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Method for producing 4-hydroxycinnamaldehyde by catalyzing recombinant strain and whole cells thereof

A technology of recombinant strains and hydroxycinnamic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of catalyzed production of 4-hydroxycinnamaldehyde without recombinant bifunctional enzyme strains, and achieve reduction and complexity Sexuality, simplification of production process, low nutrition effect

Inactive Publication Date: 2015-04-08
BEIJING FORESTRY UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of recombinant bifunctional enzyme strains to catalyze the production of 4-hydroxycinnamaldehyde

Method used

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  • Method for producing 4-hydroxycinnamaldehyde by catalyzing recombinant strain and whole cells thereof
  • Method for producing 4-hydroxycinnamaldehyde by catalyzing recombinant strain and whole cells thereof
  • Method for producing 4-hydroxycinnamaldehyde by catalyzing recombinant strain and whole cells thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] (1) Pick the recombinant strain E. coli M15 / pQE31-4CL1-CCR single colony was inoculated in 5mL LB liquid medium, and cultured overnight at 37°C and 200rpm with shaking; the obtained bacterial liquid was transferred to 50mL LB liquid culture at a volume ratio of 2:100 Shake at 37°C and 200rpm until the control OD600 is 0.6; add inducer isopropyl-β-D-thiogalactopyranoside IPTG to the culture to a final concentration of 0.4mmol / L, at 28°C, 200rpm induction culture for 8h; the formula of the LB liquid medium is: in g / L, tryptone 10, yeast extract 5, NaCl10, pH7.5, ampicillin 0.1, kanamycin 0.025; after stirring and dissolving evenly Sterilized and cooled for later use;

[0039] (2) Put the substrate coumaric acid at a concentration of 1mM directly into 50mL of the above liquid culture, and shake and react at 37°C and 200rpm in the dark for 4h;

[0040] (3) After the reaction is completed, centrifuge the cells at 4°C and 50×100 rpm to collect the reaction solution; the r...

Embodiment 2

[0042] (1) Pick the recombinant strain E. coli M15 / pQE31-4CL1-CCR single colony was inoculated in 5mL LB liquid medium, and cultured overnight at 37°C and 200rpm with shaking; the obtained bacterial liquid was transferred to 50mL LB liquid culture at a volume ratio of 2:100 Shake at 37°C and 200rpm until the control OD600 is 0.6; add inducer isopropyl-β-D-thiogalactopyranoside IPTG to the culture to a final concentration of 0.4mmol / L, at 28°C, 200rpm induction culture for 8h; the formula of the LB liquid medium is: in g / L, tryptone 10, yeast extract 5, NaCl10, pH7.5, ampicillin 0.1, kanamycin 0.025; after stirring and dissolving evenly Sterilized and cooled for later use;

[0043] (2) Put the substrate caffeic acid at a concentration of 1mM directly into 50mL of the above liquid culture, and shake and react at 37°C and 200rpm in the dark for 8h;

[0044] (3) After the reaction is completed, centrifuge the cells at 4°C and 50×100 rpm to collect the reaction solution; the re...

Embodiment 3

[0046] (1) Pick the recombinant strain E. coli M15 / pQE31-4CL1-CCR single colony was inoculated in 5mL LB liquid medium, and cultured overnight at 37°C and 200rpm with shaking; the obtained bacterial liquid was transferred to 50mL LB liquid culture at a volume ratio of 2:100 Shake at 37°C and 200rpm until the control OD600 is 0.6; add inducer isopropyl-β-D-thiogalactopyranoside IPTG to the culture to a final concentration of 0.4mmol / L, at 28°C, 200rpm induction culture for 8h; the formula of the LB liquid medium is: in g / L, tryptone 10, yeast extract 5, NaCl10, pH7.5, ampicillin 0.1, kanamycin 0.025; after stirring and dissolving evenly Sterilized and cooled for later use;

[0047] (2) Put the substrate ferulic acid at a concentration of 1mM directly into 50mL of the above-mentioned liquid culture, and shake and react at 37°C and 200rpm in the dark for 32h;

[0048] (3) After the reaction is completed, centrifuge the cells at 4°C and 50×100 rpm to collect the reaction solut...

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Abstract

The invention discloses a method for producing 4-hydroxycinnamaldehyde by catalyzing a recombinant strain and whole cells thereof. The method is characterized in that 4-hydroxycinnamic acid is catalyzed by using whole cells of a recombinant strain as a biocatalyst to prepare the 4-hydroxycinnamaldehyde, and the method comprises the following steps: (1) constructing a recombinant strain E.coli M15 / pQE31-4CL1-CCR; (2) preparing whole cells of the recombinant strain; and (3) catalyzing the whole cells of the recombinant strain to produce the 4-hydroxycinnamaldehyde, and detecting the obtained product by high performance liquid chromatography-tandem mass spectrometry. The invention provides a path for synthesizing the 4-hydroxycinnamaldehyde by using a bioconversion method for the first time; the method has the advantages of mild reaction conditions, high enzyme activity, cost conservation, convenience in operation and the like; and the invention provides a new method for synthesizing the 4-hydroxycinnamaldehyde, and also provides a new thought for producing secondary metabolites of plants.

Description

technical field [0001] The invention relates to the preparation of plant secondary metabolites by a biotransformation method. Background technique [0002] 4-Hydroxycinnamaldehyde is an important intermediate metabolite in the process of lignin biosynthesis and degradation, including coumaraldehyde, caffealdehyde, coniferaldehyde and sinapaldehyde. 4-Hydroxycinnamaldehyde is one of the research hotspots in the fields of agricultural chemistry, plant science and bioenergy. Such aromatic polymers prevent the conversion of lignocellulosic biomass into liquid fuels and are therefore the greatest obstacle to the economical use of lignocellulose in industrial and agricultural production such as biofuel production, cellulose digestion and utilization by ruminants, and pulp and paper. one. 4-Hydroxycinnamaldehyde is not only a key intermediate in the biosynthesis of lignin monomers, but also an important metabolite in the process of lignin synthesis and natural synthesis. Interes...

Claims

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Application Information

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IPC IPC(8): C12P7/24C12R1/19
CPCC12N9/0008C12N9/93C12P7/24C12Y102/01044C12Y602/01012
Inventor 盖颖刘淑欣侯佳音陆海蒋湘宁
Owner BEIJING FORESTRY UNIVERSITY
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