Linping and then use activity charcoal powder foot couple enzymolysis solution to decolour.But, a large amount of acid to be consumed in the process of acid decolorizing resin regeneration, it is more difficult that gac reclaims, and acid decolorizing resin and gac have a small amount of absorption to Nucleotide, a part of Nucleotide can be lost, and superhigh cross-linking degree resin SX-01 used in the present invention is less to four kinds of Nucleotide absorption, and decolorizing effect is better, percent of decolourization is higher, and reclaiming process is simple, and regenerator consumption is less.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of decolouring to Nucleotide enzymolysis solution.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
To the method that Nucleotide enzymolysis solution decolours, Nucleotide enzymolysis solution is carried out pigment absorption by hypercrosslinked polymeric resin SX-01, adsorb saturated after, hypercrosslinked polymeric resin SX-01 utilizes regenerator to regenerate;
Wherein, described hypercrosslinked polymeric resin SX-01 has following structural unit:
The skeleton of this resin is polystyrene, and median size is 0.3 ~ 1mm, and water content is 32% ~ 49%, and aperture is 1.5 ~ 4nm, and most probable pore size is 1.8nm, and average specific surface area is 950 ~ 1800m
2/ g, average pore volume is 0.54 ~ 0.82cm
3/ g.This structural unit is drawn by the infrared analysis spectroscopic analysis of Fig. 1: 3100-3200cm
-1place is the stretching vibration absorption peak of N-H, 2900-3000cm
-1place is the stretching vibration absorption peak of C-H; 1500-1670cm
-1place is the skeletal vibration peak of phenyl ring; 1395cm
-1place is the stretching vibration absorption peak of C-N.
The preparation method of described hypercrosslinked polymeric resin SX-01 is: chloromethylated polystyrene resin is fully swelling in the o-nitroethylbenzene of 3 ~ 8 times of weight, swelling rear stirring adds the amination reagent of 2 ~ 5 times of chlorine ball weights, fourier catalyzer made by the iron trichloride adding 10 ~ 30% chlorine ball weights again, Friedel-Crafts post-crosslinking reaction is carried out at 100 ~ 150 DEG C, the stopped reaction when resin residue cl content is 1 ~ 3%, reaction terminates rear cooling and leaches resin spheroid, with ethanol, distilled water wash, obtained hypercrosslinked polymeric resin SX-01 after vacuum-drying.
Wherein, described Nucleotide enzymolysis solution obtains with nuclease p1 enzymolysis RNA powder.Preparation process is as follows:
(1) the 6%RNA solution 600ml of pH=5.5 is prepared: first by the NaOH solution of 1mol/l, 600ml deionized water pH is adjusted to about 13, then add RNA powder (36g) to dissolve, adjust pH to 5.5 with the NaOH of 1mol/l again, be placed in 70 DEG C of water-baths and be preheated to 70 DEG C.
(2) in RNA solution, add preheating halfhour enzyme liquid 50mL under 70 DEG C of conditions, shake up, enzymolysis 3h50min under 70 DEG C of conditions.
(3) 0.2%(1.2g is added again) gac, continues enzymolysis 50min.
(4) be cooled to 45 DEG C, centrifugal 10min under 4000rpm condition, suction filtration, namely obtains RNA enzymolysis solution.
Wherein, pigment suction type is ADSORPTION IN A FIXED BED, and resin column aspect ratio is 8 ~ 12, and adsorption flow rate is 1 ~ 3BV/h, and it is 30 ~ 50 DEG C that column temperature controls.
Wherein, described regenerator is 1mol/L NaOH and dehydrated alcohol.
Wherein, regenerative process is for first to regenerate with 3BV1mol/L NaOH, and then carry out regenerating with dehydrated alcohol until effluent liquid is close to the ethanol washed away with deionized water time colourless in resin, the flow velocity of NaOH and ethanol is 1BV/h.
