CN101363016A - Methods for concentrating and purifying nuclease P1 by nanofiltration and producing nucleotide - Google Patents

Methods for concentrating and purifying nuclease P1 by nanofiltration and producing nucleotide Download PDF

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Publication number
CN101363016A
CN101363016A CNA2008100134195A CN200810013419A CN101363016A CN 101363016 A CN101363016 A CN 101363016A CN A2008100134195 A CNA2008100134195 A CN A2008100134195A CN 200810013419 A CN200810013419 A CN 200810013419A CN 101363016 A CN101363016 A CN 101363016A
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Prior art keywords
nuclease
nanofiltration
concentrated
nucleotide
membrane
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CNA2008100134195A
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乔宾福
俞慧君
霍旭辉
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DALIAN ZHEN-AO BIO-ENGINEERING STOCK Co Ltd
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DALIAN ZHEN-AO BIO-ENGINEERING STOCK Co Ltd
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Publication of CN101363016A publication Critical patent/CN101363016A/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Abstract

The invention discloses a method for carrying out nanofiltration, concentration and purification of a nuclease P1, a Penicillium citrinum strain M843 with the high activity is bred, and the nuclease P1 is obtained via the liquid depth fermentation in a culture medium; a centrifuge is used for removing bacteria and solid matters of the culture medium, thereby obtaining supernatant liquid containing the nuclease P1; part of water, inorganic salts and pigments are removed through the dialysis by a nanofiltration membrane. The invention further discloses a method for producing nucleotide by using the nuclease P1 which is prepared by nanofiltration, concentration and purification, the concentrated and purified nuclease P1 is used for degrading ribonucleic acid; degraded solution is carried out the ultrafiltration via a ceramic membrane of 20nm to 50nm; ultrafiltration liquid is dialyzed by the nanofiltration membrane with the molecular weight cutoff of 300 Daltons, thereby removing the inorganic salts, the pigments and other impurities and obtaining concentrated 5' nucleotide solution; the obtained concentrated 5' nucleotide solution is carried out the further concentration by a rising membrane concentrating tower, thereby leading the content of solid matters to be 35 to 45 percent and preparing a 5' nucleotide powder product by spray drying or microwave drying. The method has the advantages of simple procedure, high yield, low cost, stable quality, large-scale production and the like.

