WO1999024600A1 - Process for preparing sugar nucleotide - Google Patents

Abstract

A process for preparing a sugar nucleotide, characterized by phosphorylating a sugar with a kinase to prepare a sugar 1-phosphate and effecting chemical condensation between the sugar 1-phosphate and a nucleoside 5'-monophosphate. The target sugar nucleotide can be obtained in a high yield and can be isolated and purified readily.

Description

 Description Method for producing sugar nucleotides

Technical field

 The present invention relates to a novel method for producing sugar nucleotides, which are important substrates for sugar chain synthesis.

Background art

 In recent years, research on sugar chains has progressed rapidly, and as their functions have been elucidated, the development of applications of bioactive oligosaccharides as pharmaceuticals or functional materials has attracted attention. However, there are only a limited number of oligosaccharides on the market today, and they are extremely expensive. In addition, such oligosaccharides can be produced only at the reagent level, and a method for mass-producing them is not always established.

 Conventionally, oligosaccharides have been produced by extraction from natural products, chemical synthesis, or enzyme synthesis, or a combination of these methods.Enzyme synthesis is considered to be a suitable method for mass production. Have been. In other words, (1) the enzyme synthesis method does not require complicated procedures such as protection and deprotection found in the chemical synthesis method, and the desired oligosaccharide can be synthesized quickly. (2) Due to the substrate specificity of the enzyme, This method is considered to be advantageous over other methods in that it can synthesize oligosaccharides with extremely high structure specificity, etc. In addition, with the development of DNA recombination technology in recent years, various synthases can be produced at low cost and in large quantities. The ability to do so has further enhanced the advantages of enzyme synthesis.

As a method for synthesizing oligosaccharide by an enzyme synthesis method, two methods, a method using a reverse reaction of an oligosaccharide hydrolase and a method using a glycosyltransferase are considered. The former method is based on the fact that low-cost monosaccharides can be used as substrates. Despite the advantages mentioned above, the reaction itself utilizes the reverse reaction of the decomposition reaction, and is not necessarily considered to be the best method in terms of the synthesis yield and application to oligosaccharides with complex structures. .

 On the other hand, the latter is a synthesis method using a glycosyltransferase, and is considered to be more advantageous than the former method in terms of synthesis yield and application to oligosaccharides having a complicated structure. However, with the recent advances in DNA recombination technology, the mass production of various glycosyltransferases is also pushing the realization of this technology.

 However, sugar nucleotides, which are sugar donors used in glycosyltransferase-based synthetic methods, are still expensive except for some, and can be provided only in small quantities at reagent level. Is the current situation. Perysin diphosphate-galactose (UDP-Gai), a donor of galactose contained in many bioactive sugar chain cores, was also prepared using a yeast of the genus C andida (Proc. IV IFS: Ferment. Technol. Today) , p. 463 (1972), Agric, Biol. Chem., 37, 1741 (1973)), or a method of chemical synthesis (Adv. Carbohydr. Chem. Biochem., 28, 307 (1973)), Methods Enzymol. , 8, 136, (1966, J. Am. Chem. So, 83, 659 (1961)).

 When producing UDP-Ga using yeast of the genus Can dida, cultivation of the yeast cells and preparation of dried cells are indispensable, requiring a great deal of equipment and labor, and the production yield is not always satisfactory. It was not something. A method of preparing UDP-glucose using yeast and converting it to UDP-Ga ^ using epimerase has also been considered, but the conversion reaction using epimerase is an equilibrium reaction. However, UDP-Ga ^ and UDP-glucose are always mixed in the reaction solution, and it is extremely difficult to separate them.