Spectrophotometer pigment in 420nm place is to enzymolysis solution is used to detect in the present invention, the OD of enzymolysis solution
420proportional with the concentration of pigment in enzymolysis solution, the percent of decolourization of enzymolysis solution calculates with following formula:
Percent of decolourization (%)=(A
before-A
after)/A
before* 100%
Wherein A
before, A
afterbe respectively the absorbancy of the forward and backward enzymolysis solution of decolouring at 420nm place.
Use HPLC to adopt external standard method to carry out detection by quantitative to enzymolysis solution nucleotide concentration in the present invention, chromatographic condition is:
1. chromatographic column: Chinese nation Lichrospher C18(250mm × 4.6mm i.d., 5 μm);
2. moving phase: A: ultrapure water; B: methyl alcohol; C:(NH
4) H
2pO
4with the aqueous solution (20mmol/L (NH of methyl alcohol
4) H
2pO
4+ 35ml/L ethanol).Gradient elution program: 0 ~ 2min, C phase volume fraction (lower same) 100%; 2 ~ 8min, B phase is linearly increased to 11%, C phase by 0% and is linearly reduced to 89% by 100%; 9 ~ 10min, B phase is linearly reduced to 0%, C phase by 11% and is increased to 100%; 10 ~ 15min, C phase is 100%;
3. flow velocity: 1.0ml/min;
4. determined wavelength: 254nm;
5. column temperature: room temperature;
6. sampling volume: 20 μ L.
Detection method and step:
1. the balance of pillar: the mixing filtering with microporous membrane in the water prepared and 0.22 μm, damping fluid aperture, the methyl alcohol nylon micro porous filter membrane in 0.45 μm, aperture filters, and carries out supersound process 30min after filtration.Be placed on high performance liquid chromatography by the moving phase after process, open high performance liquid chromatography, with the methyl alcohol of 100%, flow velocity is 1ml/min, rinses pillar and is about 20min; After pressure stabilisation, change methyl alcohol 3% into, water 97%, flow velocity is 1ml/min, balances, about 20min, after pressure-stabilisation, then changes methyl alcohol 3% into, and damping fluid 97% balances, and turns on light simultaneously, starts to gather baseline, and when treating that baseline is tending towards straight line, balance terminates.
2. the standard substance after the process of mistake film and sample are placed on the corresponding position of automatic sampler according to sample introduction sequence, start sample introduction and also collect profile information by the detection of sample: write rinse procedures and sample introduction sequence according to chromatographic condition.
The rate of loss of enzymolysis solution nucleotide after using following formulae discovery to decolour:
Rate of loss (%)=(C
before-C
after)/C
before* 100%
Wherein C
before, C
afterbe respectively the concentration of four kinds of Nucleotide before decolouring and after decolouring in enzymolysis solution.
BV/h described in the present invention is the amount of solution flowing through resin column bed volume (Bed Volume) per hour, and BV is the bed volume in resin column.
Beneficial effect of the present invention:
1. superhigh cross-linking degree resin SX-01 is applied to the decolouring of Nucleotide enzymolysis solution, this resin price is cheap, large to the pigment adsorptive capacity in enzymolysis solution, percent of decolourization higher (reaching more than 80%), enzymolysis solution treatment capacity comparatively large (reaching 11BV), greatly can improve the appearance luster of enzymolysis solution, can not introduce new impurity in decolorization, and the rate of loss of four kinds of mononucleotides lower (lower than 2%), the difficulty of separation after reduction Nucleotide, crystallization greatly;
2. the resin SX-01 zeolite regeneration after adsorpting pigment is easier to, and regeneration is comparatively complete, and regenerator consumption less (regenerator consumption is 5BV), resin life cycle is longer.
3. the hypercrosslinked polymeric resin SX-01 used in the present invention not only may be used for the decolorization of Nucleotide enzymolysis solution, can also be applied to the decolorization of various organic acid fermentation liquid, amino acid zymotic fluid etc.
