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Method for purifying oxidized beta-nicotinamide adenine dinucleotide

A technology of nicotinamide adenine and dinucleotide, which is applied in the field of purifying coenzymes, can solve the problems of low purity, limited production capacity, low yield, etc., achieve wide application prospects and reduce production costs

Active Publication Date: 2015-09-02
BONTAC INVITROLIFE BIO TECH SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art above, the object of the present invention is to provide a method for purifying oxidized β-nicotinamide adenine dinucleotide, aiming at solving the problem of the existing purified oxidized β-nicotinamide adenine dinucleotide. The process of glycosidic acid has the problems of low purity, low yield and limited production capacity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Sample treatment: Microfiltration and nanofiltration are carried out successively on the reaction liquid obtained from the enzyme-catalyzed reaction. The hollow fiber membrane with a molecular weight of 200 concentrates the filtrate to 30-50g / L, and collects the concentrate for future use.

[0043] 2. Purification:

[0044] Purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 5cm×30cm. Mobile phase: Phase A: 20mM buffered saline solution with pH 3 prepared with formic acid and sodium hydroxide; Phase B: ethanol. Flow rate: 50-80 mL / min. Detection wavelength: 260 nm. Gradient: B%: 3% to 15% (elution time 40 min). The injection volume is 10-15g.

[0045] Purification process: add phosphoric acid or hydrochloric acid to the concentrated sample filtrate to adjust the pH to 3-5, rinse the chromatographic column with more than 30% ethanol, ...

Embodiment 2

[0048] 1. Sample treatment: Microfiltration and nanofiltration are performed successively on the reaction liquid obtained from the enzyme-catalyzed reaction. A hollow fiber membrane with a molecular weight cut-off of 200 concentrates the filtrate to 30-50 g / L, and collects the concentrate for later use.

[0049] 2. Purification:

[0050] Purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 15cm×30cm. Mobile phase: Phase A: 20mM buffered saline solution with pH 4 prepared with formic acid and sodium hydroxide; Phase B: ethanol. Flow rate: 400-500 mL / min. Detection wavelength: 260 nm. Gradient: B%: 3% to 15% (elution time 40 min). The injection volume is 80-100g.

[0051] Purification process: Add phosphoric acid or hydrochloric acid to the concentrated solution to adjust the pH to 3-5, rinse the chromatographic column with more than 30% ethano...

Embodiment 3

[0054] 1. Sample treatment: microfiltration and nanofiltration are carried out successively on the reaction liquid obtained from the enzyme-catalyzed reaction. To remove microorganisms, nanofiltration adopts a hollow fiber membrane with a molecular weight cut-off of 200 to concentrate the filtrate to 30-50g / L, and collect the concentrated solution for later use.

[0055] 2. Purification:

[0056] Purification conditions: chromatographic column: a chromatographic column with octadecylsilane bonded silica gel as the stationary phase, and the diameter and length of the column are: 30cm×30cm. Mobile phase: Phase A: 20mM buffered saline solution with pH 5 prepared with formic acid and sodium hydroxide; Phase B: ethanol. Flow rate: 2500-3000 mL / min. Detection wavelength: 260 nm. Gradient: B%: 3% to 15% (elution time 40 min). The injection volume is 400-500g.

[0057] Purification process: Add 20mM tetramethylammonium hydroxide to the concentrated solution, rinse the chromatogra...

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Abstract

The invention discloses a method for purifying oxidized beta-nicotinamide adenine dinucleotide, which comprises the following steps: carrying out microfiltration and nanofiltration on a reaction solution obtained by enzyme catalysis reaction by using filter membranes, and collecting the concentrated solution for later use; adding acid into the concentrated filtrate to regulate the pH value of the concentrated solution, and carrying out gradient elution purification by using an inversed phase chromatographic column as a stationary phase, a buffer salt solution as a phase A and ethanol as a phase B; and carrying out nanofiltration concentration on the purified solution through a filter membrane, and carrying out freeze-drying with a vacuum freeze drier. By using the inversed phase high performance liquid chromatography to purify the oxidized beta-nicotinamide adenine dinucleotide, the oxidized beta-nicotinamide adenine dinucleotide has high purity and high yield, and achieves the industrialization requirements.

Description

technical field [0001] The invention relates to a method for purifying coenzymes, in particular to a method for purifying oxidized β-nicotinamide adenine dinucleotide. Background technique [0002] Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide NAD abbreviation, also known as diphosphate pyridine nucleotide (abbreviation DPN), or co-dehydrogenase (codehydrogenase) Ⅰ or coenzyme Ⅰ. NAD accepts a hydrogen atom and an electron from the substrate through various dehydrogenases to become reduced, and the pyridine ring is reduced, and this reaction can also proceed reversibly. Therefore, NAD+ can be used as a common substrate of various dehydrogenases, acting between two dehydrogenases, and the presence of a trace amount can catalyze the oxidation-reduction reaction (electron transfer) between the two substrates. NAD can be widely used as a raw material for chemical synthesis, and has a large market demand. [0003] The current mainstream purification proces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/207C07H1/06
CPCC07H19/207C07H21/02C07H1/06
Inventor 傅荣昭戴柱张琦
Owner BONTAC INVITROLIFE BIO TECH SHENZHEN CO LTD
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