On the other hand, when UDP-Ga ^ is produced by a chemical synthesis method, it is common to first synthesize galactose 1-phosphate and then chemically condense it with peridine 5'-monophosphate. When chemically synthesizing galactose 1-phosphate, the synthesis yield Not only low, but also it was extremely difficult to isolate and purify only galactose-monophosphate from the reaction solution. In addition, when low-purity galactose-monophosphate is used in the subsequent condensation reaction, the reaction does not proceed well, the synthesis yield of the resulting sugar nucleotide is low, and its isolation is extremely troublesome. Had disadvantages. Disclosure of the invention

 The present inventors have conducted research to find a simple and practical method of UDP-G a £. As a result of combining enzymatic reaction and chemical reaction, galactokinase synthesizes galactose monophosphate from galactose. And UDP-Ga are efficiently synthesized by a two-step process consisting of a reaction that chemically reacts the prepared galactose-monophosphate with peridine 5'-monophosphate (UMP). The present inventors have found that the method can be performed, and further confirmed that the method is not limited to the production of UDP-Ga ^ but can be applied to the production of other sugar nucleotides, thereby completing the present invention.

 That is, the present invention provides a method for phosphorylating a sugar using a kinase to prepare a sugar monophosphate, and converting a sugar nucleotide from the obtained sugar 1-phosphate and nucleoside 5′-monophosphate by a chemical condensation method. The present invention relates to a method for producing a sugar nucleotide, characterized by being prepared. BEST MODE FOR CARRYING OUT THE INVENTION

The kinase used in the present invention is not particularly limited as long as the sugar 1-phosphate can be prepared from a phosphate donor such as adenosine 5'-triphosphate (ATP) and a sugar. Illustrative examples include, but are not limited to, enzyme, glucuronokinase, galacturonokinase, arabinokinase, and fucokinase. The origin of the kinase is not limited to a specific origin such as animal origin, plant origin, or microbial origin, and any origin can be used. However, it is convenient to use an enzyme derived from a microorganism from the viewpoint of simplicity of enzyme preparation. In addition, simple preparation of various enzymes In order to enhance the preparation efficiency as well as the convenience, the enzyme production using the so-called recombinant DNA technique, in which the enzyme gene is cloned, expressed in a large amount in a microbial cell, and the enzyme is prepared in large quantities, is most efficient.

 The kinase added to the reaction system may be in any form as long as it has the activity. Specific examples include microbial cells, processed products of the microbial cells, enzyme preparations obtained from the processed products, and the like.

 The cells of the microorganism can be prepared by using a medium in which the microorganism can grow, culturing by a conventional method, and collecting cells by centrifugation or the like. To be more specific, using E. coli as an example, the culture medium may be broth medium, LB medium (1% tryptone, 0.5% first extract, 1% salt) or 2XYT medium (1.6% Triton, 1% yeast extract, 0.5% salt) can be used.After inoculating the culture medium with the inoculum, stir at 30 to 50 ° C for about 10 to 50 hours if necessary. Microbial cells having kinase activity can be prepared by centrifuging the obtained culture solution and collecting the microbial cells.

 Microbial cell processed products include mechanical destruction of the above microbial cells (using a Waring blender, French press, homogenizer, mortar, etc.), freeze-thawing, self-digestion, drying (freeze-drying, air-drying, etc.), enzymes Destruction of cells or denaturation of cell walls or cell membranes obtained by treatment according to general treatment methods such as treatment (eg, with lysozyme), ultrasonic treatment, or chemical treatment (eg, with acid or alkali). Can be exemplified.

 As the enzyme preparation, a fraction having the enzyme activity from the treated cells is purified by a conventional enzyme purification method (salt-out treatment, isoelectric point precipitation, organic solvent precipitation, dialysis, various types of chromatography) And the like).

Examples of the sugar used in the present invention include galactose, glucuronic acid, galacturonic acid, arabinose, fucose and the like depending on the target sugar nucleotide. These can use a commercial thing. The concentration used can be appropriately set within a range of about 1 to 200 mM, preferably about 10 to 10 OmM.

 ATP can be used as a phosphoric acid donor. ATP may also be a commercially available product, and the concentration used can be appropriately set in the range of about 1 to 20 OmM, preferably about 10 to 10 OmM, corresponding to the sugar concentration. Since ATP is generally expensive, an ATP regeneration system using polyphosphate kinase using polyphosphate as a phosphate donor can also be used.

