CN101012469A - Method for preparing 5'-nucleotides using malt root complex phospho-esterase - Google Patents
Method for preparing 5'-nucleotides using malt root complex phospho-esterase Download PDFInfo
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- CN101012469A CN101012469A CN 200710063910 CN200710063910A CN101012469A CN 101012469 A CN101012469 A CN 101012469A CN 200710063910 CN200710063910 CN 200710063910 CN 200710063910 A CN200710063910 A CN 200710063910A CN 101012469 A CN101012469 A CN 101012469A
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Abstract
The invention discloses a making method of 5'-nucleic acid through enzymolyzing RNA by composite phospho-esterase of malt root, which is characterized by the following: adopting liquid RNA as substrate; inhibiting the activity of phosphomonoesterase and 3'-phosphodiesterase through the activity of composite phospho-esterase; obtaining the production of 5'-nucleic acid more than 15mg/ml.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of utilizing root of Cornu Cervi Pantotrichum phosphoesterases complex preparation 5 '-Nucleotide particularly.
Background technology
The research of Nucleotide has been reached the stage of two industryization productions both at home and abroad, suitability for industrialized production Nucleotide is mainly enzymolysis process and fermentation method, and the emphasis of nearly decades mainly is an enzymolysis process, and enzymolysis process mainly is nucleic acid P
1Enzyme liberating also has the phosphoesterases complex enzymolysis that utilizes the by product root of Cornu Cervi Pantotrichum lixiviate of producing beer in the last few years.Nucleic acid P
1The enzyme preparation is compared with the phosphoesterases complex of root of Cornu Cervi Pantotrichum preparation, exists the microorganism culturing cycle long, and facility investment is big, and expense is than problems such as height; Utilize the phosphoesterases complex and preparation nucleic acid P of root of Cornu Cervi Pantotrichum preparation
1Enzyme is compared, and vigor is suitable, and preparation is easy, low cost and other advantages.Produce the by product root of Cornu Cervi Pantotrichum of beer and the utilization of yeast rna solution, both met the recycling economy comprehensive utilization, reach the purpose of preserving the ecological environment again.
Utilize the phosphoesterases complex of root of Cornu Cervi Pantotrichum preparation, the existing report [1 of technology of enzymolysis yeast rna preparation 5 '-Nucleotide, 2], but study not deep enough, technology is not very ripe, the enzyme activity of lixiviate low (200～300u/ml), enzymatic hydrolyzation low (about 50%), defective such as the Nucleotide productive rate is not high, product purity is low.
Summary of the invention
The objective of the invention is at above-mentioned deficiency, a kind of method of utilizing root of Cornu Cervi Pantotrichum phosphoesterases complex efficient production 5 '-Nucleotide is provided.
The present invention prepares the root of Cornu Cervi Pantotrichum phosphoesterases complex by specific preparation technology's lixiviate, root of Cornu Cervi Pantotrichum phosphoesterases complex enzyme liquid, liquid RNA is carried out pre-treatment respectively reach the purpose that improves 5 '-Nucleotide output.Be concrete preparation method below:
1, the preparation of root of Cornu Cervi Pantotrichum phosphoesterases complex and pre-treatment: get fresh root of Cornu Cervi Pantotrichum and add water, pulverize according to mass ratio 1: 7～12, behind the lixiviate 4-8h, solid-liquid separation, enzyme liquid is measured enzyme 450-550u/ml alive according to the method for GB mensuration phosphoesterases complex.Enzyme liquid be heated to 30 ℃-60 ℃ standby.Be preferably and get the water that fresh root of Cornu Cervi Pantotrichum adds 10 times of amounts, pulverize, behind the lixiviate 6h, solid-liquid separation, enzyme liquid is measured enzyme 500u/ml alive according to the method for GB mensuration phosphoesterases complex.Enzyme liquid be heated to 45 ℃ standby.
2, liquid yeast RNA pre-treatment: liquid yeast RNA thin up is regulated pH8.0-10.0 to the concentration 2.5%-5.5% that needs, and is configured to RNA solution, with RNA solution be warming up to 70 ℃-100 ℃ standby.Be preferably: liquid yeast RNA pre-treatment: liquid yeast RNA thin up is regulated pH10.0 to the concentration 4% that needs, and is configured to RNA solution, with RNA solution be warming up to 90 ℃ standby.Wherein, adopt 10mol/L NaOH to regulate pH.
