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Method for converting indole by phenol hydroxylase genetic engineering strains to prepare indigotine

A technology of phenol hydroxylase and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of limited practical application research, unsuitable practical application, slow reaction speed and the like, and achieve the effects of simple extraction steps, short production cycle and low cost

Inactive Publication Date: 2010-10-06
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2000, Li et al. used the pure enzyme of pigment cell P450Ph87Leu188Ala74 to convert indole. Only when NADPH exists, the enzyme can convert indole into indigo. NADPH is a coenzyme, usually used as a reducing agent for biosynthesis, but it is expensive. not suitable for practical application
[0005] Existing research on the preparation of indigo by phenol hydroxylase involves relatively single enzyme resources, and most of them focus on the transformation of genes and transformation mechanisms, and the practical application research is limited.
In 2005, Kim et al. obtained the gene of phenol hydroxylase from Pseudomonas sp.KL33, and recombined the gene into an engineered bacterium formed in Escherichia coli, which can convert indole into indigo, and the conversion reaction is to suspend the whole cell in 50mmol / L phosphate buffer solution, and adding 20mmol / L glucose and 1mmol / L substrate, the reaction time is 48h, the reaction speed is slow, the efficiency is low, and the product concentration is 19mg / L

Method used

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  • Method for converting indole by phenol hydroxylase genetic engineering strains to prepare indigotine
  • Method for converting indole by phenol hydroxylase genetic engineering strains to prepare indigotine
  • Method for converting indole by phenol hydroxylase genetic engineering strains to prepare indigotine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1, phenol hydroxylase genetically engineered bacteria biotransform indole to produce indigo, and investigate the influence of some parameters on the concentration of indigo.

[0022] The formula of LB medium for the expanded cultivation of phenol hydroxylase genetically engineered bacteria is: 10g / L NaCl, 10g / L peptone, 5g / L yeast extract, adjust the pH to 7.0-7.2. Sterilize at 121°C for 20 minutes, and the medium After cooling, add kanamycin at a final concentration of 10-20mmol / L as an antibiotic. The formula of solid plate medium is 15-20g / L agar added on the basis of LB medium. The steps and parameters are as follows:

[0023] 1. Inoculate the phenol hydroxylase genetically engineered bacteria on the LB medium of the solid plate, culture it statically at 37°C for 2 days, store it in the refrigerator at 4°C, and use it as the activated seed of the plate;

[0024] 2. Use the plate obtained in step 1 to activate the seeds, inoculate them into liquid LB medium...

Embodiment 2

[0034] Example 2, phenol hydroxylase genetically engineered bacteria biotransform indole to produce indigo.

[0035]The formula of LB medium and steps 1 to 5 are the same as in Example 1, wherein the dry weight concentration of whole cells of the phenol hydroxylase genetically engineered bacteria prepared in step 3 is 1.0 g / L;

[0036] In a 50mL Erlenmeyer flask, add 20mL of the whole cell of the strain phenol hydroxylase genetically engineered bacteria prepared in step 3, 20 μL of the indole / acetone solution prepared in step 4, and 10 μL of the glucose stock solution prepared in step 5 to make the indole in the reaction solution The concentration of indole is 100mg / L, and the concentration of glucose is 0.5mmol / L; shake at 50r / min for 2 hours at 20°C.

[0037] According to the measurement method described in Example 1, the indigo concentration recorded is 37.86mg / L.

Embodiment 3

[0038] Example 3, phenol hydroxylase genetically engineered bacteria biotransform indole to produce indigo.

[0039] The formula of LB medium and steps 1 to 5 are the same as in Example 1, wherein the dry weight concentration of whole cells of the phenol hydroxylase genetically engineered bacteria prepared in step 3 is 1.2 g / L;

[0040] In a 50mL Erlenmeyer flask, add 20mL of whole cells of the strain phenol hydroxylase genetically engineered bacteria prepared in step 3, 30 μL of indole / acetone solution prepared in step 4, and 20 μL of glucose stock solution prepared in step 5 to make the indole in the reaction solution The concentration of indole was 150 mg / L, the concentration of glucose was 1.0 mmol / L, and the reaction was carried out at 25° C. with shaking at 100 r / min for 4 hours.

[0041] According to the measurement method described in Example 1, the indigo concentration recorded is 77.86mg / L.

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Abstract

The invention relates to a method for converting indole to prepare indigotine by engineering strains formed by recombining phenol hydroxylase genes into colon bacillus, which belongs to the technical field of biological engineering. The method comprises the following steps: obtaining phenol hydroxylase gene full-length sequences with the total length of 5490bp and the Genbank registration number of FJ610336 in high-efficient phenol-degrading bacteria Arthrobacter sp.W1 with the GenBank registration number of EU339930; connecting the phenol hydroxylase gene full-length sequences to carrier plasmids (pET28a (+) Vector); and converting the phenol hydroxylase gene full-length sequences into host cell colon bacillus to construct phenol hydroxylase genetic engineering strains. The full-cell dry weight concentration of the phenol hydroxylase genetic engineering strains in a biological conversation reaction solution is between 1 and 2 g / L, the indole concentration is between 100 and 600 mg / L, the glucose concentration is between 0.5 and 2 mmol / L, the rotation speed of a shaking table is controlled between 50 to 200 r / min at a temperature between 20 and 40 DEG C, and the oscillating reaction lasts 2 to 14 hours. The biological conversation method of the invention has the advantages of mild reaction condition, high product concentration and short production period, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for recombining phenol hydroxylase gene into Escherichia coli to form engineering bacteria for transforming indole to prepare indigo. Background technique [0002] Indigo is a pigment with a history of more than 4,000 years and is widely used in printing and dyeing, medicine and food industries. Since 1890, chemically synthesized indigo has gradually replaced plant-derived indigo, but the raw materials and catalysts used in the chemical synthesis process have certain damage to the operator’s respiratory tract, central nervous system and liver, and even have potential carcinogenic toxicity. The rinsing wastewater containing a large amount of toxic substances such as ferric chloride, ferric sulfate, sodium sulfate, sodium chloride, aniline and nitrobenzene is produced, which has serious pollution to the environment. [0003] Biological preparation of indigo can simpl...

Claims

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Application Information

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IPC IPC(8): C12P17/16C12N15/53C12N1/21C12R1/19
Inventor 曲媛媛李慧周集体时胜男项学敏乔森吕红张爱丽
Owner DALIAN UNIV OF TECH
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