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Method for preparing beta-nicotinamide mononucleotide through immobilized whole-cell catalysis by taking modified diatomite as carrier

An immobilized cell and single nucleotide technology is applied in the field of immobilized whole cells to catalyze the preparation of β-nicotinamide mononucleotides, which can solve the problems of poor mechanical strength of immobilized whole cells, fewer cycles of use, and high cost of adsorption carriers. problems, to achieve the effects of being not easily degraded by microorganisms, increasing the number of cycles, and not easily falling off cell adsorption

Pending Publication Date: 2022-04-12
INNER MONGOLIA KINGDOMWAY PHARMA LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the invention is to address the defects of the current chemical synthesis method, such as large environmental hazards, high production costs, and complicated preparation processes, as well as problems such as poor overall mechanical strength of immobilized whole cells, fewer cycles, and high cost of adsorption carriers.

Method used

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  • Method for preparing beta-nicotinamide mononucleotide through immobilized whole-cell catalysis by taking modified diatomite as carrier
  • Method for preparing beta-nicotinamide mononucleotide through immobilized whole-cell catalysis by taking modified diatomite as carrier
  • Method for preparing beta-nicotinamide mononucleotide through immobilized whole-cell catalysis by taking modified diatomite as carrier

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Preparation of fermentation broth

[0045] (1) Strain activation: In a sterile environment, the production strains (Pseudomonas stutzeri) of polyphosphokinase, ribulose-5-phosphate isomerase, phosphoribosyl pyrophosphate synthase and nicotinamide phosphoribosyltransferase bacteria, Penicillium chrysogenum, Bacillus amyloliquefaciens and Pichia pastoris) were added to the plate medium containing 15-25mg / L kanamycin (recipe: tryptone 10g / L, yeast extract 5g) according to the gradient dilution method. / L, sodium chloride 10g / L, agar 12g / L, kanamycin 15-25mg / L, pH=7.0), cultured at 35°C for 20h, and activated.

[0046] (2) Seed liquid preparation: pick any single colony from the cultured plate, and then insert it into the medium containing the 3% inoculum (recipe: tryptone 10g / L, yeast extract 5g / L) , sodium chloride 10g / L, agar 12g / L, pH=7.0) in a shaker flask, put it into a shaker, and cultivate for 15h at 35°C and 180r / min. Then, 3% of the inoculum is insert...

Embodiment 2

[0048] Example 2 Diatomite modification treatment

[0049] Acid treatment: prepare 15% hydrochloric acid solution and diatomite (the diatomite used is commercially available FS-500 industrial grade, see the scanning electron microscope picture of the surface structure of diatomite particles figure 1 c) Mix it according to the ratio of 5mL:1g, stir well and soak for 3h. Then filter, rinse the filter cake with ultrapure water until the filtrate is neutral, dry at 110°C and pass through an 80-mesh sieve for use. The SEM image of the surface structure of the obtained diatomite particles is shown in figure 1 the a.

[0050] Alkali treatment: configure 10% sodium hydroxide solution, mix it with the filtrate after drying according to the ratio of 12mL:4g, and soak for 3h. Then filter, rinse the filter cake with ultrapure water until the filtrate is neutral, dry at 110° C. and pass through a 100-mesh sieve to obtain modified diatomite. The SEM image of the surface structure of the...

Embodiment 3

[0056] Example 3 Preparation of immobilized cells

[0057] The bacterial strain fermentation broth obtained in Example 1 was centrifuged for 5 min under the condition of 5000 r / min, respectively, and the lower bacterial sludge was collected, washed with an appropriate amount of deionized water, and centrifuged for 6 min under the same conditions to remove residual bacteria in the bacterial sludge. Fermentation broth and other impurities. After centrifugation, the wet bacterial mud and 0.6% NaCl solution were mixed uniformly in proportion to prepare bacterial suspensions with a concentration of 25% (v / v). Add polyethyleneimine (PEI) solution and stir for 30-50min, then add glutaraldehyde solution, stir and crosslink for 30min. Finally, according to the dry matter content of bacteria mud and the mass ratio of diatomite, it is 1g: 0.5g The modified diatomite obtained in Example 2 was added to carry out adsorption treatment, and after standing for 1-2h, 8-15mM / L MgCl was used 2 ...

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Abstract

The invention discloses a method for preparing beta-nicotinamide mononucleotide through immobilized whole-cell catalysis by taking modified diatomite as a carrier. Modified diatomite subjected to acid and alkali treatment is used as an immobilization carrier, strains respectively containing polyphosphate kinase (Ppk), ribulose-5-phosphate isomerase (RKI1), ribose phosphate pyrophosphate synthase (Prps) and nicotinamide ribose phosphate transferase (Nampt) are used as objects to prepare immobilized cells, and then D-ribose, ATP and nicotinamide are used as raw materials to prepare the immobilized cells. The combination of four enzymes including polyphosphate kinase, ribulose-5-phosphate isomerase, ribose phosphate pyrophosphate synthase and nicotinamide ribose phosphate transferase is added for catalytic reaction synthesis of beta-nicotinamide mononucleotide (beta-NMN), and the conversion rate is high. Not only are the efficiency of synthesizing beta-NMN by an enzyme method and the substrate conversion rate improved, but also the immobilized cells can be repeatedly used for multiple times.

Description

technical field [0001] The invention relates to the field of cell technology and biocatalysis, in particular to a method for preparing beta-nicotinamide mononucleotide by catalysis of immobilized whole cells using modified diatomite as a carrier. Background technique [0002] β-Nicotinamide mononucleotide (β-NMN) is the product of phosphoribosyl pyrophosphate and nicotinamide catalyzed by nicotinamide phosphoribosyltransferase. It plays an important role in the body and is also NAD. + one of the key precursors. The study found that when the NAD in mice + When levels were increased, signs of aging were reversed in some tissues and muscles in older mice, suggesting that NAD + It has certain effects in delaying aging and maintaining muscle activity. But due to NAD + The molecular weight is too large to be taken into cells orally, and it mainly depends on the self-synthesis of cells in the body, and the synthesis amount is very low. But with the NAD + Research on the precu...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N1/14C12N1/20C12P19/30C01B25/42C01B33/12C01G9/06C12R1/38C12R1/82C12R1/07C12R1/84
Inventor 王东王炳荣陈忠发徐鲁明吴轶
Owner INNER MONGOLIA KINGDOMWAY PHARMA LTD
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