Method of producing mixture of 5 prime-ribonucleatides from nucleic acid solutions containing desoxy

专利摘要:
Verfahren zur Herstellung von 5'-Ribonucleotiden, bei dem man eine Ribonucleinsäure enthaltende Lösung von Rohnucleinsäuren mit einer an einem polymeren Trägerimmobilisierten 5'-Phosphodiesterase selektiv hydrolysiert und aus dem Hydrolysat die unveränderte Desoxyribonucleinsäure und die 5'-Ribonucleotide durch bekannte Reinigungs- und Trennverfahren isoliert oder das Hydrolysat unmittelbar weiter umsetzt.
公开号:SU1435158A3
申请号:SU823494005
申请日:1982-09-16
公开日:1988-10-30
发明作者:Келлер Райнхольд；Шлингманн Мертен
申请人:Хехст Аг (Фирма)；
IPC主号:

专利说明:

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I The invention relates to the field of unitins, in particular, to methods for the synthesis of 5-ribonucleotides. I The purpose of the invention is to prevent the decomposition of deoxyribonuc- bic acid ii simplifying the method and by using natural nucleic acids containing deoxy-fibonucleic acid mol. bo-fiee 100,000, and ribonucleic acid, mol.m. less than 50,000.
The method is carried out as follows.
Example 1. The enzyme 5-phosphodiesterase is immobilized by forming a covalent bond between the enzyme and the polymer 1m carrier, as used such polymer porous media, which have channels with a diameter of 100 them and a volume of 1 then 2-3 ml / g, preferably 2.5 ml / g. An especially good carrier for 5-phosphodiesterase is Eupongit C epoxy carrier (R), which is a copolymer of glycidyl methacrylate, allyl glycidyl ether, methacrylamide and methylene bis. meth acrylamide. The enzyme is combined with a polymeric carrier under conditions which do not affect the stability of the enzyme. The result of the compound is determined by measuring the enzyme activity on the polymer and in the wash water. The process is carried out as follows.
2 g of 5-phosphodieste times (nucleases IR p, Amano company) are dissolved in 80 ml of L M phosphate buffer solution with pH; 7.8. The solution is fed to the column, containing 20 g of epoxy carrier (Oi-pergit s) with channels with a diameter of up to ilOO nm and a pore volume of 2–3 l / g, lightly stirred and left for 3 days 1 at room temperature. Polymer: the resin is filtered off and washed with the following liquids: bidistil tol; 1 S with a H 2 O 3 solution; 0.5 M Nad solution; bidistil tom; 1 M solution of HCi; bidistil tom The washed resin is placed in a 0.05 M acetate-buffer solution and loaded into a double-jacket glass column with a diameter of 2.6 and a height of 20 cm. To measure the activity of the immobilized; 5-phosphodiesterase, the substrate-solution is from 4% RNA and 0.1 mN solution of zinc sulfate and 0.05 M acetate buffer solution with pI 4.5,
,,

