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Biosynthesizing method of imidazole-4-acetic acid

A biosynthesis and imidazole technology, applied in the field of biochemical engineering, can solve the problems of harsh reaction conditions, large environmental pollution, high cost of raw materials, etc., and achieve the effect of low equipment requirements, high substrate conversion rate, and simple steps

Active Publication Date: 2017-09-19
NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned chemical synthesis method has harsh reaction conditions, high cost of raw materials, complex products and great environmental pollution.

Method used

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  • Biosynthesizing method of imidazole-4-acetic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Construction of membrane-linked L-amino acid deaminase engineering bacteria

[0025] According to the gene sequence of Proteus vulgaris IAM12003 membrane-linked L-amino acid deaminase (its sequence accession number is BAA90864), the whole gene synthesis was carried out, and the synthetic gene (LAAD) was linked into pET20 ( In b)+, Escherichia coli DH5α strain was transformed, and positive clones were screened to obtain recombinant plasmid pET20b-laad. In addition, the LAAD gene was linked into pET28a(+) through NcoI and BamHI restriction sites, transformed into Escherichia coli DH5α strain, and positive clones were screened to obtain the recombinant plasmid pET28a-laad.

[0026] The two recombinant plasmids were transformed into Rosstta(DE3), wherein the LAAD expressed by Rosstta(DE3) / pET20b-laad contained the PelB signal peptide, and was expressed in the periplasm of the cell using the conserved secretory pathway, while Rosstta(DE3) / pET20b-laad pET28a-laa...

Embodiment 2

[0027] Example 2 Synthesis of imidazole-4-acetic acid by membrane-linked L-amino acid deaminase engineering bacteria

[0028] Specific steps are as follows:

[0029] (1) Pick engineering bacteria (E.coli Rosstta(DE3) / pET28a-laad) from the activated LB solid medium, insert the monoclonal bacteria into -1 ) in LB liquid medium, 37°C, 200r / min shaking culture overnight, to obtain seed liquid.

[0030] (2) Inoculate the seed solution with a volume fraction of 2% inoculum into a seed containing kanamycin (50 μg·mL -1 ) of LB medium, cultured at 37°C, 200r / min until OD 600 At 0.6, add IPTG to make the final concentration of IPTG 0.5 μM, induce culture at 30° C., 150 r / min for 6 h.

[0031] (3) Collect the engineering bacteria cells by centrifugation, discard the supernatant, and wash the cells with 0.2mM phosphate buffer (pH 7.5). Add the cleaned engineered bacteria into a phosphate buffer (0.2mM, pH 7.5) containing 50mM L-histidine, and the density of the bacteria in the solut...

Embodiment 3

[0033] Example 3 Utilize membrane-linked L-amino acid deaminase engineering bacteria to synthesize imidazole-4-acetic acid

[0034]Adjust the L-histidine concentration of the reaction solution in step (3) of Example 2 to 100 mM, and keep other conditions unchanged. Cultivate for 37 hours at 37° C. and 200 r / min to obtain a solution containing 3-(4-imidazole)-2-oxopropionic acid. To this solution was added 1% H 2 o 2 , and reacted for 10 minutes to obtain an imidazole-4-acetic acid solution with a content of 35.6 mM, and the conversion rate of histidine to imidazole-4-acetic acid reached 37%.

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Abstract

The invention discloses a biosynthesizing method of imidazole-4-acetic acid. The biosynthesizing method comprises the following steps of using L-histidine as a substrate, and performing deaminizing reaction under the catalyzing action of membrane-linked L-amino acid deaminases, so as to obtain reaction liquid containing an intermediate product, namely 3-(4-imidazole)-2-oxo propionic acid; adding a hydrogen peroxide solution into the reaction liquid, and reacting, so as to obtain the imidazole-4-acetic acid. The biosynthesizing method has the characteristics that the steps are simple, the reaction conditions are mild, the sources of raw materials are wide, the preparation of a biological catalyst is easy, the requirement on equipment is low, and the conversion rate of the substrate is high; the organic solvent and the toxic reagent are not needed; compared with the existing chemical method, the efficiency is high, the cost is low, and the environment-friendly effect is realized.

Description

technical field [0001] The invention relates to the field of biochemical engineering, in particular to a biosynthesis method of imidazole-4-acetic acid. Background technique [0002] Imidazole-4-acetic acid (IAA) is an important imidazole derivative. It is a conformationally restricted analogue of the main inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system, and has pharmacological effects such as hypnosis, antihypertensive and antioxidation. At the same time, imidazole-4-acetic acid has also been used to synthesize protease inhibitors, anti-blocking agents for pregnant urine pannus receptors, and anti-cancer compounds. In addition, IAA can also form coordination compounds with transition metals through its own carboxyl group and imidazole group. Recently, IAA has also been used to prepare imidazole-modified chitosan nanoparticles, which are considered to be good siRNA drug carriers. [0003] At present, commercial IAAs are prepared by che...

Claims

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Application Information

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IPC IPC(8): C12P17/10C12R1/19
CPCC12P17/10
Inventor 梅乐和赵伟睿丁焕茹胡升黄俊金志华
Owner NINGBO INST OF TECH ZHEJIANG UNIV ZHEJIANG
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