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Rhodococcus rhodochrous and use thereof as catalyst for use in preparation of iminodiacetic acid from iminodiacetonitrile

A technology of iminodiacetic acid and Rhodococcus rhodococcus, applied in the field of strains with nitrilase activity, can solve the problems of high equipment requirements, harsh reaction conditions, high energy consumption, etc., and achieve the effect of efficient production

Active Publication Date: 2013-04-24
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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  • Rhodococcus rhodochrous and use thereof as catalyst for use in preparation of iminodiacetic acid from iminodiacetonitrile
  • Rhodococcus rhodochrous and use thereof as catalyst for use in preparation of iminodiacetic acid from iminodiacetonitrile
  • Rhodococcus rhodochrous and use thereof as catalyst for use in preparation of iminodiacetic acid from iminodiacetonitrile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the initial screening of producing nitrilase bacterial classification

[0024] Each 1 L of primary screening medium was prepared as follows: yeast extract 6 g, NaCl 1 g, K 2 HPO 4 1 g, MgSO 4 1 g, 20 g of agar powder, pH 7.0 water to make up to 1000 mL, after sterilization, cool to 50 °C, add 5 g of iminodiacetonitrile and 1 g of bromocresol violet, and mix well to the plate.

[0025] 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 color circles in the colonies, and inoculate them on LB slopes (25~28°C);

[0026]The microbial strains with yellow discoloration circles are: Pseudomonas marginalis ZJB09121, Pseudomonas putia ZJB09134, Pseudomonas putia ZJB09129, Pseudomonas putia ZJB09129, Pseudomonas putia ZJB09129 (Pseudomonas putia ZJB09135)、荧光假单胞菌ZJB09137(Pseudomonas fluorescens ZJB09137)、产酸克雷伯氏菌ZJB09123(Kelbsiella ocytoca ZJB09123)、弗氏红球菌ZJB0912...

Embodiment 2

[0027] Embodiment 2: the double screening of producing nitrilase bacterial classification

[0028] Inoculate the strains screened in Example 1 that can produce discoloration reactions and have large yellow discoloration circles into the medium for re-screening, at 30-32°C, 100-200 r / min, in a 500-mL triangular flask with a liquid volume of 150 mL, After 3 days of fermentation, the fermentation liquid was centrifuged to obtain crude enzyme liquid or wet bacteria. Take 1g of wet bacteria each, put it in a 500ml Erlenmeyer flask, add 50ml of 2% (w / w) reaction substrate iminodiacetonitrile aqueous solution, react at 30°C for 60min on a shaker at 150r / min, and remove by centrifugation Bacteria, the production amount of iminodiacetic acid was measured, and the conversion rate was calculated. The results are shown in Table 1.

[0029] Each 1 L of re-screening medium was prepared as follows: glycerol 8 g, yeast extract 6 g, NaCl 1 g, K 2 HPO 4 5g, MgSO 4 0.2 g, 5 g n-butyronitr...

Embodiment 3

[0032] Example 3: Cultivation and biocatalytic preparation of iminodiacetic acid produced by nitrilase microorganisms

[0033] Each 1 L of enzyme production medium was prepared as follows: glycerol 8 g, yeast extract 6 g, NaCl 1 g, K 2 HPO 4 5 g, MgSO 4 0.2 g, made up to 1000 mL with water; add 5 g of inducer n-butyronitrile.

[0034] The enzyme-producing medium was divided into 500 mL Erlenmeyer flasks, with a liquid volume of 100 mL / bottle, and heat sterilized in a sterilizing pot for 20 min at a temperature of 121 °C.

[0035] The strains obtained in Example 1 were respectively inoculated with 1 ring of strains activated on a slant to the enzyme-producing medium added with an inducer, and cultured at a shaker flask with a rotation speed of 200 r / min and a temperature of 30°C for 3 d, collecting wet thallus as nitrilase crude enzyme. Add 5 g of wet bacteria to 100 mL of 3% (w / w) iminodiacetonitrile aqueous solution for hydrolysis reaction, control the temperature at 30...

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Abstract

The invention discloses a method for preparing iminodiacetic acid from iminodiacetonitrile in presence of a biocatalyst and a strain. The method comprises: performing nitrilase production culture of nitrilase-producing strain to obtain nitrilase; and preparing iminodiacetic acid from iminodiacetonitrile under the biocatalytic action of the nitrilase serving as the biocatalyst. The method providesa basis for producing iminodiacetic acid from iminodiacetonitrile in the presence of the biocatalyst and has a bright application prospect.

Description

(1) Technical field [0001] The invention relates to a method for producing iminodiacetic acid by catalyzing iminodiacetonitrile with nitrilase-producing microorganisms, and the strain obtained by screening with nitrilase activity. (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 generally used to prepare iminodiacetic acid. According to dif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/04C12R1/01
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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