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allowed through a column with a flow rate (SV) of 20-30h
At 55 s, the amount of mononucleotides is determined photometrically using liquid chromatography on a column (reversed phase silica gel, hydrophobized aliphatic compounds with a carbohydrate chain containing up to 18 carbon atoms). The elution was carried out with water acidified with phosphoric acid to a pH of 2.1, at a flow rate of 2 ml / min. The development is carried out using UV radiation with a wavelength of 254 nm.
PRI mme R 2. An aqueous solution containing 0.9% (w / v) of naturally occurring nucleic acid, in which the amount of RNA and DNA is 4: 1 and the DNA is mol.m. more than 100,000, and RNA less than 50,000, adjusted to pH 4.5 with glacial acetic acid and passed through a column with an immobilized 5-phosphodiesterase (according to example 1) at 55 C. The flow rate through the column is adjusted so that 100 % conversion of RNA. In order to isolate 5-ribonucleotides, the destroyed solution of nucleic acids is streamed through a column filled with slio-basic but with DUOLIIE A 7 resin in an acetate form. To purify and isolate the DNA, the eluate from the column is concentrated using ROMICON hollow fiber holders (polysulfone fiber, isolation limit 100000) and dialyzed against salt-free water. Molecules with a molecular weight less than 50,000 can pass through the membrane. DNA is precipitated, the pH of the solution is adjusted to 2.0 and, after drying, is obtained as a white powder. Separation of the 5.-ribonucleotide mixture is carried out by selective desorption with acetate solutions of the following concentration: SMR: 0.1N. acetic acid; AMP: 0.3 n. acetic acid; VMP: n ammonium acetate; GMP: 0.5 n. ammonium acetate. Total 5-ribonucleotides 75-80%.
P r. Ime R Z. An aqueous solution containing 3% (w / v) of natural nucleic acid, in which the amount of RNA and DNA is in the B ratio of 4: 1 and the DNA has a mol.m. more than 100,000, and RNA less than 50,000, adjusted to
p no 4,5 with glacial acetic acid 1 and at 55 ° C pass through a column with immobilisation with 5-phosphoric ester according to example 1. The flow rate through the column is adjusted so that 100% conversion of RNA is performed (for example, with an activity of 265 g of RNA per 1 liter of fixed enzyme per hour, the solution is passed through the column with a pump at a speed of 2.12 l / h). The resulting solution also contains 5-adenosine monophosphate. To obtain a 5-inosine monophosphate from it, the solution is passed through a second column with immobilized 5-deaminase (also according to example 1). The eluate from the column is concentrated using hollow fiber membranes and the part passed through the membrane is adjusted to pH 1.5-2.0 with concentrated hydrochloric acid, then passed through a column filled with granular activated carbon, adsorbed 5-nucleotides. DNA is precipitated from the remainder at pH 2.0 and then dried. Isolation of a mixture of 5-ribonucleotides is carried out by selective
ten
 kitty And so, the precipitated DIC is dried and dried.
The overall use of the S3S-ribonucleotide DNA containing DNA is 75-80Z. . . Thus, the method of obtaining 5-ribonucleotides does not require preliminary purification of RNA. And thus does not entail DNA destruction. In addition, the solution obtained by hydrolysis, along with 5-ribonucleotides and intact DNA, also contains impurities of proteins and salts of enzymes that can be isolated by purification.
The solution obtained by the hydrolysis of natural nucleic acids, also containing 5-adenosinionophosphate, can be used directly to obtain 5-inosine monophosphate, used as a flavor enhancer, without prior selection of DNA.
25 Form u, l and inventions
15
20
1. A method of obtaining a mixture of 5-nucleotides from a nucleic acid solution containing deoxyribon desorption with alkaline methanol plant gluic acid by hydrolysis by thieves of the following composition: 50 ob.h. ribonucleic acid followed by methanol, 49 ob.h. water and 1 ob.ch. concentrated aqueous solution by the product amionic separation by ion exchange or adsorption chromatography, about the fact that, in order to prevent the decomposition of deoxyribriucleic acid and simplify the process, a solution of natural nucleic acid (w / v) natural nucleic acid acid containing deoxyribo - lots (RNA), DNA 1: 1, mol.m. DNA more nucleic acid with mpl.m. more YuOORO, RNA - less than 50,000) are treated with 100,000 and ribonucleic acid with wate., as in example 2 and for decomposition: mol.m. less than 50,000, pass through
miak. The total yield of 5-ribonucleotides is 75-80%.
 PRI mede 4. Aqueous solution of 2%
passed through a column with and supplemented 5-phosphodiesterase. The eluate is adjusted to pH 8.5 with NaOH and
in order to adsorb nucleotides, they are passed through a column filled with strong

0
 kitty And so, the precipitated DIC is dried and dried.
The overall use of the S3S-ribonucleotide DNA containing DNA is 75-80Z. . . Thus, the method of obtaining 5-ribonucleotides does not require preliminary purification of RNA. And thus does not entail DNA destruction. In addition, the solution obtained by hydrolysis, along with 5-ribonucleotides and intact DNA, also contains impurities of proteins and salts of enzymes that can be isolated by purification.
The solution obtained by the hydrolysis of natural nucleic acids, also containing 5-adenosinionophosphate, can be used directly to obtain 5-inosine monophosphate, used as a flavor enhancer, without prior selection of DNA.
five
25 Form u, l and inventions
isolation of the product by ion-exchange or adsorption chromatography, about the fact that, in order to prevent the decomposition of deoxy ribonucleic acid and simplify the method, a solution of natural nucleic acids containing deoxyribonucleic acid with mll.m. more than 100,000 and ribonucleic acid mol.m. less than 50,000, pass through
pores 2-3 ml / g.
a column with 5 phosphodiesterase, previously immobilized on a macroporous polymeric carrier with channels up to 100 nm in diameter and