3, enzymolysis: above-mentioned pretreated enzyme liquid and RNA solution are mixed, and enzymolysis solution RNA final concentration is at 2%-4%, pH between 4.5-6.0, the enzymolysis solution enzyme 50-150u/ml that lives, temperature is incubated 1.5-3.0 hour between 65 ℃-75 ℃, 90 ℃ of-100 ℃ of deactivations.Enzymolysis solution detects: adopt methylamine fixing phosphorus method and HPLC method, enzymatic hydrolyzation 〉=70%, 5 '-Nucleotide output 〉=15mg/ml.Be preferably: above-mentioned pretreated enzyme liquid and RNA solution are mixed, and control enzymolysis solution RNA final concentration is 2%, and pH is controlled at 5.2, control enzymolysis solution enzyme 100u/ml alive, and temperature is controlled at 70 ℃, is incubated 2 hours, 100 ℃ of deactivations.
4, separation and purification.Progressively purifying of following method is adopted in separation and purification:
1) micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation.Obtain the solution clarification, no suspended substance, the product rate of recovery＞95%;
2) ultrafiltration: adopt ultra-filtration membrane, remove macromole impurity.The albumen clearance is more than 95%, product recovery rate＞95%;
3) nanofiltration: selecting the molecular retention amount for use is that the nanofiltration membrane of 100-300D concentrates product recovery rate＞90%.
5 '-Nucleotide behind the purifying promptly obtains dry finished product after drying.
Utilize the inventive method preparation 5 '-Nucleotide to have following advantage:
1. directly use liquid RNA (through acid heavy still washing with alcohol of no use and exsiccant RNA), with OD
260Calculating feeds intake, and can reduce raw materials cost more than 20%;
2. enzyme liquid and RNA are through thermal pretreatment, effectively utilize the activity of phosphodiesterase, suppressed the activity of phosphomonoesterase, 3 '-assorted enzymes such as phosphodiesterase, can also reach the optimum temperuture of enzymolysis fast, thereby having improved enzymatic hydrolyzation, enzymatic hydrolyzation can reach more than 70%;
3. ceramic membrane substitutes solid-liquid separation, product recovery rate＞95%;
4. the use of ultra-filtration membrane can be removed macromole impurity, reaches the preliminary purification product, albumen clearance＞95%, product recovery rate＞95%;
5. nanofiltration can concentrate 5 times, product recovery rate＞90%.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Embodiment
Following embodiment is used for further specifying of the present invention, but is not used for limiting invention which is intended to be protected.
Embodiment 1
1. pre-treatment and enzymolysis
1. pre-treatment: liquid RNA, with OD
260Absorbancy is calculated OD
260Reading is 0.375 * 2000, and (readings after 2000 times of the liquid RNA dilutions are got in expression, down with) amounts to the pure rna 3750g that feeds intake, and regulates pH9.0; Root of Cornu Cervi Pantotrichum 6kg adds water 60L, and colloidal mill is pulverized back lixiviate 4h, the centrifugal enzyme liquid 40L that gets.RNA solution is warming up to 93 ℃, and enzyme liquid heat to 40 is ℃ standby;
2. enzymatic hydrolysis condition: after enzyme liquid and RNA mix, 74 ℃ of controlled temperature, pH5.2, RNA ultimate density 2.5%, the final enzyme 120u/ml that lives, insulation enzymolysis 2h;
3. termination reaction: after enzymolysis is finished, be warming up to 90 ℃-100 ℃, be incubated 30 minutes;
4. methylamine is decided phosphorus: 5 '-Nucleotide 21mg/ml, enzymatic hydrolyzation are 84%;
2. micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation.Obtain the solution clarification, no suspended substance, the rate of recovery 97%;
3. ultrafiltration: adopt ultra-filtration membrane, remove macromole impurity.Product recovery rate 96%, albumen clearance 96%;
4. nanofiltration: nanofiltration membrane concentrates, and concentrates 5 times, amounts to the 32L concentrated solution, product recovery rate 93%;
5. spraying drying: the 22L concentrated solution, altogether dry powder 6100g.
Dry powder index: 5 '-Nucleotide mass content 44%, albumen＜0.1%, moisture 2.78%.
