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Culture of bacillus alcaligenes and method for preparing glycolic acid by using the same to hydrolyzing nitrile

A technology for catalyzing hydroxyacetonitrile and glycolic acid with nitrilase, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, hydrolytic enzymes, etc., and can solve problems such as poor tolerance of hydroxyacetonitrile

Inactive Publication Date: 2008-05-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing nitrilase has poor tolerance to hydroxyacetonitrile, and it is urgent to screen new nitrilase strains

Method used

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  • Culture of bacillus alcaligenes and method for preparing glycolic acid by using the same to hydrolyzing nitrile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 microbial shake flask culture

[0053] Inoculate 50mL seed medium (10g of glycerol, 5g of peptone, 5g of yeast extract, KH 2 PO 4 2g, NaCl 1g, MgSO 4 0.2g, water 1000mL, pH 8.0) in the pre-cultivation for 15h, with the culture solution as the seed solution, inoculated to 200mL fermentation medium (ammonium acetate 15g, peptone 10g, yeast extract 6g, KH 2 PO 4 2g, NaCl2g, MgSO 4 0.4g, Auxin FeSO 4 ·7H 2 O 0.5g, inducer phenylacetonitrile 0.2g, water 1000mL, pH 8.0), cultured on a shaker at 160rpm at 30°C for 48h.

Embodiment 2

[0054] Embodiment 2 nitrilase bacterial strain activity comparison

[0055] The isolated Alcaligenes sp.ECU0401 was compared with the nitrilase activity of Rhodococcus erythropolis1.2362, Rhodococcus rhodochrous ECU1201 and Rhodococcus ruber 4.1187. Suspend the resting cells of the above-mentioned four strains in 100mL potassium phosphate buffer solution, add hydroxyacetonitrile at a final concentration of 20mM, shake and react at 30°C and 160rpm for 30min, and immediately add to the supernatant 2 MH 2 SO 4 Acidify to a pH of 1.0-2.0 to terminate the reaction, centrifuge the reaction solution at 10,000×g for 15 minutes, remove cells, and analyze by HPLC. It is found that Alcaligenes sp.ECU0401 has a higher activity in nitrile hydrolysis of p-hydroxyacetonitrile. The reaction solution was analyzed by ion chromatography, and no possible by-products of glyoxylic acid, formic acid and oxalic acid were found, and the product glycolic acid was not metabolized.

[0056] Table 1 Ni...

Embodiment 3

[0058] Example 3 Utilize Alcaligenes sp.ECU0401 to grow cells

[0059] Catalytic hydrolysis of hydroxyacetonitrile to produce glycolic acid

[0060] Prepare seed medium (glycerol 10g, peptone 5g, yeast extract 5g, KH 2 PO 4 2g, NaCl 1g, MgSO 4 0.2g, water 1000mL, pH 8.0), put 200mL seed medium into a 1L shake flask, and cultivate the seed liquid. Then prepare the fermentation medium (ammonium acetate 15g, peptone 10g, yeast extract 6g, KH 2 PO 4 2g, NaCl 2g, MgSO 4 0.4g, Auxin FeSO 4 ·7H 2 (00.5g, inducer benzyl nitrile 0.2g, water 1000mL, pH 8.0). Put 3L fermentation medium in a 5L fermenter, insert 5% seed liquid after disinfection, carry out fermentation and cultivation for 24 hours, add 100mM hydroxyacetonitrile, take samples for HPLC analysis at different times, and continue to supplement when the yield of glycolic acid analysis is 96%. Add 100mM of hydroxyacetonitrile solution neutralized by sodium hydroxide and 10g of ammonium acetate. When the cumulative c...

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Abstract

The invention relates to cultivation of nitrilase producing bacterium Alcaligenes sp. ECU0401 which is recently separated from soil and a process for catalyzing glycolonitrile to be hydrolyzed and preparing glycollic acid. The invention employs nitrilase to be starting material, and Alcaligenes sp. ECU0401 cell which is achieved by proceeding enlarging culture in the fermentation culture medium is utilized as biocatalyst, and the product glycollic acid is achieved by the hydrolysis reaction of the glycolonitrile under certain condition. The invention has the advantages of mild reaction condition, non-pollution, simple technological route and the like.

Description

technical field [0001] The invention relates to a strain of Alcaligenes and its application, especially a method for preparing glycolic acid. technical background [0002] Glycolic acid (HOCH 2 COOH) is the simplest α-hydroxy acid, widely used in daily chemical, textile and pharmaceutical industries, not only as household and industrial cleaning agents, but also as adhesives, welding agents, demulsifiers and coatings The ingredients and synthesis of various pesticides, pharmaceuticals and chemical additives have a large market demand. There are two ways to synthesize glycolic acid by hydrolysis: chemical method and biological method. The enzyme systems involved in nitrile hydrolysis in the biological method mainly include nitrilase, nitrile hydratase and amidase; nitrilase can hydrolyze nitrile to generate corresponding carboxylic acid and ammonia, which has the advantages of good selectivity, mild reaction conditions and high product purity. , significantly better than c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N9/14C12R1/05
Inventor 许建和何玉财张志钧
Owner EAST CHINA UNIV OF SCI & TECH
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