Accompanying drawing explanation
Fig. 1 is the infrared spectrogram of hypercrosslinked polymeric resin SX-01 of the present invention;
Fig. 2 is the scanning electron microscope (SEM) photograph (SEM figure) of hypercrosslinked polymeric resin SX-01 of the present invention;
Fig. 3 is the BET figure of hypercrosslinked polymeric resin SX-01 of the present invention;
Fig. 4 is the graph of pore diameter distribution of hypercrosslinked polymeric resin SX-01 of the present invention;
The consumption of Fig. 5 different eluent when being resin column regeneration in the invention process 5.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, concrete material proportion, processing condition and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1: the preparation of Nucleotide enzymolysis solution.
The enzymolysis process of RNA, adopt nuclease p1 to act on RNA solution and obtain 4 kinds of 5 '-mononucleotides, be 5'-AMP (AMP), 5'-CMP (CMP), 5'-GMP (GMP) and 5'-UMP (UMP).Concrete steps are as follows:
(1) the 6%RNA solution 600ml of pH=5.5 is prepared: first by the NaOH solution of 1mol/L, pH is adjusted to about 13, then add RNA powder (36g) to dissolve, adjust pH to 5.5 by the NaOH solution of 1mol/L again, be placed in 70 DEG C of water-baths and be preheated to 70 DEG C.
(2) in RNA solution, add 70 DEG C of preheating halfhour enzyme liquid 50ml, shake up, enzymolysis 3h50min under 70 DEG C of conditions.
(3) 0.2%(1.2g is added again) gac, continues enzymolysis 50min.
(4) be cooled to 45 DEG C, centrifugal 10min under 4000rpm condition, suction filtration, namely obtains RNA enzymolysis solution, and after enzymolysis, the pH of enzymolysis solution reduces to 4.5.
Embodiment 2: the acquisition of hypercrosslinked polymeric resin SX-01.
In cleaning, in dry 2000mL there-necked flask, add the chloromethylated polystyrene resin (chlorine ball) of 200g drying, add 1200g o-nitroethylbenzene, stirring and evenly mixing, abundant swelling more than 2h under room temperature, swelling rear stirring adds the methylamine of 4 times of chlorine ball weights, add catalyzer FERRIC CHLORIDE ANHYDROUS 20g again, under agitation temperature programming to 135 ~ 150 DEG C, Friedel-Crafts post-crosslinking reaction certain hour is carried out again at this temperature, control residual chlorine content 1 ~ 3%, stopped reaction, sucking-off mother liquor after cooling, with industrial spirit repeatedly extracting resin to extraction effluent liquid colorless clear, using 4% hydrochloric acid and distilled water washing resin to neutral successively, dry for standby.
Products therefrom is brown rounded grain shape, and median size is 0.3 ~ 1mm, and water content is 32% ~ 49%, and aperture is 1.5 ~ 4nm, and most probable pore size is 1.8nm, and average specific surface area is 950 ~ 1800m
2/ g, average pore volume is 0.54 ~ 0.82cm
3/ g.Derived the chemical structural formula of resin by the infrared spectrogram of Fig. 1, calculate resin specific surface area by Fig. 2 and Fig. 3, obtained aperture and the most probable pore size of resin by Fig. 4.
Embodiment 3: the pre-treatment of hypercrosslinked polymeric resin SX-01.
The HCl solution rinsing resin → wash away with deionized water ethanol → 2BV0.1mol/L with the ethanol of 2BV with the flow velocity of 2BV/h rinses resin → be washed to neutrality → rinse resin → be washed to neutrality → with the HCl solution of 2BV0.1mol/L with the flow velocity of 1BV/h flushing resin → be washed to neutrality with the flow velocity of 1BV/h by the NaOH solution of 2BV0.1mol/L with the flow velocity of 1BV/h.
Embodiment 4: fixed-bed resin post decolours.