Embodiment 2
1. pre-treatment and enzymolysis
1. pre-treatment: liquid RNA, with OD
260Absorbancy is calculated OD
260Reading is 0.453 * 2000 to amount to the pure rna 3500g that feeds intake, and regulates pH9.0; Root of Cornu Cervi Pantotrichum 7kg adds water 70L, and colloidal mill is pulverized back lixiviate 4h, the centrifugal enzyme liquid 55L that gets.RNA solution is warming up to 95 ℃, and enzyme liquid heat to 40 is ℃ standby;
2. enzymatic hydrolysis condition: after enzyme liquid and RNA mix, 72 ℃ of controlled temperature, pH5.2, RNA ultimate density 2.5%, the final enzyme 100u/ml that lives, insulation enzymolysis 2h;
3. termination reaction: after enzymolysis is finished, be warming up to 90 ℃-100 ℃, be incubated 30 minutes;
4. methylamine is decided phosphorus: 5 '-Nucleotide 18mg/ml, enzymatic hydrolyzation are 72.8%;
2. micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation.Obtain the solution clarification, no suspended substance, the rate of recovery 96.4%;
3. ultrafiltration: adopt hollow fiber ultrafiltration membrane, remove macromole impurity.Product recovery rate 97.6%, albumen clearance 97%;
4. nanofiltration: nanofiltration membrane concentrates, and concentrates 5 times, amounts to the 28L concentrated solution, product recovery rate 95%;
5. spraying drying: the 28L concentrated solution, altogether dry powder 4500g.
Dry powder index: 5 '-Nucleotide mass content 50%, albumen＜0.1%, moisture 2.88%.
Embodiment 3
1. pre-treatment and enzymolysis
1. pre-treatment: liquid RNA, with OD
260Absorbancy is calculated OD
260Reading is 0.412 * 2000 to amount to the pure rna 3000g that feeds intake, and regulates pH8.0; Root of Cornu Cervi Pantotrichum 8kg adds water 100L, and colloidal mill is pulverized back lixiviate 8h, the centrifugal enzyme liquid 70L that gets.RNA solution is warming up to 70 ℃, and enzyme liquid heat to 30 is ℃ standby;
2. enzymatic hydrolysis condition: after enzyme liquid and RNA mix, 65 ℃ of controlled temperature, pH4.5, RNA ultimate density 2.0%, the final enzyme 150u/ml that lives, insulation enzymolysis 3h;
3. termination reaction: after enzymolysis is finished, be warming up to 90 ℃-100 ℃, be incubated 30 minutes;
4. methylamine is decided phosphorus: 5 '-Nucleotide 15.3mg/ml, enzymatic hydrolyzation are 76.7%;
2. micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation.Obtain the solution clarification, no suspended substance, the rate of recovery 96.2%;
3. ultrafiltration: adopt ultra-filtration membrane, remove macromole impurity.Product recovery rate 96.7%, albumen clearance 96.5%;
4. nanofiltration: concentrate 5 times, amount to the 31L concentrated solution, product recovery rate 94%;
5. spraying drying: the 31L concentrated solution, altogether dry powder 3700g.
Dry powder index: 5 '-Nucleotide mass content 53%, albumen＜0.1%, moisture 2.00%.
Embodiment 4
1. pre-treatment and enzymolysis
1. pre-treatment: liquid RNA, with OD
260Absorbancy is calculated OD
260Reading is 0.880 * 2000 to amount to the pure rna 4800g that feeds intake and regulate pH10.0; Root of Cornu Cervi Pantotrichum 8kg adds water 90L, and colloidal mill is pulverized back lixiviate 6h, the centrifugal enzyme liquid 60L that gets.RNA solution is warming up to 100 ℃, and enzyme liquid heat to 60 is ℃ standby;
2. enzymatic hydrolysis condition: after enzyme liquid and RNA mix, 75 ℃ of controlled temperature, pH6.0, RNA ultimate density 4.0%, the final enzyme 50u/ml that lives, insulation enzymolysis 3h;
3. termination reaction: after enzymolysis is finished, be warming up to 90 ℃-100 ℃, be incubated 30 minutes;
4. methylamine is decided phosphorus: 5 '-Nucleotide 32mg/ml, degradation rate are 80.7%;
2. micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation.Obtain the solution clarification, no suspended substance, the rate of recovery 95.5%;
3. ultrafiltration: adopt ultra-filtration membrane, remove macromole impurity.Product recovery rate 95.8%, albumen clearance 95.5%;
4. nanofiltration: concentrate 5 times, amount to the 29L concentrated solution, product recovery rate 94%;
5. spraying drying: the 29L concentrated solution altogether dry powder 6350g.
Dry powder index: 5 '-Nucleotide mass content 52%, albumen＜0.1%, moisture 2.50%.
Reference:
[1] Qiao Shiwei, the extraction of flavour nucleotide, Chinese seasonings, calendar year 2001 (2): 29-31.
