Unlock instant, AI-driven research and patent intelligence for your innovation.

Preparation method for caffeic acid and vanillin and preparation method of reaction catalysts of caffeic acid and vanillin

A catalyst and caffeic acid technology, applied in biochemical equipment and methods, enzymes, oxidoreductases, etc., can solve the problems of large environmental pollution, high cost, and difficulty entering the European market in chemical synthesis methods, and reduce production costs and simplify production processes. Process, the effect of reducing environmental pollution

Pending Publication Date: 2020-09-18
上海仁酶生物科技有限公司 +1
View PDF5 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In the above two preparation methods of vanillin, there are deficiencies respectively, the chemical synthesis method has great environmental pollution, and is difficult to enter the European market
Biosynthesis uses plant resources as raw materials, and the cost is relatively high

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method for caffeic acid and vanillin and preparation method of reaction catalysts of caffeic acid and vanillin
  • Preparation method for caffeic acid and vanillin and preparation method of reaction catalysts of caffeic acid and vanillin
  • Preparation method for caffeic acid and vanillin and preparation method of reaction catalysts of caffeic acid and vanillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of prokaryotic expression system

[0042] The ortho-methyltransferase (OMT) gene fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and recombined into the pET21a vector. The positive recombinant plasmid pET21a(+) was transformed into the expression host strain BL21(DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain OMT-pET21a(+) / BL21(DE3) was obtained as a subsequent catalytic reaction primary strain.

[0043]Aromatic carboxylic acid reductase (ACAR) gene fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and recombined into pET21a vector. The positive recombinant plasmid pET21a(+) was transformed into the expression host strain BL21(DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain ACAR-pET21a(+) / BL21(DE3) was obtained as a subsequent catalytic reaction primary strain.

Embodiment 2

[0044] Embodiment 2 Fermentation preparation of enzyme

[0045] The expression strains OMT-pET21a(+) / BL21(DE3) and ACAR-pET21a(+) / BL21(DE3) constructed above were added to 5mL LB liquid medium [10g / L pancreatic Peptone (OXIOD), 5g / L Yeast Powder (OXIOD), 10g / L Sodium Chloride (Sinopharm Reagents)], shake culture overnight at 37°C and 200rpm, inoculate in a 1% (V / V) ratio containing the final concentration In 500mL LB liquid medium containing 100ug / mL ampicillin, shake culture at 37°C and 200rpm. 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 8000rpm at 4°C for 20min, and the supernatant was taken for later use.

Embodiment 3

[0047] Add phosphate to 800ml of aqueous solution containing 50g of 3-dehydroshikimic acid to a final concentration of 50mM, and the pH of the solution reaches 7.0. After stirring and dissolving, add 200mL of 3-dehydroshikimate dehydratase enzyme solution to make the final volume 1L. 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 .

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a biological production method of caffeic acid and vanillin. The biological production method comprises the following steps: firstly, glucose and the like are taken as initialraw materials, and fermentation is performed by Escherichia coli to produce an intermediate 3-dehydroshikimic acid, the concentration of the intermediate is higher than 50 g / L, and the sugar mass conversion rate (A / G) is 30%-40%, and the maximum theoretical conversion rate can be reached; secondly, an enzyme catalysis process is adopted, 3-dehydroshikimic acid obtained in the fermentation processtaken as a raw material is subjected to bio-enzyme catalysis by an independently developed suitable enzyme, and an intermediate protocatechuic acid is obtained; thirdly, an enzyme catalysis process isadopted, the protocatechuic acid obtained in the previous step is used as a raw material to be subjected to bio-enzyme catalysis by an independently developed suitable enzyme, and an intermediate caffeic acid is obtained; and fourthly, an enzyme catalysis process is adopted, the caffeic acid obtained in the previous step is used as a raw material to be subjected to biological enzyme catalysis byan independently developed suitable enzyme, and the product vanillin is obtained. One-step fermentation and three-step enzyme catalysis are adopted in the whole process to synthesize vanillin. The method has the beneficial effects that glucose is taken as the raw material, and is affordable and widely sourced; the whole process is simplified, and the product concentration is high and can reach 20-80 g / L, so that the production cost of vanillin and environmental pollution are reduced; the reaction routes have fewer side reactions, simple post-treatment and high total yield, and the yield can reach 25%; and reaction time is short, and the whole process is completed in 72-96 h.

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 biological production of vanillin. Background technique [0002] Vanillin is commonly known as vanilla powder, vanillin, vanilla powder, vanilla extract, vanillin, etc. Extracted from the Rutaceae vanilla bean. White to light yellow crystal or crystalline powder, slightly sweet. Soluble in hot water, glycerin and alcohol, not easily soluble in cold water and vegetable oil. The aroma is stable and not easy to volatilize at higher temperature. It is easy to oxidize in the air, and it is easy to change color when it encounters alkaline substances. [0003] The chemical name is 3-methoxy-4-hydroxybenzaldehyde. It has vanilla bean aroma and strong milky aroma. It acts as an aroma enhancer and fixer. It is widely used in cosmetics, tobacco, cakes, candies, and baked goods, etc. The industry is on...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P7/24C12N9/10C12N9/02C12N15/70
CPCC12P7/42C12P7/24C12N9/1007C12N9/0008C12N15/70
Inventor 林涛徐明文蒋丽丽曹学松
Owner 上海仁酶生物科技有限公司