权利要求:
Claims (3)
[1]
1. The method of obtaining a mixture of 5 ¹ -ribonucleotides from a nucleic acid solution containing deoxyribonucleic acid by hydrolysis of ribonucleic acid with subsequent isolation of the product by ion exchange or adsorption chromatography is characterized in that, in order to prevent decomposition of deoxyribonucleic acid and simplify the method, a solution of natural nucleic acids containing deoxyribonucleic acid with a mol.m. more than 100,000 and ribonucleic acid c: mod. less than 50,000, pass through a column of 5 ¹ -phosphodiesterase previously immobilized. on a macroporous polymer carrier with channels with a diameter of up to 100 nm and a pore volume of 2-3 ml / g.
[2]
2. The method according to claim 1 / about t l and h and rout and y. with i so for
[3]
5'-inosine monophosphate: the hydrolyzate is passed through a column with 5'-deaminase fixed on the carrier.

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同族专利:

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AU551838B2|1986-05-15|

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ES8306163A1|1983-05-01|

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AT24019T|1986-12-15|

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US3139385A|1960-12-20|1964-06-30|Takeda Chemical Industries Ltd|Method for the production of 5'-nucleotides|

---

GB940660A|1961-02-04|1963-10-30|Waldhof Zellstoff Fab|Improvements in and relating to the production of 5-nucleotides|

---

DE2215539C2|1972-03-30|1984-08-02|Bayer Ag, 5090 Leverkusen|New water-insoluble enzyme, in particular penicillin acylase or enzyme inhibitor preparations|

---

US4044196A|1972-03-30|1977-08-23|Bayer Aktiengesellschaft|Crosslinked copolymers of α,β-olefinically unsaturated dicarboxylic anhydrides|

---

FR2308636B1|1975-04-23|1977-11-25|Choay Sa|

---

JPS5729153B2|1975-08-04|1982-06-21|

---

US4206243A|1976-07-27|1980-06-03|Hoechst Aktiengesellschaft|Process for reducing the contents of lipids and nucleic acid in microbial cell masses|

---

US4208309A|1977-05-20|1980-06-17|Rohm Gmbh|Pearl polymer containing hollow pearls|

---

DE2722751C2|1977-05-20|1990-06-21|Röhm GmbH, 6100 Darmstadt|Bead polymer with a particle diameter of 5 to 1000 μm and an internal cavity|

---

US4342833A|1977-05-31|1982-08-03|Bethesda Research Laboratory|Immobilized restriction endonucleases|

---

DE2732301C3|1977-07-16|1980-12-11|Roehm Gmbh, 6100 Darmstadt|Process for the production of stabilized carrier-bound proteins|

---

JPS5722313B2|1979-01-05|1982-05-12|IL90600D0|1988-06-16|1990-01-18|Du Pont|Polynucleotide phosphorylase immobilized on epoxy-activated beads|

---

AU5943690A|1989-07-03|1991-01-17|E.I. Du Pont De Nemours And Company|Polynucleotide phosphorylase immobilized on trismethylacrylamide polymer beads|

---

US6277562B1|1993-01-26|2001-08-21|Robert-A. Ollar|Method for paraffinophilic isolation and identification from a body specimen|

---

KR100538502B1|1999-03-05|2005-12-23|미쯔비시 레이온 가부시끼가이샤|Carriers Having Biological Substance|

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US7968705B2|2003-01-27|2011-06-28|Dsm Ip Assets B.V.|Production of 5'-ribonucleotides|

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WO2004078342A1|2003-03-04|2004-09-16|Manac Inc.|Scavenger for substance having anionic substituent|

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AU2003902303A0|2003-05-14|2003-05-29|Protech Research Pty Ltd|Extracting and purifying enzymes|

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US20100056671A1|2007-04-12|2010-03-04|Designer Molecules, Inc.|Polyfunctional epoxy oligomers|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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DE19813136940|DE3136940A1|1981-09-17|1981-09-17|"METHOD FOR PRODUCING 5'-RIBONUCLEOTIDES"|

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