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Bacillus subtilis and application thereof in preparation of niacin by biocatalysis

A technology of Bacillus subtilis and nicotinic acid, which is applied in the field of Bacillus subtilis and its application in biocatalytic production of nicotinic acid, achieving the effect of large industrial application potential, high product purity and good selectivity

Inactive Publication Date: 2010-05-19
NANJING HUAZHOU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this technology is that the reaction also requires a high temperature and high pressure environment, which puts forward higher requirements on the safety performance and corrosion resistance of the equipment.
At the same time, the chemical reactions in near-critical aqueous media are still in the research and exploration stage, and there is still a considerable distance from large-scale industrial production.

Method used

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  • Bacillus subtilis and application thereof in preparation of niacin by biocatalysis
  • Bacillus subtilis and application thereof in preparation of niacin by biocatalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 strain screening

[0041] The Bacillus subtilis KR2 (CGMCC NO.3242) involved in the present invention is that the members of the biochemical industry research group of our company screen and obtain from the pesticide factory soil that has been polluted by nitrile compounds for more than 15 years, and its screening method is as follows:

[0042] More than 100 batches of sludge samples were taken from different angles of the sewage pool of Nanjing No. 1 Pesticide Factory, and screened and identified after enrichment and cultivation in batches:

[0043] Weigh 10 grams of sludge, add 50 mL of sterile water, shake it with glass beads, inoculate 5 mL of the suspension into 45 mL of enrichment medium, place on a shaker at 30°C, and shake at 120 rpm for 3 days. Afterwards, the culture solution was taken for gradient dilution, and 10 -6 、10 -7 、10 -8 Gradient dilutions were spread on the plate containing the first screening solid medium, and cultured in a constan...

Embodiment 2

[0049] The preparation of embodiment 2 free cells

[0050] Scrape 3 loops of Bacillus subtilis KR2 (CGMCC NO.3242) slant strains with an inoculation loop in a sterile ultra-clean workbench, and inoculate them into the primary liquid seed medium (glucose: 15, yeast extract: 5, peptone: 5. Sodium chloride: 2, potassium dihydrogen phosphate: 1, dipotassium hydrogen phosphate: 1, magnesium sulfate: 0.5, caprolactam: 5, isovaleronitrile: 1, cobalt chloride: 10ppm, pH: 7.0, unit: g / L). At a temperature of 25°C and a rotating speed of 150rpm, shake and cultivate for 48 hours to obtain the first-grade Bacillus subtilis KR2 (CGMCC NO.3242) seed liquid, which is transferred to a similar The culture is continued in the sterilized medium, and the secondary seed liquid of Bacillus subtilis KR2 (CGMCC NO.3242) can be obtained after 48 hours.

[0051] Inoculate the secondary seed solution of Bacillus subtilis KR2 (CGMCC NO.3242) into 35L sterilized liquid fermentation medium according to ...

Embodiment 3

[0052] Example 3 Enzyme Activity Determination

[0053] In this example, the fermentation broth of Bacillus subtilis CGMCC NO.3242 was used to measure the enzyme activity. Definition of enzyme activity: at 25°C, the number of micrograms of nicotinic acid produced after catalytic reaction of 1 mL of fermentation broth with 1 microgram of 3-cyanopyridine for 1 hour, which is the number of units of total enzyme activity. Nicotinic acid is detected by liquid chromatography, and detection parameters are: chromatographic column: CLC-ODS post, 150mm * 6.0mm I.D; Mobile phase: acetonitrile / water / sodium heptanesulfonate / triethylamine (50 / 450 / 0.8 g / 2ml) (pH3); flow rate: 1.2mL / min; column temperature: room temperature; detection wavelength: UV 268nm. Under the conditions of the present invention, the biomass per milliliter of fermented liquid can reach up to 47mg wet bacteria, and the total enzyme activity can reach up to 1050U.

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Abstract

The invention belongs to the field of biochemical engineering and discloses bacillus subtilis and application thereof in preparation of niacin by biocatalysis. The niacin is prepared by reacting 3-cyanopyridine serving as a substrate with bacillus subtilis KR2 (CGMCC NO.3242) serving as a biocatalyst at 5 to 35 DEG C and 100 to 300rpm for 15 to 96h, wherein the final yield of the niacin is over 90 percent and the purity of the obtained niacin is over 98 percent. The method of the invention is simple in operation and mild in reaction condition, and is suitable for application in scale industrial production.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a bacillus subtilis and its application in biocatalytic production of nicotinic acid. Background technique [0002] Nicotinic acid is also known as 3-pyridinecarboxylic acid, pyridine-3-carboxylic acid, nicotinic acid, etc., and it belongs to the B vitamins with niacinamide, collectively referred to as vitamin PP, molecular formula: C 6 h 5 NO 2 , heat resistance, can sublimate. This compound is a water-soluble vitamin, one of the 13 vitamins necessary for the human body; it is converted into nicotinamide in the human body, and is a component of coenzyme I and coenzyme II, and participates in lipid metabolism in the body, oxidation process of tissue respiration and sugar anaerobic decomposition process. It can be used as an anti-pellagra, as blood dilating drug, food and feed additive, etc.; it can also be used as a pharmaceutical intermediate for the production of isoniazid...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/12C12R1/125
Inventor 杨寿海孔国平杨玉林顾福海唐旺全
Owner NANJING HUAZHOU PHARMA
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