[2] Li Xiang, the preparation of 5 '-phosphodiesterase and the application in yeast extract is produced thereof, Chinese seasonings, (4): 12-14. in 2000
Claims (6)
1, a kind of method of preparation 5 '-Nucleotide is characterized in that, comprises the steps:
1) preparation of root of Cornu Cervi Pantotrichum phosphoesterases complex and pre-treatment: get fresh root of Cornu Cervi Pantotrichum and add water, pulverize according to mass ratio 1: 7～12, behind the lixiviate 4-8h, solid-liquid separation, enzyme liquid be heated to 30 ℃-60 ℃ standby;
2) liquid yeast RNA pre-treatment: liquid yeast RNA thin up to concentration is 2.5%-5.5%, regulates pH 8.0-10.0, is configured to RNA solution, with RNA solution be warming up to 70 ℃-100 ℃ standby;
3) enzymolysis: above-mentioned pretreated enzyme liquid and RNA solution are mixed, and enzymolysis solution RNA final concentration is at 2%-4%, pH between 4.5-6.0, the enzymolysis solution enzyme 50-150u/ml that lives, temperature is incubated 1.5-3.0 hour between 65 ℃-75 ℃, 90 ℃ of-100 ℃ of deactivations;
4) separation and purification.
2, the method for claim 1, wherein the preparation of root of Cornu Cervi Pantotrichum phosphoesterases complex and pretreatment process are: it is broken with pigment according to mass ratio 1: 10 to get fresh root of Cornu Cervi Pantotrichum, behind the lixiviate 6h, solid-liquid separation, enzyme liquid be heated to 45 ℃ standby.
3, the method for claim 1, wherein the pretreated method of liquid yeast RNA is: liquid yeast RNA thin up to 3.7%, regulate pH 9.0, be configured to RNA solution, with RNA solution be warming up to 90 ℃ standby.
4, the method for claim 1, wherein enzymatic hydrolysis condition is: pretreated enzyme liquid and RNA solution are mixed, and enzymolysis solution RNA final concentration is 3%, and pH is 5.2, the enzymolysis solution enzyme 120u/ml that lives, temperature is incubated 2 hours at 72 ℃, 90 ℃ of-100 ℃ of deactivations.
5, as each described method of claim 1～4, wherein said separation and purification comprises step:
1) micro-filtration: adopt inorganic ceramic film, enzymolysis solution is carried out solid-liquid separation;
2) ultrafiltration: adopt ultra-filtration membrane, remove macromole impurity;
3) nanofiltration: selecting the molecular retention amount for use is that the nanofiltration membrane of 100-300D concentrates.
6, as each described method of claim 1～4, it also comprises step: 5 '-Nucleotide behind the purifying is carried out drying.
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| CN 200710063910 CN101012469A (en) | 2007-02-14 | 2007-02-14 | Method for preparing 5'-nucleotides using malt root complex phospho-esterase |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845422A (en) * | 2010-05-07 | 2010-09-29 | 上海斯贝生物科技有限公司 | Method for preparing 5'-phosphodiesterase and method for preparing 2'-deoxyribonucleotide by using same |
| CN102051349B (en) * | 2009-10-30 | 2012-07-25 | 安琪酵母股份有限公司 | Method for preparing nuclease P1 |
| CN101418327B (en) * | 2008-11-21 | 2012-09-05 | 大连珍奥生物技术股份有限公司 | Novel production process of high-purity 5' nucleotide |
| CN105112384A (en) * | 2015-09-24 | 2015-12-02 | 青岛黄海制药有限责任公司 | Extracting and drying method of phosphodiesterase complex |
-
2007
- 2007-02-14 CN CN 200710063910 patent/CN101012469A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418327B (en) * | 2008-11-21 | 2012-09-05 | 大连珍奥生物技术股份有限公司 | Novel production process of high-purity 5' nucleotide |
| CN102051349B (en) * | 2009-10-30 | 2012-07-25 | 安琪酵母股份有限公司 | Method for preparing nuclease P1 |
| CN101845422A (en) * | 2010-05-07 | 2010-09-29 | 上海斯贝生物科技有限公司 | Method for preparing 5'-phosphodiesterase and method for preparing 2'-deoxyribonucleotide by using same |
| CN105112384A (en) * | 2015-09-24 | 2015-12-02 | 青岛黄海制药有限责任公司 | Extracting and drying method of phosphodiesterase complex |
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Open date: 20070808 |