 In the synthesis reaction of sugar monophosphate, inorganic ions corresponding to enzymes such as magnesium ion and zinc ion are added in the range of about 5 to 2 OmM in addition to the above kinase, sugar and ATP or ATP regeneration system, and the pH is adjusted to about pH. It can be carried out in a suitable buffer in the range of 5.0 to 9.0 at about 10 to 50 ° C, preferably in the range of 30 to 40 ° C.

 The sugar 1-phosphate thus obtained can be easily isolated and purified by ordinary isolation and purification means (eg, ion exchange chromatography, adsorption chromatography, salting out, etc.).

Next, the chemical condensation reaction between sugar 1-phosphate and nucleoside 5'-monophosphate may be carried out according to a known method. Specifically, a method using dicyclohexyl carpoimide (DCC) (Am. Chem. So, 80, 3756 (1958)), a morpholidate method (Am. Chem. So, 83, 659 (1961)) , Anion exchange method (Biochim. Biopys. Acta, 91, 1-13 (1964), Japanese Patent Publication No. 49-10674, Japanese Patent Publication No. 51-39227), etc. Among them, the anion exchange method is particularly preferable. The reaction conditions and the like for the condensation reaction may be performed according to the description in the above-mentioned literature. The sugar nucleotide thus obtained can be isolated and purified by ordinary isolation and purification means (eg, ion exchange chromatography, adsorption chromatography, salting out, etc.). Example Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1

 (1) Preparation of Escherichia coli galactokinase

 One platinum loop of the ga1K gene (Nucleic Acid) containing 1 Om ^ of 100 g / m £ ampicillin in 2 x YT (1.6% tryptone, 1% yeast extract, 0.5% salt) medium Res., 13, 1841 (1985)), and inoculated with Escherichia coli JM105 (obtained from Pharmacia) harboring pDR540 (Gene, 20, 231 (1982), obtained from Pharmacia). And cultured with shaking at 37 ° C for 16 hours. This was used as a precultured cell and inoculated into 50 Om of 2 x YT medium in a 200 Om ^ flask, and cultured with shaking at 30 ° C for 2 hours. Next, isopropyl-D-thiogalactobilanoside was added to a final concentration of 0.5 mM, and shaking culture was continued for further 5 hours at 30 ° C. The cells are collected from the culture by centrifugation, suspended in 5 Om ^ buffer (5 OmM Tris-HCl (pH 7.8), 1 mM EDTA), and then disrupted by sonication. Was crushed. The supernatant obtained by centrifugation was used as an enzyme solution.

 The recovered 5 Om crude enzyme solution contained 15 units / m of galactokinase activity, and the activity per protein was 1.3 units / mg. The unit of activity (unit) is measured and calculated by the following method.

 (Measurement of galactokinase activity and calculation of units)

 Add an appropriate amount of enzyme preparation to 100 mM Tris-HCl buffer (pH 7.8) containing 5 mM magnesium chloride, 1 OmM galactose, and 10 mM ATP.

After incubating at 37 ° C, the enzyme is inactivated by heat treatment at 100 ° C for 1 minute. Quantify the concentration of galactose 1-phosphate in the reaction solution using a DI ONEX sugar analyzer. Under these conditions, 1 micromol of galactose-monophosphate per minute The generated activity was defined as one unit.

 (2) Synthesis of galactose 1-phosphate (Ga _g— 1—P)

 Add about 1.0 ^ of deionized water to a 5.0-volume vial, add 96.91 g of tris (hydroxymethyl) aminoaminomethane, 50 OmM magnesium chloride solution 2 Om_g, 36.0 3 g Galactose and 29.7 g of ATP were added, and after dissolution, the mixture was adjusted to pH 7.9 with 6 N hydrochloric acid and filled up to 2.0 ^. After pre-incubation in a 37 ° C water bath, a galactokinase solution was added thereto to a concentration of 0.5 UZm ^ and incubated in a 37 ° C water bath. The reaction rate was confirmed with a Dionex sugar analyzer, and after 2 hours and 30 minutes, the enzyme reaction was stopped when the temperature reached 80 ° C. The denatured protein precipitate was filtered off by 3.0 βπι MF filtration to obtain 2.2 £ of an enzyme reaction solution.