Description

The concentrated and purified nuclease P of nanofiltration 1And the method for producing Nucleotide
Technical field:
The present invention relates to a kind of nuclease P 1Purifying method and with the nuclease P of purifying 1Produce the method for Nucleotide.Especially use modern molecular level isolation technique (nanofiltration), will have the nuclease P that degraded Yeast Nucleic Acid becomes 5 ' Nucleotide 1Concentrated, purifying becomes the liquid enzymes of high reactivity, stability; Use this kind of enzyme liquid to prepare 5 ' Nucleotide solution, and further use molecular level isolation technique (ultrafiltration and nanofiltration) purifying, concentrated 5 ' Nucleotide solution, to produce 5 ' Nucleotide finished product.
Background technology:
5 ' Nucleotide comprises 5 ' adenylic acid (AMP), 5 ' guanylic acid, 5 ' cytidylic acid, 5 ' uridylic acid, they are not only the genetic material Yeast Nucleic Acid (RNA) of synthetic life in the genetically engineered and the basic raw material of oligonucleotide, and be the precursor of many antitumor, antiviral, in animal-feed, add 5 ' Nucleotide and have the immunologic function of raising, promotion growth, metabolic function, on agricultural, spray, irrigate, soak seed and use, the effect that have the promotion plant-growth, bloom, the result increases.External a large amount of report 5 ' Nucleotide are the functional complementary food of a class after the nineties in last century, have significant immunologic function, and domestic market demand enlarges, and output obviously improves.
Produce four kind of 5 ' Nucleotide simultaneously and can only use narrow spectrum enzyme (nuclease P 1) degraded Yeast Nucleic Acid method.Up to now, on industrial production, to obtain nuclease P 1Have only two approach: the firstth, cultivate a kind of bacterial strain at cell exocrine nuclease P 1The secondth, this enzyme of lixiviate from Fructus Hordei Germinatus.It is bulky that lixiviate relates to source, preservation, the extracting of Fructus Hordei Germinatus resource from Fructus Hordei Germinatus, and operation is complicated, enzyme activity is not high, and using in industrial production is not preferred plan.For nuclease P 1Use, developed into immobilized enzyme, immobilized enzyme (dry powder enzyme) and three kinds of modes of liquid enzymes in the world.With immobilized enzyme and solid enzyme, not only the technology of preparing requirement is very high, and production cost is also very high, with dry powder nuclease P 1Be example, the import price per kilogram is used to produce 5 ' Nucleotide at 2500 yuan～3000 yuans, and it will account for about 30% of main raw material cost price, be unfavorable for the scale operation of 5 ' Nucleotide.
At present, with liquid nuclease P 1The method of producing 5 ' Nucleotide all is directly with not purified liquid nuclease P 1Degraded Yeast Nucleic Acid obtains 5 ' Nucleotide solution, and then further separates through the separating technology of ion exchange resin, removes impurity and obtains 5 ' adenylic acid (AMP), 5 ' guanylic acid, 5 ' cytidylic acid, 5 ' uridylic acid monomer.Though product purity is very high, can reach 98%～102%, but but exist cost height, problem that yield is low, also be unfavorable for the scale operation of 5 ' Nucleotide, especially for not needing 5 ' nucleotide monomer and do not need the production of high purity 5 ' oligonucleotide product, this kind method is more inapplicable.
Summary of the invention:
The present invention be directed to existing in prior technology cost height, yield low, be not suitable for problem such as scale operation, provide that a kind of operation is simple, yield is high, cost is low, steady quality, be fit to the concentrated and purified nuclease P of nanofiltration of scale operation 1And the method for producing Nucleotide.
Technical solution of the present invention is: the concentrated and purified nuclease P of a kind of nanofiltration 1Method, it is characterized in that carrying out as follows:
A. the highly active Penicillium citrinum bacterial strain of seed selection M843 obtains nuclease P through liquid submerged fermentation in substratum 1
B. remove the solid substance of thalline and substratum with whizzer, must contain nuclease P 1Supernatant liquor;
C. portion water, inorganic salt and pigment are removed in the nanofiltration membrane dialysis.
Described a step is to be carbon source with glucose 2%～10%, and peptone 0.3%～0.8% is a nitrogenous source.
The condition of described a step is: substratum ingredient and weight percent 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; Culture condition: 28 ℃～30 ℃, the ventilation stir culture is cultivated 25hr.
Described c step is to be the dialysis of 300～10000 daltonian nanofiltration membrane through molecular weight cut off.
Described c step is to add deionized water to carry out repeatedly, and making enzyme liquid cycles of concentration is 5～16 times.
A kind of concentrated and purified nuclease P that obtains with preceding method 1Produce the method for Nucleotide, it is characterized in that carrying out as follows:
D. use concentrated and purified nuclease P 1Degraded Yeast Nucleic Acid, substrate Yeast Nucleic Acid mass concentration 2%～5%, condensed nucleic acid enzyme P 1Consumption be cumulative volume 1.4%～4%, PH5.5～6.2,65 ℃～70 ℃ of temperature, degradation time 3～5 hours degradation solution;
E. with the ceramic membrane ultrafitration of degradation solution through 20nm～50nm;
F. be 300 daltonian nanofiltration membrane dialysis with ultrafiltrated through molecular weight cut off, the deionized water that adds cumulative volume 30%～50% carries out repeatedly, and foreign material such as inorganic salt, pigment are removed in nanofiltration, concentrate and obtain 3～5 times 5 ' Nucleotide solution;
G. resulting 5 ' the Nucleotide solution that concentrates 3～5 times is further concentrated through rising the membrane concentration tower, making solid content is 35～45%, and 5 ' Nucleotide powder-like product is made in spraying drying or microwave drying.
