Preparation method of gallic acid and protocatechuic acid, and preparation method of reaction catalyst thereof

A technology of gallic acid and enzyme catalysts, applied in biochemical equipment and methods, oxidoreductases, enzymes, etc., can solve the problems of rising prices of plant-derived tannins and increased production costs of gallic acid, and achieve reduced production costs and simplified processes , the effect of reducing environmental pollution

Pending Publication Date: 2019-08-30
南京趣酶生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the above three preparation methods of gallic acid, each has its own advantages, but their common disadvantage is that they use plant resource tannin as

Method used

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  • Preparation method of gallic acid and protocatechuic acid, and preparation method of reaction catalyst thereof
  • Preparation method of gallic acid and protocatechuic acid, and preparation method of reaction catalyst thereof
  • Preparation method of gallic acid and protocatechuic acid, and preparation method of reaction catalyst thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of prokaryotic expression system

[0042] The aroZ fragment of 3-dehydroshikimate dehydratase gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and recombined into pET21a vector. The positive recombinant plasmid aroZ-pET21a(+) was transformed into the expression host strain BL21(DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain aroZ-pET21a(+) / BL21(DE3) was obtained as a follow-up Catalyzed reaction primary strains.

[0043] The p-hydroxybenzoic acid hydroxylase gene pobA fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and recombined into the pET21a vector. The positive recombinant plasmid pobA-pET21a(+) was transformed into the expression host strain BL21(DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain pobA-pET21a(+) / BL21(DE3) was obtained as a follow-up Catalyzed reaction primary st...

Embodiment 2

[0044] Embodiment 2 Fermentation preparation of enzyme

[0045] The expression strain constructed above aro Z -pET21a(+) / BL21(DE3), pobA-pET21a(+) / BL21(DE3) was added with a final concentration of 100ug / mL ampicillin in 5mL LB liquid medium [10g / L tryptone (OXIOD), 5g / L yeast powder (OXIOD), 10g / L chlorine Sodium chloride (Sinopharm Reagent)] was shaken at 37°C and 200rpm overnight, inoculated in 500mL LB liquid medium containing ampicillin with a final concentration of 100ug / mL at a ratio of 1% (V / V), and incubated at 37°C, 200rpm shaking culture. When the OD600 was between 0.8-1.0, the inducer IPTG (isopropyl-β-D-thiogalactopyranoside, IPTG) was added at a final concentration of 0.1 mM, and induced overnight at 25°C. The bacteria were collected by centrifugation at 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 10min), centrifuged at 12000rpm at 4°C for 20min, and the supernatant was taken for use.

Embodiment 3

[0047] In 800ml of aqueous solution containing 50g of substrate I (3-dehydroshikimic acid), add phosphate to a final concentration of 50mM, and the pH value of the solution reaches 7.0. After stirring and dissolving, add 200mL of 3-dehydroshikimate dehydratase enzyme solution so that The final volume is 1 L. The reaction solution was placed in a constant temperature water bath at 37°C and mechanically stirred for reaction. After reacting for 1h, carry out HPLC detection, the conversion rate of substrate>98%, see figure 1 .

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Abstract

The invention discloses a preparation method of gallic acid and protocatechuic acid as well as a preparation method of a reaction catalyst thereof. According to the preparation method of the gallic acid and the protocatechuic acid, a catalytic enzyme solution is prepared by carrying out construction of aroZ fragments of 3-dehydroshikimate dehydratase genes or pobA fragments of hydroxybenzoate hydroxylase genes; and then, biosynthesis reaction is carried out so as to synthesize the gallic acid and the protocatechuic acid. The preparation method of the gallic acid and the protocatechuic acid hasthe following beneficial effects: by taking glucose, which is affordable in price and wide in source, as a raw material, the whole process is simplified so as to have environmental pollution reducedwith production cost of gallic acid decreased.

Description

technical field [0001] The invention relates to the technology of preparing aromatic ring compound derivatives by using an enzyme-catalyzed method, in particular to the technology of biologically producing gallic acid. Background technique [0002] Gallic acid, also known as gallic acid, gallic acid (gallic acid), chemical name is 3,4,5-trihydroxybenzoic acid (3,4,5-trihydroxybenzoic acid), white needle crystal, is a widely used Chemicals. [0003] At present, there are three main production processes of gallic acid: [0004] Chemical hydrolysis method: use gallnut as raw material, use strong acid or strong base as catalyst to hydrolyze gallnut tannin to produce gallic acid, and then pass through a series of chemical processes such as filtration, concentration, decolorization and drying. This process is simple to operate, but due to the large environmental pollution caused by strong acid and strong alkali, the source of raw materials is greatly affected by the environment,...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12P7/42C12N9/88C12N9/02
CPCC12N15/70C12N15/66C12P7/42C12N9/88C12Y402/01118C12N9/0071
Inventor 林涛于丽珺徐明文蒋丽丽
Owner 南京趣酶生物科技有限公司
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