Production method of oxidized nicotinamide adenine dinucleotide phosphate

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

Active Publication Date: 2018-07-24
HEZE RUIZHI TECH DEV
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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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  • 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 and particularly relates to a production method of oxidized nicotinamide adenine dinucleotide phosphate. The production method comprises the following steps of by taking bacillus subtilis-bacillus megaterium as a raw material, performing resuspension on bacillus subtilis-bacillus megaterium with deionized water, performing crushing, performing flocculation with a flocculant, performing filtering, and collecting filtrate; removing protein in the filtrate with an ultrafiltration membrane to obtain a product I; separating the product I with cationic resin by two steps near an isoelectric point, and performing desalting with a nanofiltration membrane to obtain a product II; dropwise adding an organic solvent in the product II,performing low temperature crystallization, and drying crystals at low temperature to obtain product III. The production method has the beneficial effects that the production method fills up the technology gap in domestic production and meets the demands on industrial production, scientific research and the like at different levels, and the production cost is effectively controlled; and by utilizing the amphoterism characteristic of oxidized NADP+ molecules, precision extraction separation is performed on the product near the isoelectric point by utilizing two different kinds of cationic resin.

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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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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