 Next, 2.2_g of the enzyme reaction mixture was aliquoted by anion exchange resin column chromatography, washed with water and eluted with potassium acetate buffer (pH 4.7). The solution was added dropwise, adjusted to pH 7.0, and concentrated. Next, alcohol was added to the concentrated solution, followed by stirring to precipitate potassium galactose 1-phosphate. The crystals were separated by membrane filtration and dried in vacuo to obtain 41 g of potassium galactose-11-phosphate (purity 99.9%).

 (3) Synthesis of UDP—Ga ^

 Galactose 1-phosphate potassium salt was dissolved in water, and this was subjected to cation exchange resin column chromatography and washed with water. Tributylamine was added to the obtained passing and washing solution, neutralized by stirring, and then concentrated under reduced pressure. After drying, pyridine was added to redissolve and concentrated under reduced pressure. Further, pyridine was added again and redissolved to obtain a pyridine solution of Ga_g-1 -—- tri-η-butylamine.

To a solution of UMP tri-η-butylamine (8 Omm 0) in N, N-dimethylacetamide (40 m ^) was added triptylamine (101 mm 0 ^) and dioxane. After adding 60 μm of phosphoric acid chloride and reacting for 60 minutes, the P ¹ -peridine-5′P ² -diphenylpyrophosphate solution was added. Obtained. A pyridine solution of G a _g-1 -P-tri-n-butylamine salt was added to the solution, and the mixture was reacted at room temperature for 60 minutes. The reaction was stopped by adding water. After concentration under reduced pressure, ethyl acetate was added and stirred, and an aqueous layer was collected as a liquid separation and concentrated.

 (4) Purification of UDP-Ga ^

 The synthesis solution thus obtained was diluted, applied to an anion-exchange resin, Lamchromatography, washed with water, and eluted UDP-Ga ^ with a NaC solution. The obtained UDP-Ga fraction was adjusted to pH 3.0 by adding 1 N hydrochloric acid, passed through a granular activated carbon column, washed with water, and eluted with a NaOH solution. The fractions containing UDP-Ga ^ in the water washing and eluate were collected, adjusted to pH 6.2, and concentrated under reduced pressure. Alcohol was added to this solution to precipitate UDP-Ga ^. The supernatant was removed by decantation, and the precipitate was dissolved in water. After concentration under reduced pressure, the residue was freeze-dried to obtain 22.4 g of UDP-Ga_g.

Example 2

 (1) Cloning of Escherichia coli polyphosphate kinase gene

 Chromosomal DNA of Escherichia coli K12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). Using this DNA as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the Escherichia coli polyphosphate kinase (ppk) gene was amplified by PCR.

 Primer (A): 5'-TACCATGGGTCAGGAAAAGCTATA-3 '

 Primer (B): 5'-ATGGATCCTTATTCAGGTTGTTCGAGTGA-3 '

Amplification of the ppk gene by PCR is performed in a reaction mixture of 10 Om ^ (5 OmM chloride, 1 OmM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.01% gelatin, temperate 0.1 µg of DNA, 0.2 µM each of primer DNA (A) and (B), 2.5 units of Ampli Taq DNA polymerase) were added to Perkin—ElmerCetus Instrument Thermal denaturation (94 ° C, 1 minute), annealing (55 ° C, 1.5 minutes), polymerase (72 ° (1.5 minutes)) using DNA Thermal Cyc 1 er ) Was repeated 25 times.

 After gene amplification, the reaction solution was treated with a mixed solution of phenol Z-cloth form (1: 1), and two times the volume of ethanol was added to the water-soluble fraction to precipitate DNA. The DNA collected by precipitation was separated by agarose gel electrophoresis according to the method described in the literature (Molecular Cloning, supra), and a DNA fragment corresponding to 1.0 kb was purified. The DNA was digested with restriction enzymes NcoI and BamHI and ligated with plasmid pTrc99A (obtained from Pharmacia Biotech), also digested with restriction enzymes NcoI and BamHI, using T4 DNA ligase. . Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pTrc-PPK was isolated from the obtained ampicillin-resistant transformant. pTr c -PPK is obtained by inserting an Nco I -BamHI DNA fragment containing the E. coli ppk gene into the Nco I -BamHI cleavage site downstream of the "trc promoter of pTr c 99A.

