Bacillus subtilis strain and application thereof in synthesis of iminodiacetic acid

A technology of Bacillus subtilis and iminodiacetic acid, which is applied in the application field of biocatalytic synthesis of iminodiacetic acid, can solve the problems of low product concentration, difficult separation of substrate and product, and low substrate solubility, and achieve product Effects of high purity, enhanced solubility, and high product concentration

Inactive Publication Date: 2012-05-30
NANJING HUAZHOU PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the chemical hydrolysis method is the current mainstream process, this route needs to consume a large amount of strong acid and strong alkali, and also requires high temperature, high pressure and other reaction conditions. There are many by-products, low output, large amount of waste water, and serious environmental pollution.
[0008] Recently, there are also reports on the synthesis of iminodiacetic acid by biocatalysis, but they all react in the aqueous phase. Although many shortcomings of the chemical method are overcome, there are also poor mass transfer effects caused by low substrate solubility and low product concentration. The separation of substrate and product is difficult, the utilization rate of the device is not high, and it is difficult to industrialize and other unfavorable factors

Method used

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  • Bacillus subtilis strain and application thereof in synthesis of iminodiacetic acid
  • Bacillus subtilis strain and application thereof in synthesis of iminodiacetic acid
  • Bacillus subtilis strain and application thereof in synthesis of iminodiacetic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1 strain screening

[0047] The screening method of bacterial classification among the present invention is as follows:

[0048] 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:

[0049] 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...

Embodiment 2

[0055] The preparation of embodiment 2 free cells

[0056] 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, isovaleric acid: 0.1~5, caprolactam: 5, cobalt chloride: 10ppm, pH: 7.0, unit : g / L). At a temperature of 25°C and a rotating speed of 150rpm, the seed liquid of the first-grade Bacillus subtilis KR2 (CGMCC No.3242) can be obtained by shaking and culturing for 48 hours, and the first-grade seed liquid is transferred to the same 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.

[0057] Inoculate the secondary seed liquid of Bacillus subtilis KR2 (CGMCC No.3242) into 35L steril...

Embodiment 3

[0058] Example 3 Determination of Enzyme Activity

[0059] 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 iminodiacetic acid produced after catalytic reaction of 1 mL of fermentation broth with 1 microgram of iminodiacetonitrile for 1 hour, which is the number of units of total enzyme activity. Iminodiacetic acid is detected by liquid chromatography, and the detection parameters are: Spherisorb SAX strong anion exchange column, 150mm × 4.6mm I.D; mobile phase: 0.005mol / L potassium dihydrogen phosphate buffer solution (pH2.5); flow rate : 1.0mL / min; column temperature: room temperature; detection wavelength: UV 210nm. 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 560U.

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Abstract

The invention discloses a bacillus subtilis strain and an application thereof in synthesis of iminodiacetic acid, which belongs to the field of biochemical industry. The application method comprises the following steps: in homogeneous solvent system comprising water and water soluble solvent, iminodiacetic acid is obtained after reaction for 15-50 hours, with iminodiacetonitrile as the substrate and flocculent cell sap of bacillus subtilis KR2 (CGMCC NO. 3242) as the biocatalyst, at a temperature of 5-35 DEG C, and at a stirring speed of 100-300 rpm. The purity of the finished product of the technique is above 96 percent, and the yield rate is over 90.0 percent. The method has the advantages that the operation is simple, the reaction condition is mild, the wastewater amount is small, and the technique is suitable for large-scale industrial production and application.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a new bacterial strain—Bacillus subtilis (Bacillus subtilis) KR2, and the application of the bacterial strain in biocatalytic synthesis of iminodiacetic acid. Background technique [0002] Iminodiacetonitrile: NH(CH 2 EN) 2 , Soluble in organic solvents such as acetone, mainly used in the synthesis of herbicide glyphosate. In addition, as an important fine chemical intermediate, it also has certain applications in the fields of dyes, electroplating, water treatment, and synthetic resins. [0003] Iminodiacetic acid: NH(CH 2 COOH) 2 , specific gravity 1.56, soluble in water, hardly soluble in alcohol, acetone and ether, melting point 247.5°C (decomposition), forms salts with acids and bases, and forms chelates with various metals. Iminodiacetic acid, also known as aminodipic acid, or IDA for short, is an important fine chemical product, mainly used in the manufacture of glyp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/04C12R1/125
Inventor 杨寿海孔国平王红明刘善和葛九敢
Owner NANJING HUAZHOU PHARMA
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