A kind of separation and purification method of deoxynucleoside triphosphate

Abstract

The invention discloses a separation and purification method for deoxynucleoside triphosphate. When the temperature is below 4°C, the deoxynucleoside triphosphate biosynthesis reaction solution is directly settled with an organic solvent, and the deoxynucleoside triphosphate biosynthesis reaction solution is mixed with the deoxynucleoside triphosphate biosynthesis reaction solution. The volume ratio of the organic solvent is 1:1 to 1:25 to obtain a solid mixture of deoxynucleoside triphosphate; when the temperature is below 4°C, dissolve the solid mixture with ultrapure water, and adjust the pH value to 0.1 to 0.1 with an acidic aqueous solution. 2. Re-precipitate with an organic solvent to obtain a solid deoxynucleoside triphosphate; then dissolve the solid in ultrapure water, adjust the pH to 5-11 with an alkaline aqueous solution of sodium hydroxide, and freeze in liquid nitrogen And carry out freeze-drying to obtain deoxynucleoside triphosphate sodium salt product. The invention has the advantages of rapid separation, better purification effect, lower production cost, less water resource pollution and process energy consumption, and can be used in industrialized production.

Description

Technical field

[0001] The invention relates to the field of biochemical industry, in particular to a method for separating and purifying widely used deoxynucleoside triphosphate (dNTP) enzymatically synthesized products.

Background technique

[0002] Usually, the biosynthesis reaction liquid of deoxynucleoside triphosphate (dNTP) is synthesized from deoxynucleoside monophosphate (dNMP), and the dNTP generated is separated in the form of barium salt by sedimentation operation, such as using barium bromide aqueous solution to make dNTP from Sedimentation separation separated from the reaction solution, and then use ion exchange resin to elute dNTP, making it a separation and purification method for repeated sedimentation and elution of dNTP aqueous solution, or directly elute the dNTP biosynthesis reaction solution with ion exchange resin packed column , and then use high-pressure liquid chromatography to separate and purify the method.

[0003] The existing method for s...

Examples

Embodiment 1

[0018] Separation and purification of adenine triphosphate deoxyribonucleoside dATP:

[0019] Cool 50ml of the enzyme synthesis reaction solution containing about dATP100-mM with a pH value of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC is regularly performed to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask. Two volumes of absolute ethanol (about 200 mL) was slowly added in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dATP peak was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value below 2.0 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully we...

Embodiment 2

[0021] Separation and purification of guanine triphosphate deoxyribonucleoside dGTP:

[0022] Cool the 50mL enzyme synthesis reaction solution containing about 20-mM dGTP at a pH of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dGMP, dGTP, dGDP and pyruvate), and transferred to a 1L three-necked flask , slowly added isopropanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dGTP peak was shown. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 1.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed and dissolved in water (~...

Embodiment 3

[0024] Separation and purification of cytosine triphosphate deoxyribonucleoside dCTP:

[0025] Cool 100mL enzyme synthesis reaction solution with a pH value of 7.0 to 8.0 containing about dCTP 10-mM to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask , slowly added absolute ethanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no peak shape of dCTP was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 0.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed a...

Technical field

[0001] The invention relates to the field of biochemical industry, in particular to a method for separating and purifying widely used deoxynucleoside triphosphate (dNTP) enzymatically synthesized products.

Background technique

[0002] Usually, the biosynthesis reaction liquid of deoxynucleoside triphosphate (dNTP) is synthesized from deoxynucleoside monophosphate (dNMP), and the dNTP generated is separated in the form of barium salt by sedimentation operation, such as using barium bromide aqueous solution to make dNTP from Sedimentation separation separated from the reaction solution, and then use ion exchange resin to elute dNTP, making it a separation and purification method for repeated sedimentation and elution of dNTP aqueous solution, or directly elute the dNTP biosynthesis reaction solution with ion exchange resin packed column , and then use high-pressure liquid chromatography to separate and purify the method.

[0003] The existing method for s...

Examples

Embodiment 1

[0018] Separation and purification of adenine triphosphate deoxyribonucleoside dATP:

[0019] Cool 50ml of the enzyme synthesis reaction solution containing about dATP100-mM with a pH value of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC is regularly performed to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask. Two volumes of absolute ethanol (about 200 mL) was slowly added in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dATP peak was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value below 2.0 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully we...

Embodiment 2

[0021] Separation and purification of guanine triphosphate deoxyribonucleoside dGTP:

[0022] Cool the 50mL enzyme synthesis reaction solution containing about 20-mM dGTP at a pH of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dGMP, dGTP, dGDP and pyruvate), and transferred to a 1L three-necked flask , slowly added isopropanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dGTP peak was shown. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 1.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed and dissolved in water (~...

Embodiment 3

[0024] Separation and purification of cytosine triphosphate deoxyribonucleoside dCTP:

[0025] Cool 100mL enzyme synthesis reaction solution with a pH value of 7.0 to 8.0 containing about dCTP 10-mM to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask , slowly added absolute ethanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no peak shape of dCTP was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 0.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed a...