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A kind of production method of oxidized nicotinamide adenine dinucleotide phosphate

A nicotinamide adenine and dinucleotide technology, applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low content, low product yield, complicated procedures, etc. The process is simple, the separation purity is high, and the cost of product use is minimized.

Active Publication Date: 2021-10-12
HEZE RUIZHI TECH DEV
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  • Abstract
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Problems solved by technology

Coenzyme II exists widely in organisms, but its content is extremely low. Coenzyme II extracted from organisms exists in the form of oxidation, and the market price per kilogram is tens of thousands of yuan. In industrial production, oxidized products, namely NADP+, and reduced ones are used. Product is more expensive
The literature reports on NADP+ / NADPH-related production technology have been blank in China, and only one literature report on the extraction method of coenzyme Ⅰ (NAD+) published by the Shanghai Institute of Biochemistry and other units of the Chinese Academy of Sciences in 1978 was found. The coenzyme I extraction process route provided is: hot and cold breaking of yeast cells, centrifugation to collect the supernatant, anion resin removal of impurities, weak acid cation resin adsorption, desorption, strong acid cation resin removal of impurities, strong alkaline anion resin adsorption, desorption Activated carbon adsorption, water / organic solvent washing, desorption, crystallization, and drying. This process uses 4 steps of resin treatment and 1 step of activated carbon adsorption and desorption before and after the process. The process is very complicated, the product yield is low, and the wastewater generated by adsorption, desorption and regeneration Will bring great pressure to environmental protection
[0005] The publication number is: 104876993A, and the patent titled "A Purification Method for Oxidized β-Nicotinamide Adenine Dinucleotide Phosphate" only provides the part of the purification method in the NADP+ production technology, and the reverse phase chromatography resin used Expensive, unaffordable for general enterprises

Method used

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  • A kind of production method of oxidized nicotinamide adenine dinucleotide phosphate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Propagate with Bacillus megaterium or Bacillus subtilis, insert 5% of the inoculum into 30L of TB medium sterilized at 121°C for 20 minutes, use ammonia water to control pH7.0 throughout the process; initial speed 300 rpm Minutes, 600 rpm in the middle and late stages; initial flow rate 1:0.6, 1:1.2 in the middle and late stages; tank pressure 0.05MPa; after the dissolved oxygen rebounds, start to add 80% glycerin solution at a rate of 165ml / h; when OD600 ≥ 60 , the temperature was lowered from 37°C to 30°C, and the culture period was 42h. The OD600 of the tank is 98, and the volume of the tank is 34.2L.

[0033] (2) The NADP+ content of the fermentation broth was detected by HPLC to be 256mg / L.

[0034] (3) The fermentation broth was centrifuged at 8000 rpm to collect 6.82kg of bacterial cells.

[0035] (4) The bacteria were resuspended in 24L of deionized water, and homogeneously crushed twice under 60MPa pressure.

[0036] (5) Add 6.4g of chitosan to the crush...

Embodiment 2

[0039] (1) Product I in Example 1 was concentrated to 1.46L with a 300-600Da nanofiltration membrane.

[0040](2) Add 50g of strong acid cationic resin to the concentrated solution, the resin is Na+ type, adjust the pH to 2.8-3.0 with hydrochloric acid, stir for 0.5-4h, and adjust the pH at any time to keep it within the range of 2.8-3.0.

[0041] (3) Filtrate, collect 1.42L of filtrate, lower the pH to 1.5.0-2.0 with hydrochloric acid, put on a weakly acidic cationic resin column Φ35*350, the resin loading capacity is 150ml, the resin is pre-transformed into H+ type, and the sample loading flow rate is 2BV / h; After loading the column, rinse with 2BV deionized water.

[0042] (4) Desorb with 0.2M sodium hydroxide solution, and intercept 345ml of the intermediate desorption solution according to the detection index.

[0043] (5) The desorption liquid is adjusted to PH6.5 with hydrochloric acid, and the above-mentioned desorption liquid is desalted and concentrated with a nanof...

Embodiment 3

[0045] (1) Add 300ml of acetone dropwise to the product II in step (5) of Example 2 for 0.5h.

[0046] (2) After the dropwise addition, crystallize at -5--5°C for 6 hours.

[0047] (3) Vacuum filter with a Buchner funnel, top wash with 50ml of acetone, and collect the solid.

[0048] (4) The solid was vacuum-dried at ≤50°C for 4 hours to obtain 6.86 g of solid, namely product III. The content of NADP+ sodium salt was detected by HPLC to be 99.4%. Yield 88.6%.

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Abstract

The invention belongs to the field of biological product extraction, in particular to a production method of oxidized nicotinamide adenine dinucleotide phosphate. This method uses Bacillus subtilis¸ Bacillus megaterium as raw material, resuspended with deionized water, crushed, added flocculant to flocculate, and filtered to collect the filtrate; after the filtrate was deproteinized by an ultrafiltration membrane, product I was obtained; After two-step cationic resin separation near the electric point and desalination with nanofiltration membrane, product II is obtained; organic solvent is added dropwise to product II, crystallized at low temperature, and crystal is dried at low temperature to obtain product III. The beneficial effects of the present invention are: the production method of the present invention fills the blank of domestic production technology; meets the needs of different levels of industrial production and scientific research, and effectively controls the production cost; utilizes the amphoteric characteristics of oxidized NADP+ molecules, Using two different cationic resins, the products were precisely extracted and separated.

Description

(1) Technical field [0001] The invention belongs to the field of biological product extraction, in particular to a production method of oxidized nicotinamide adenine dinucleotide phosphate. (2) Background technology [0002] Nicotinamide adenine dinucleotide phosphate, also known as coenzyme II, is divided into oxidized and reduced forms, generally abbreviated as NADP+ / NADPH, and the molecular structure is shown in the appendix figure 1 . Nicotinamide adenine dinucleotide phosphate is an important coenzyme in cells, which mainly participates in cell anabolism and metabolism, such as most of the redox reactions in the metabolism of sugar, lipid and protein. It is the most abundant oxidation reaction in the microbial metabolic network. One of the reducing coenzymes, it plays an important role in the metabolism of all organisms; secondly, nicotinamide adenine dinucleotide phosphate is an important component of the intracellular antioxidant defense system, which plays a role in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/36C07H1/06C07H19/207C12R1/125C12R1/11
CPCC07H1/06C07H19/207C12P19/36
Inventor 彭继先于海勤刘艳平孟中英乔久生李辉吴桂平
Owner HEZE RUIZHI TECH DEV
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