Enzymolysis solution is flowed through resin column and carry out pigment absorption, the aspect ratio of resin column is 10, and the pH of enzymolysis solution is 4.5, and enzymolysis solution flow velocity is 1BV/h, and resin column column temperature is controlled as 30 DEG C, 40 DEG C, 50 DEG C, as the OD at column outlet place
420close to the OD of stock liquid
420time terminate upper prop, calculate upper prop percent of decolourization, four kinds of mononucleotide rate of loss and enzymolysis solution treatment capacity, percent of decolourization under each temperature condition, Nucleotide rate of loss and enzymolysis solution treatment capacity are as table 1, temperature is little on the impact of percent of decolourization, Nucleotide rate of loss and enzymolysis solution treatment capacity, but when 30 DEG C, enzymolysis solution agglutination, there will be the phenomenon of gambling pillar in the process of upper prop decolouring, and consumed energy is many when 50 DEG C, therefore select 40 DEG C as best upper prop bleaching temperature.
Table 1
Embodiment 5: fixed-bed resin post decolours.
Enzymolysis solution is flowed through resin column and carry out pigment absorption, the aspect ratio of resin column is 10, and the pH of enzymolysis solution is 4.5, and it is 40 DEG C that resin column column temperature controls, and enzymolysis solution flow velocity is respectively 1BV/h, 2BV/h, 3BV/h, as the OD at column outlet place
420close to the OD of stock liquid
420time terminate upper prop, calculate upper prop percent of decolourization, four kinds of mononucleotide rate of loss and enzymolysis solution treatment capacity, under different in flow rate condition, the treatment capacity of enzymolysis solution is as table 2.Under three kinds of flow velocitys, Nucleotide rate of loss is all very low as can be seen from the table, and when flow velocity is 1BV/h, percent of decolourization is higher and treatment capacity is maximum, is respectively 84.8% and 11.6 times of bed volume.
Table 2
Embodiment 6: fixed-bed resin column regeneration.
Regenerator is flowed through the regeneration that the resin column after adsorpting pigment carries out resin, regenerant flow rate is 1BV/h, and regenerator kind used is respectively following one:
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 first carry out wash-out with 3BV1mol/LNaOH solution, and then carry out wash-out with dehydrated alcohol.
Until column outlet drop terminates wash-out close to time colourless, calculate eluent consumption, the consumption of various eluent is as Fig. 5.Wherein the 8th kind of eluent, namely first carries out wash-out with 3BV1mol/LNaOH solution, and then it is lower to carry out wash-out eluent consumption with dehydrated alcohol, is approximately 5BV.
Claims (4)
1. to the method that Nucleotide enzymolysis solution decolours, it is characterized in that, Nucleotide enzymolysis solution carried out pigment absorption by hypercrosslinked polymeric resin SX-01, adsorb saturated after, hypercrosslinked polymeric resin SX-01 utilizes regenerator to carry out resin regeneration;
Wherein, described hypercrosslinked polymeric resin SX-01 has following structural unit:
The skeleton of this resin is polystyrene, and median size is 0.3 ~ 1mm, and water content is 32% ~ 49%, and aperture is 1.5 ~ 4nm, and most probable pore size is 1.8nm, and average specific surface area is 950 ~ 1800m
2/ g, average pore volume is 0.54 ~ 0.82cm
3/ g;
Described regenerator is 1mol/L NaOH and dehydrated alcohol.
2. method of decolouring to Nucleotide enzymolysis solution according to claim 1, is characterized in that, described Nucleotide enzymolysis solution obtains with nuclease p1 enzymolysis RNA powder.
3. method of decolouring to Nucleotide enzymolysis solution according to claim 1, is characterized in that, pigment suction type is ADSORPTION IN A FIXED BED, resin column aspect ratio is 8 ~ 12, the pH of enzymolysis solution is 4.5, and adsorption flow rate is 1 ~ 3BV/h, and it is 30 ~ 50 DEG C that column temperature controls.
4. the method that Nucleotide enzymolysis solution is decoloured according to claim 1, it is characterized in that, regenerative process is for first to regenerate with 3BV 1mol/L NaOH, then carry out regenerating with dehydrated alcohol until effluent liquid is close to the ethanol washed away with deionized water time colourless in resin, the flow velocity of NaOH and ethanol is 1BV/h.
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