Methods used for producing glucuronic acid, and special-purpose engineering bacteria of method

A technology of glucuronic acid and engineering bacteria, applied in the field of glucuronic acid production, can solve the problems of poor selectivity, low product yield, high energy consumption, etc., and achieve the effect of significant promotion and application value and huge economic benefits

Active Publication Date: 2019-03-05
ZHUCHENG HAOTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process has problems such as high energy consumption, poor selectivity, low product yield, and serious environmental pollution.

Method used

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  • Methods used for producing glucuronic acid, and special-purpose engineering bacteria of method
  • Methods used for producing glucuronic acid, and special-purpose engineering bacteria of method
  • Methods used for producing glucuronic acid, and special-purpose engineering bacteria of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the construction of recombinant bacteria

[0047] 1. Insert DNA molecule A (DNA molecule A is a double-stranded DNA molecule, composed of nucleotide C and sequence 2 in the sequence table from upstream to downstream) into the vector pBAD / HisB for XhoI and EcoRI digestion Between the sites, recombinant plasmid A was obtained. According to the sequencing results, the structure of the recombinant plasmid A is described as follows: DNA molecule A is inserted between the XhoI and EcoRI restriction sites of the vector pBAD / HisB. DNA molecule A and part of nucleotides on the carrier form fusion gene A to express fusion protein A.

[0048] 2. Insert DNA molecule B (DNA molecule B is a double-stranded DNA molecule, composed of nucleotide C and sequence 4 in the sequence table from upstream to downstream) into the vector pBAD / HisB for XhoI and EcoRI digestion Between the sites, the recombinant plasmid B was obtained. According to the sequencing results, the struc...

Embodiment 2

[0053] Embodiment 2, the preparation of albumen

[0054] The test strains were respectively: recombinant bacteria I, recombinant bacteria II or recombinant bacteria III prepared in Example 1.

[0055] 1. Take a single clone of the test strain, inoculate it into liquid LB medium containing 50 μg / mL streptomycin, and culture it with shaking at 37°C and 220 rpm until OD 600nm =0.6~0.8.

[0056] 2. After completing step 1, add L-arabinose to the culture system to make the concentration 0.2g / 100mL, shake and culture at 30°C and 200rpm for 12 hours.

[0057] 3. After completing step 2, take the entire culture system, centrifuge at 4°C and 6000rpm for 15min, and collect the bacterial precipitate.

[0058] 4. Take the bacterial pellet obtained in step 3, resuspend in buffer A and perform ultrasonic disruption, then centrifuge at 10,000rpm for 30min, and collect the supernatant.

[0059] Buffer A (pH8.0): contains 50mmol / L Tris-HCl and 100mmol / LNaCl, and the balance is water.

[00...

Embodiment 3

[0068] Embodiment 3, the enzyme activity assay of protein

[0069] The protein solution to be tested is the protein solution I, protein solution II or protein solution III prepared in Example 2.

[0070] 1. Detect the protein concentration of the protein solution to be tested.

[0071] 2. Detect the enzyme activity of the protein solution to be tested

[0072] The reaction system consists of the protein solution to be tested, L-cysteine, inositol, ferrous sulfate (providing Fe 2+ ) and buffer composition. The buffer was Tris-HCl buffer, pH 8.0, 50 mM. In the reaction system, the protein concentration is 100μg / mL (the only source of protein is the protein solution to be tested), the concentration of L-cysteine ​​is 2mmol / L, Fe 2+ The concentration of inositol is 1mmol / L, and the concentration of inositol is 20mmol / L.

[0073] Reaction conditions: stand at 37°C for 30 minutes.

[0074] After the reaction was finished, a sample was taken, diluted to 10 times the volume with...

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Abstract

The invention discloses a methods used for producing glucuronic acid, and a special-purpose engineering bacteria of the method. A first method comprises following steps: inositol is taken as a raw material, under the effect of the engineering bacteria, glucuronic acid is produced; the engineering bacteria is a recombinant bacteria capable of expressing function protein, and is obtained through introduction of functional genes used for coding the function protein into starting bacteria. A second method comprises following steps: inositol is taken as a raw material, under the effect of the function protein, glucuronic acid is produced; the function protein is one selected from (a1), or (a2), or (a3); (a1) the protein is represented by sequence table 3; (a2), the protein is a fusion protein obtained through connecting the N terminal or / and the C terminal of the protein in (a1) with labels; and (a3), the protein is represented by sequence table 7. Great economical benefit is achieved in glucuronic acid production, and high popularization application value is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for producing glucuronic acid and special engineering bacteria. Background technique [0002] Glucuronic acid (D-glucuronic acid) is the uronic acid formed by the oxidation of the hydroxyl group at the C-6 position of glucose to a carboxyl group. Its molecular formula is C 6 h 10 o 7 , the molecular weight is 194.14. Glucuronic acid is a white needle-like crystal or powder, with a melting point of 155°C-157°C, and its structure diagram is shown in figure 1 . Glucuronic acid widely exists in plants and animals, and rarely exists in nature in free form. Glucuronic acid is not only a traditional liver detoxifier and immune function regulator, but also an important intermediate in the synthesis of many medicines, and is often used as an additive in functional drinks, weight loss drugs, cosmetics, etc. [0003] Myo-inositol, also known as cyclohexanhexol, has th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/02C12R1/19
CPCC07K14/00C07K2319/00C12N15/70C12P19/02
Inventor 胡美荣王雷滕斐陶勇
Owner ZHUCHENG HAOTIAN PHARMA
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