 (2) Preparation of E. coli polyphosphate kinase

Escherichia coli JM109 carrying the plasmid pTrc-PPK was inoculated into 2Om ^ of a 2 x YT medium containing 100 µg of ampicillin and cultured with shaking at 37 ° C. Once at the 4 X 1 0 ⁸ bacteria Zm ^, was added I PTG to a final concentration of 1 mM to the culture solution was continued for an additional 5 hours with shaking cultured at 30 ° C. After completion of the culture, the cells were collected by centrifugation (9,000 xg, 10 minutes), and a 60-m buffer (50 mM Tris-HCl (ρΗ7.5), 5 mM EDTA, 0.1% Triton X-100, 0.2 mgZm lysozyme). After incubating at 37 ° C for 1 hour, the cells were sonicated to disrupt the cells, and centrifuged (20,000 xg, 10 minutes) to remove the cell residues. The supernatant fraction thus obtained was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 5 mM magnesium chloride and 1 mM 2-mercaptoethanol to obtain a crude enzyme solution. The specific activity of polyphosphate kinase in the crude enzyme solution is 0.19 units / mg protein, which is the specific activity of the control bacterium (Escherichia coli JM109 carrying pTrc99A).

(0.00000 unit mg protein). Next, the crude enzyme solution was fractionated using DEAE Toyopearl 65 M (Tosoichi Co., Ltd.) with a concentration gradient of 0 to 0.5 M NaCp to obtain a polyphosphate kinase fraction. This fraction was used as a polyphosphate kinase enzyme preparation. The specific activity of polyphosphate kinase in this enzyme preparation was 0.6 unit / mg protein.

 (3) Cloning of E. coli adenylate tokinase

 Chromosomal DNA of Escherichia coli 12 strain JM109 (obtained from Takara Shuzo Co., Ltd.) was prepared by the method of Saito and Miura (Biochim. Biophys. Acta., 72, 619 (1963)). Using this DNA as a template, the following two primer DNAs were synthesized according to a conventional method, and the E. coli adenylate tokinase (adk) gene was amplified by PCR.

 Primer (C): 5'-ATGGATCCCGTTTCAGCCCCAGGTGCC-3 '

 Primer (D): 5'-ATAAGCTTGGCCTGAGATTGCTGATAAG-3 '

The amplification of the adk gene by PCR was performed in a reaction mixture of 1 O Om ^ (50 mM chloride, 1 OmM tris-hydrochloride (ρΗ8.3), 1.5 mM magnesium chloride, 0.01% gelatin, Belate DNA 0.1 g, Primer DNA (C) (D) 0.1 M each, Ampli Taq DNA polymerase 2.5 units> ¾rPerkin—ElmerCetυS I Thermal denaturation (94 ° C, 1 minute), annealing (56 ° C, 1.0 minute), polymerase (72 ° C) using DNA Thermal Cyc 1 er manufactured by nstrument (C, 3.0 minutes) was repeated 25 times.After gene amplification, the reaction solution was treated with a mixture of phenol Z-cloth form (1: 1) and doubled to the water-soluble fraction. The precipitated DNA was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and the DNA was precipitated. A DNA fragment corresponding to kb was purified. The DNA was cleaved with restriction enzymes BamHI and Hind1, and plasmid pUC18 (obtained from Takara Shuzo Co., Ltd.) and T4DNA ligase digested with restriction enzymes BamHI and Hindill were also used. Connected. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pUC-ADK was isolated from the resulting ampicillin-resistant transformant. pUC-ADK is obtained by inserting a BamHI-HindI DNA fragment containing the E. coli adk gene into the BamHI-HindIE cleavage site downstream of the 1 ac promoter of pUC18.