The present invention is seed selection one strain Penicillium citrinum bacterial strain M843 (Penicillium citrinum), produces liquid nuclease P in submerged fermentation 1, cell balling not in the fermentation culture process, thus the expression of enzymes of high vigor is arranged, and enzyme activity can reach 1150～1400 units per ml; , purification of nucleic acid enzyme P concentrated with nano filtering process 1, can remove portion water, inorganic salt and pigment, concentrate back unit enzyme activity and improve, be easy to preserve, enzyme lives stable; With this concentrated, purification of nucleic acid enzyme P 1Yeast Nucleic Acid is effectively degraded and ultrafiltration, the nanofiltration separation technology of used molecular level, both can remove impurity such as foreigh protein removing, residual nucleus ribosomal ribonucleic acid, inorganic salt, pigment, need not to use again the separating technology of the high ion exchange resin of cost, can prepare 5 ' the Nucleotide mixture that satisfies purity requirement fully, have that operation is simple, yield is high, cost is low, steady quality, advantage such as can be mass-produced.
Embodiment:
Embodiment 1:
A. liquid nuclease P 1Preparation:
Bacterial classification: Penicillium citrinum M843 (Penicillium citrinum).
The composition of substratum and weight percent: glucose 5%, peptone 0.5%, K 2HPO 40.05%, KH 2PO 40.05%, MgSO 40.04%, CaCl 20.04%, ZnSO 40.02%, PH 6.0.
Culture condition: 28 ℃～30 ℃, the ventilation stir culture is cultivated 25hr, enzyme activity 1294U/ml.
B. nanofiltration condensed nucleic acid enzyme P 1:
With cultured fermented liquid, with obtaining containing nuclease P behind the conventional centrifugal basket drier bactofugation filament 1Supernatant liquor; Get the 2000ml supernatant liquor and dialyse, remove moisture, inorganic salt and pigment, every membrane area 0.02m with 10000 daltonian nanofiltration membrane 2The film bag, control intake pressure 0.15Mpa, top hole pressure 0.05Mpa, must concentrate enzyme liquid 120ml through nanofiltration in 30 minutes, volume has concentrated 16.7 times, enzyme activity 1188U/ml before concentrating, concentrate back enzyme activity 16740U/ml, every milliliter of enzyme activity is 14.1 times of former enzyme activity.
The enzyme activity that the nanofiltration membrane molecular weight cut off should guarantee to reach satisfied reclaims, and concentrated enzyme is preserved 3 months enzyme activities at normal temperatures and is kept at more than 95%.Be 300～10000 daltonian nanofiltration membrane with molecular weight cut off preferably, can add an amount of deionized water continuation nanofiltration in order to reach purifying and to concentrate purpose, enzyme liquid cycles of concentration can be controlled in 5～16 times of scopes.
With prepared concentrated type nuclease P 1But efficient degradation Yeast Nucleic Acid becomes 5 ' Nucleotide.
Degradation condition:
RNA strength of solution 2.5%, enzyme concentration 1.4% (V/V), 65 ℃～70 ℃ of operative temperatures, effect PH5.5～6.2, degradation time 4 hours.
Degradation solution is through the HPLC assay determination, and degradation rate is 78.65%.
Embodiment 2:
A. nuclease P 1Preparation
Bacterial classification: Penicillium citrinum M843
Substratum and culture condition were cultivated enzyme activity 1314U/ml 23 hours with embodiment 1.
B. nanofiltration condensed nucleic acid enzyme P 1
With cultured fermented liquid, with obtaining containing nuclease P behind the conventional centrifugal basket drier bactofugation filament 1Supernatant liquor; Getting the 685L supernatant liquor is 30m with 300 dalton, membrane area 2Collecting and filtering apparatus carry out nanofiltration.3 hours times spent, average flux 240L/hr adds pure water 70L midway, control pressure 1～1.2kg/cm 2, electricity is led 800us/cm 2, nanofiltration liquid 110L, volume has concentrated 6.23 times, every milliliter of enzyme activity is 5.9 times of former enzyme activity, reaches 7776U/ml.
Concentrate enzyme at 5 ℃～20 ℃ storages, nuclease P 1Vigor is preserved percentage such as following table:
The result shows concentrated type nuclease P 1Preserved at normal temperatures three months, vigor is preserved percentage greater than 95%.
C. the degraded of Yeast Nucleic Acid
Nuclease P with concentrated 6.23 times of gained 1, enzyme concentration is 3.7% (V/V), presses embodiment 1 degradation condition, and degraded is in four batches analyzed through HPLC, and degradation results sees the following form:
The degraded batch 1 2 3 4
The concentrated enzyme shelf time (my god) 1 13 23 51
Degradation rate % 79.78 82.87 86.08 86.05
Producing of embodiment 3:5 '-Nucleotide mixture product.
A. nuclease P 1Preparation is with embodiment 1;
B. concentrated and purified nuclease P 1With embodiment 1 or embodiment 2;
C. the degraded of Yeast Nucleic Acid (RNA):
Pure 17.5kgRNA is dissolved to 674L, adds to concentrate purifying enzyme nuclease P 126L (dosage accounts for cumulative volume 3.7%), 65 ℃～70 ℃, degrading 4 hours in PH5.5～6.2, is warming up to 90 ℃～95 ℃, makes enzyme deactivation, is cooled to 30 ℃～40 ℃, analyzes degradation rate 82.87% through HPLC.
D. the ultrafiltration of degradation solution:
The degradation solution membrane area is 8m 2Ceramic membrane (50nm), remove remaining nucleic acid and anaenzyme albumen, during ultrafiltration control intake pressure be 4kg/cm 2, use the pure washing film of 400L, average flux 137.5L/hrcm after the ultrafiltration 2, altogether ultrafiltrated 1100L, 60 minutes times spent.
E. nanofiltration:
Ultrafiltrated is again through 300 dalton, membrane area 30m 2Collecting and filtering apparatus carries out nanofiltration, removes portion water, pigment and inorganic salt, and nanofiltration 3hr can add pure water 500L midway, control pressure 1～1.2kg/cm 2, average flux 433L/hr, electricity is led 400us/cm, gets nanofiltration liquid 315L.
F. further concentrate and drying:
With resultant 5 '-Nucleotide mixture solution that concentrates, with rising the membrane concentration tower, control admission pressure 0.3kg, holding tank solution temperature＜70 ℃, time spent 5.5hr, with 315L5 '-Nucleotide mixture solution concentration to 42L, its solid content about 40%, advance spray-drying tower and carry out spraying drying, get powdery 5 '-Nucleotide mixture 16.8kg, analyze content 77.71% through HPLC, moisture 6.0%, pure yield 70.13%.

