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Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes

A technology for catalyzing iminodiacetonitrile and nitrilase catalyzing iminodiacetonitrile is applied in the field of strains with nitrilase activity, and can solve the problems of high equipment requirements, harsh reaction conditions and high energy consumption, and achieve the effect of efficient production

Active Publication Date: 2010-01-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical hydrolysis method needs to be carried out at about 100°C, and must be equipped with heating and temperature control devices. The reaction conditions are harsh, the energy consumption is high, and the equipment requirements are high.

Method used

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  • Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes
  • Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes
  • Method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the initial screening of producing nitrilase bacterial classification

[0037] Prepare the primary screening medium per 1L as follows: yeast extract 6g, NaCl 1g, K 2 HPO 4 1g, MgSO 4 1g, 20g of agar powder, pH 7.0 water to make up to 1000mL, after sterilization, cool to 50°C, add 5g of iminodiacetonitrile and 1g of bromocresol violet, and mix well to the plate.

[0038] Collect the bacteria for screening, smear them on a plate, and culture them at a temperature of 28-30°C for 3-4 days, pick the strains that produce yellow discoloration circles in the colonies, and inoculate them on LB slopes (25-28°C);

[0039] The microbial strains in the yellow color circle are: Pseudomonas marginalis ZJB09121 (Pseudomonas marginalis ZJB09121), Pseudomonas putia ZJB09134 (Pseudomonas putia ZJB09134), Pseudomonas putia ZJB09129 (Pseudomonas putia ZJB09129), Pseudomonas putia ZJB09129 (Pseudomonas putia ZJB09129), Pseudomonas putia ZJB0913as putia ZJB09135)、荧光假单胞菌ZJB0...

Embodiment 2

[0040] Embodiment 2: the double screening of producing nitrilase bacterial classification

[0041] Inoculate the strains screened in Example 1 that can produce discoloration reactions and have large yellow discoloration circles into the re-screening medium, 30-32°C, 100-200r / min, 500mL triangular flask with a liquid volume of 150mL, and ferment for 3 days , centrifuging the fermented liquid to obtain crude enzyme liquid or wet bacteria. Take 1g of wet bacteria respectively, place them in a 500ml Erlenmeyer flask, add 50ml of 2% (w / w) reaction substrate iminodiacetonitrile aqueous solution, react for 60min under the condition of 30°C shaker at 150r / min, and remove the bacteria by centrifugation. The production amount of iminodiacetic acid was measured, and the conversion rate was calculated. The results are shown in Table 1.

[0042] Each 1L of re-screening medium is prepared as follows: glycerol 8g, yeast extract 6g, NaCl 1g, K 2 HPO 4 5g, MgSO 4 0.2g, 5g n-butyronitrile...

Embodiment 3

[0045] Example 3: Cultivation and biocatalytic preparation of iminodiacetic acid produced by nitrilase microorganisms

[0046] Each 1L of enzyme production medium is prepared as follows: glycerin 8g, yeast extract 6g, NaCl 1g, K 2 HPO 4 5g, MgSO 4 0.2g, make up to 1000mL with water; add 5g of inducer n-butyronitrile.

[0047] The enzyme production medium was divided into 500mL Erlenmeyer flasks with a liquid volume of 100mL / bottle, and was sterilized by heat in a sterilizing pot for 20min at a temperature of 121°C.

[0048] The strains obtained in Example 1 were respectively inoculated with 1 ring of strains activated on a slant to the enzyme production medium added with an inducer, and cultured for 3 days at a shaker flask with a rotation speed of 200 r / min and a temperature of 30°C. Wet cells were collected as crude nitrilase enzyme. Add 5g of wet bacteria to 100mL of 3% (w / w) iminodiacetonitrile aqueous solution for hydrolysis reaction, control the temperature at 30°C...

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Abstract

The invention provides a method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with microbes, which comprises the following steps: using bacterial strains which produce nitrilase to obtain the nitrilase through enzyme production cultivation; and taking the nitrilase as a biological catalyst to biologically catalyze the iminodiacetonitrile to prepare the iminodiacetic acid. The invention also relates to a screening method of the microbes which produce the nitrilase and the bacterial strains with nitrilase activity obtained by the screening method. The invention provides the basis for producing the iminodiacetic acid by biologically catalyzing the iminodiacetonitrile, thereby having important application prospect.

Description

(1) Technical field [0001] The invention relates to a method for preparing iminodiacetic acid by catalyzing iminodiacetonitrile with a nitrilase-producing microorganism, a screening method for the microorganism, and a bacterial strain with nitrilase activity obtained by screening the method. (2) Background technology [0002] Iminodiacetic acid (IDA) is a relatively important chemical raw material. It has a strong complexing ability and can complex with a variety of metal ions to form a chelate. It can form a blue chelate with copper ions. It is commonly used As a complexing agent and surfactant, it is often used in organic synthesis and is also an important intermediate in the production of glyphosate. Because its molecule contains imino group and carboxyl group, its chemical properties are very active, and it is widely used in electroplating, dyes, medicine, electronics and other fields. It is an important chemical intermediate. [0003] At present, chemical methods are ...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12Q1/04C12N1/20C12R1/38C12R1/01C12R1/22C12R1/40C12R1/265C12R1/13C12R1/39C12R1/385C12R1/06
Inventor 郑裕国柳志强张涛徐建妙薛亚平郑仁朝沈寅初
Owner ZHEJIANG UNIV OF TECH
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