Bacillus subtilis and application thereof in biocatalysis production of niacinamide

A technology of Bacillus subtilis and biocatalysis, which is applied in Bacillus subtilis and its application field in the production of nicotinamide by biocatalysis, can solve the problems of reduced enzyme activity, reduced reaction speed, difficulty in obtaining high-concentration products, etc., and achieves maintenance Effects of activity, reduction of accumulation time, and reduction of toxic effects

Active Publication Date: 2012-01-11
ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the above traditional biocatalysis process is that the biocatalyst is added at one time. With the prolongation of the reaction time, the inhibition effect of the cells by the substrate and the product gradually increases, the reaction speed decreases, and the enzyme activity decreases. When the product concentration is selected, it is difficult to obtain a high-concentration product

Method used

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  • Bacillus subtilis and application thereof in biocatalysis production of niacinamide
  • Bacillus subtilis and application thereof in biocatalysis production of niacinamide
  • Bacillus subtilis and application thereof in biocatalysis production of niacinamide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Screening of Bacillus subtilis (Bacillus subtilis) CGMCC NO.3242

[0044] 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:

[0045] 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 constant temperature incubator at 30°C for 3 to 6 days. Pick the grown single colony and inoculate it on the new primary screening solid medium for expanded culture, and the culture conditions are the same as above. After the bacteria grow out, scrape 3 to 5 rings and inoculate the...

Embodiment 2

[0050] The preparation of embodiment 2 biocatalysts

[0051] Inoculate the slant strain of Bacillus subtilis KR2 (CGMCC No.3242) into the liquid seed medium (glucose: 15, yeast extract: 5, peptone: 5, sodium chloride: 2, potassium dihydrogen phosphate: 1, hydrogen phosphate Dipotassium: 1, magnesium sulfate: 0.5, urea: 10, caprolactam: 5, cobalt chloride: 10ppm, pH 7.0, unit: g / L). At a temperature of 25°C and a rotating speed of 150rpm, the first-grade seed liquid of Bacillus subtilis KR2 (CGMCC No.3242) can be obtained by shaking and culturing for 48 hours. 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.

[0052] Inoculate the secondary seed liquid of Bacillus subtilis KR2 (CGMCC No.3242) into 35L sterilized liquid fermentation medium according to the inoculum size of 10%, (glucose: 30, yeast extract: 10, peptone: 10, chlorinated Sodium: 2, potassium dihydrogen phosph...

Embodiment 3

[0054] Example 3 Enzyme Activity Determination

[0055] 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 nicotinamide produced after catalyzing the reaction of 1 mL of fermentation broth with 1 microgram of 3-cyanopyridine for 1 hour, the number of micrograms is the number of units of total enzyme activity. Niacinamide is detected by liquid chromatography, and the detection parameters are: chromatographic column: CLC-ODS column, 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: UV268nm. 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 1255U.

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Abstract

The invention belongs to the biochemical industry field and discloses a Bacillus subtilis and an application thereof in biocatalysis production of niacinamide. The nitrile hydratase produced by the Bacillus subtilis with CGMCC NO. 3342 can catalyze 3-cyanopyridine for hydration to generate niacinamide. The technology using Bacillus subtilis with CGMCC NO.3342 for biocatalysis production of niacinamide is used, and 3-cyanopyridine serves as a substrate, Bacillus subtilis with CGMCC NO. 3342 serves as a biocatalyst, and the mode of coupling and replenishing the substrate and the biocatalyst is adopted for catalytic hydration to obtain the niacinamide. The invention effectively lowers toxic action on cells by high-concentration substrates and products and is favorable for obtaining high-concentration niacinamide hydration liquid; the concentration of the niacinamide is not lower than 25%, which is favorable for product separation.

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 nicotinamide. Background technique [0002] Niacinamide common name VB 3 , also known as Niacinamide (Nicotinamide, Niacinamide), chemical name 3-pyridine carboxamide, pyridine-3-carboxamide, etc., molecular formula C 6 h 6 N 2 O, molecular weight 122.13, white needle crystal or powder, melting point 129-131°C, specific gravity 1.400. Soluble in water, soluble in ethanol and glycerin, insoluble in ether, odorless, slightly bitter, slightly toxic. [0003] Niacin and nicotinamide belong to B vitamins, collectively referred to as vitamin PP, which can be replaced in equal amounts in practical applications. Niacin in nature exists in liver, kidney, yeast and rice bran, and is a component of dehydrocoenzyme I or II in organisms , involved in the metabolism of carbohydrates, fats and proteins. They can be use...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/12C12R1/125
Inventor 杨寿海孔国平陈金福韦琛鸿赵广福
Owner ANHUI RUIBANG BIOLOGICAL SCI & TECH CO LTD
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