Claims (6)

1. concentrated and purified nuclease P of nanofiltration 1Method, it is characterized in that carrying out as follows:
A. the highly active Penicillium citrinum bacterial strain of seed selection M843 obtains nuclease P through liquid submerged fermentation in substratum 1
B. remove the solid substance of thalline and substratum with whizzer, must contain nuclease P 1Supernatant liquor;
C. portion water, inorganic salt and pigment are removed in the nanofiltration membrane dialysis.
2. the concentrated and purified nuclease P of a kind of nanofiltration according to claim 1 1Method, it is characterized in that described a step is is carbon source with glucose 2%～10%, peptone 0.3%～0.8% is a nitrogenous source.
3. the concentrated and purified nuclease P of a kind of nanofiltration according to claim 2 1Method, it is characterized in that the condition of described a step is:
Substratum ingredient and weight percent 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;
Culture condition: 28 ℃～30 ℃, the ventilation stir culture is cultivated 25hr.
4. according to claim 2 or the concentrated and purified nuclease P of 3 described a kind of nanofiltrations 1Method, it is characterized in that described c step is is the dialysis of 300～10000 daltonian nanofiltration membrane through molecular weight cut off.
5. the concentrated and purified nuclease P of a kind of nanofiltration according to claim 4 1Method, it is characterized in that described c step is to add deionized water to carry out repeatedly, making enzyme liquid cycles of concentration is 5～16 times.
6. concentrated and purified nuclease P who obtains with method as described in claim 1～5 1Produce the method for Nucleotide, it is characterized in that carrying out as follows:
D. with concentrated and purified nuclease P 1 degraded Yeast Nucleic Acid, substrate Yeast Nucleic Acid mass concentration 2%～5%, condensed nucleic acid enzyme P 1Consumption be cumulative volume 1.4%～4%, PH5.5～6.2,65 ℃～70 ℃ of temperature, degradation time 3～5 hours degradation solution;
E. with the ceramic membrane ultrafitration of degradation solution through 20nm～50nm;
F. be 300 daltonian nanofiltration membrane dialysis with ultrafiltrated through molecular weight cut off, the deionized water that adds cumulative volume 30%～50% carries out repeatedly, and foreign material such as inorganic salt, pigment are removed in nanofiltration, concentrate and obtain 3～5 times 5 ' Nucleotide solution;
G. resultant 5 ' the Nucleotide solution that concentrates 3～5 times is further concentrated through rising the membrane concentration tower, making solid content is 35～45%, and 5 ' Nucleotide powder-like product is made in spraying drying or microwave drying.
CNA2008100134195A 2008-09-27 2008-09-27 Methods for concentrating and purifying nuclease P1 by nanofiltration and producing nucleotide Withdrawn CN101363016A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112851720A (en) * 2020-12-30 2021-05-28 音芙医药科技(上海)有限公司 Method for preparing high-purity NMN (N-methyl pyrrolidone) by using ultrafiltration and nanofiltration technologies

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112851720A (en) * 2020-12-30 2021-05-28 音芙医药科技(上海)有限公司 Method for preparing high-purity NMN (N-methyl pyrrolidone) by using ultrafiltration and nanofiltration technologies

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