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A method for enzymatically preparing 4-hydroxyphenylacetaldehyde

A technology of hydroxybenzene and acetaldehyde, which is applied in the field of enzymatic production of 4-hydroxyphenylacetaldehyde, can solve problems such as difficult scale-up production, difficult large-scale production, severe reaction conditions, etc., and achieve high reaction yield, low production cost, specific effect

Active Publication Date: 2021-06-04
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Description
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AI Technical Summary

Problems solved by technology

[0004] 4-Hydroxyphenylacetaldehyde can be prepared by chemical synthesis, using phenylephrine as raw material through hot acid treatment, the reaction conditions are severe, the reaction time is long, the product yield is low, the environment is not friendly, the cost is high, and it is difficult to scale up production
However, the catalytic reaction of biological enzymes in the aqueous phase is often limited by the poor solubility of the substrate and the toxicity of the product to the biological enzymes, making it difficult to scale production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: construction of engineering bacteria

[0027] According to the Escherichia coli K12 tynA gene sequence (SEQ ID No.1), the upstream primer F-tynA containing the BamH I restriction site was designed: 5'-AAGGATCCATGGGAAGCCCCCTCTCTGTATTC-3', and the downstream primer R- containing the EcoR I restriction site tynA: 5'-AACTCGAGTCACTTATCTTTCTTCAGCGCC-3'. The genomic DNA of Escherichia coli K12 was extracted by the bacterial genomic DNA extraction kit, and the genomic DNA was used as a template for PCR amplification. The PCR reaction system included 10×ExTaq buffer (Mg 2+ Plus) 5 μL, dNTPs mixture 4.0 μL, TaKaRa ExTaq 0.5 μL, template 1.0 μL, upstream and downstream primers 1.0 μL each, with dd H 2 O to make up to 50 μL. The PCR reaction parameters were: 94°C for 30s, 60°C for 30s, 72°C for 1min, and after 30 cycles, 72°C for 10min. PCR amplified products were detected with 1% agarose gel, and the target DNA fragment was purified with AxyPrep DNA Gel Recovery K...

Embodiment 2

[0028] Example 2 Production of primary amine oxidase by fermentation of recombinant bacteria and enzymatic conversion of tyramine to produce 4-hydroxyphenylacetaldehyde

[0029] (1) Insert the constructed genetically engineered bacteria into LB slant medium and cultivate at 37°C for 16h;

[0030] (2) Connect 1 ring of slant strains to LB liquid seed medium, shake and culture at 37°C and 200r / min for 6h;

[0031] (3) 3.0L medium is loaded into the 5L fermenter, the seed liquid is inserted into the fermentation medium with 8% seed quantity, the initial speed is 200r / min, the initial ventilation flow rate is 1.5L / min, and the speed is adjusted as the cell concentration increases In order to maintain the dissolved oxygen value above 20% with the ventilation flow rate, adjust the pH value at 7.0 with 25% ammonia water, culture at 37°C for 6 hours and cool down to 30°C for expression. During the fermentation process, 500g / L glycerol solution was added to maintain the glucose concen...

Embodiment 3

[0034] Example 3 Production of primary amine oxidase by fermentation of recombinant bacteria and enzymatic conversion of tyramine to produce 4-hydroxyphenylacetaldehyde

[0035] (1) Insert the genetically engineered bacteria constructed in Example 1 into LB slant medium and cultivate at 37° C. for 15 hours;

[0036] (2) Connect 1 ring of slant strains to LB liquid seed medium, shake and culture at 37°C and 200r / min for 8h;

[0037] (3) 3.0L medium is loaded into the 5L fermenter, the seed solution is inserted into the fermentation medium with a volume ratio of 7%, the initial rotation speed is 200r / min, and the initial ventilation flow rate is 1.5L / min. Adjust the rotation speed and ventilation flow to maintain the dissolved oxygen value above 20% air saturation, adjust the pH value to be stable at 7.0 with 25% ammonia water in mass to volume ratio, incubate at 37°C for 7 hours and cool down to 28°C for expression. Add 500g / L glycerol solution to maintain glycerol concentrati...

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Abstract

The invention discloses a method for enzymatically producing 4-hydroxyphenylacetaldehyde, which belongs to the field of biotechnology. The method comprises cloning a highly active primary amine oxidase gene, connecting an expression vector and transforming Escherichia coli competent cells, and constructing a primary amine oxidase gene. Amine oxidase engineering bacteria, and then use the fermented primary amine oxidase to catalyze the oxidation of tyramine to produce 4-hydroxyphenylacetaldehyde. The invention adopts an enzymatic method to produce 4-hydroxyphenylacetaldehyde, the reaction conditions are mild, the environment is friendly, and the specificity of the biological enzyme catalytic reaction is strong. The two-phase catalytic system is used to reduce the inhibitory effect of the product on the enzyme, the reaction yield is high, and the catalysis is compatible with the enzyme. Separation and coupling are beneficial to product extraction, and at the same time, the raw materials used in the production method of the present invention are cheap and easy to obtain, and the production process is simple and easy, and the production cost is low.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for enzymatically producing 4-hydroxyphenylacetaldehyde. Background technique [0002] Aromatic aldehydes are a very important class of fine chemicals. Because their molecules contain active aldehyde groups, they can be extended to synthesize a variety of products and are widely used in the fields of medicine, food and agriculture. As an important aromatic aldehyde, 4-hydroxyphenylacetaldehyde can be used as an important precursor for the synthesis of benzylisoquinoline alkaloids, tyrosol and 4-hydroxyphenylacetic acid, etc. It has wide application value and a very broad market prospect . [0003] Primary amine oxidases (PrAOs, EC 1.4.3.21) are important copper-containing amine oxidases that play an important role in the metabolism of biogenic amines, which can convert primary amines to corresponding aldehydes and generate equimolar ammonium and peroxide hydrogen. Primary...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/24C12N15/70C12N1/21C12N15/53C12R1/19
CPCC12N9/0022C12N15/70C12P7/24C12Y104/03021
Inventor 冯志彬张娟程仕伟张兴晓
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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