 (4) Preparation of E. coli adenylate kinase

Escherichia coli JM109 carrying plasmid pUC-ADK was inoculated into 30 Om ^ of 2 x YT medium containing 100 µg of ampicillin, and cultured at 37 ° C with shaking. When the culture reached 4 × 10 ⁸ Zm, IPTG was added to the culture solution to a final concentration of ImM, and shaking culture was further continued at 30 ° C. for 5 hours. After completion of the culture, the cells were collected by centrifugation (9,000 xg, 10 minutes), and a 60-m buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% Triton X—100 , 0.2 mg Zm ^ lysozyme). After incubating for 1 hour at 37, the cells were sonicated to disrupt the cells, and centrifuged (20,000 xg, 10 minutes) to remove cell residues.

The supernatant fraction thus obtained was dialyzed against 5 OmM Tris-HCl (pH 7.8) containing 5 mM magnesium chloride and ImM2-mercaptoethanol to obtain a crude enzyme solution. The specific activity of adenylate kinase in the crude enzyme solution is 134 units Zmg protein, which is about the same as the specific activity (1.9 units / mg protein) of the control bacterium (Escherichia coli JM109 carrying pUC18). It was 85 times. Next, the crude enzyme solution was fractionated using DEAE Toyopearl 650 M (Tosoichi Co., Ltd.) with a concentration gradient of 0.5 to 0.5 M Na, and a fraction having adenylate kinase activity was collected. This fraction was used as an adenylate kinase enzyme preparation. In addition, this enzyme preparation The specific activity of polyphosphate kinase in was 344 units / mg protein.c

(5) ATP regeneration by polyphosphate kinase and adenylate kinase and synthesis of galactose 1-phosphate by galactokinase

 0.1 unit / polyphosphoric acid in 100 mM Tris-HCl buffer (pH 7.8) containing 1 OmM magnesium chloride, 10 OmM ammonium sulfate, polyphosphoric acid (75 mM as inorganic phosphoric acid) and 4 mM AMP Acid kinase and 2.5 units of Zm ^ T denylate kinase enzyme preparation were added, and the mixture was incubated at 37 ° C for 120 minutes. The nucleotide concentrations in the reaction solution at the end of the AMP phosphorylation reaction were ATP 1.4 m, ADP 1.7 mM, and AMP 0.9 mM. To this reaction solution, D (+) galactose was added to a final concentration of 4 OmM, and galactokinase was further added to 1.0 unit /, followed by incubation at 37 ° C for 4.5 hours. When the reaction-terminated liquid was analyzed using a sugar analyzer (Dionex), the production of 28.4 mM galactose 1-phosphate was confirmed.

 (6) UDP-Ga synthesis

 The thus prepared galactose monophosphate was treated in the same manner as in Example 1 to prepare UDP-Ga. Industrial applicability

 By synthesizing sugar 1-phosphate by an enzyme reaction using a kinase as in the method of the present invention, the desired sugar 1-monophosphate can be prepared simply and in good yield, and Sugar monophosphate can be easily isolated and purified by simple means, and high purity sugar 1-phosphate can be obtained. When a chemical condensation reaction is performed using such high-purity sugar 1-phosphate, the target sugar nucleotide can be obtained in good yield, and the obtained sugar nucleotide can be easily isolated and purified. .

 Therefore, the method of the present invention can be said to be a practical and practical method for sugar nucleotides.

Claims

 Scope of the request. Sugar is phosphorylated using a kinase to prepare sugar monophosphate, and then the sugar nucleotide is chemically condensed from the obtained sugar monophosphate and nucleoside 5'-monophosphate. Or a method for producing a sugar nucleotide. Kinase is galactokinase, sugar is galactose, sugar 1-phosphate is galactose 1-phosphate, nucleoside 5'-monophosphate is peridine 5'-monophosphate, and sugar nucleotide is peridine 2. The method according to claim 1, which is mono-galactose diphosphate. The step of preparing a sugar monophosphate by phosphorylating a sugar using a kinase uses an ATP regeneration system using a polyphosphate kinase using polyphosphate as a phosphate donor. The method of paragraph 1. The method according to claim 1, wherein the chemical condensation method is a method selected from a method using dicyclohexylcarpoimide, a morpholidate method and an anion exchange method.