CN110499343B - Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method - Google Patents
Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method Download PDFInfo
- Publication number
- CN110499343B CN110499343B CN201910856791.0A CN201910856791A CN110499343B CN 110499343 B CN110499343 B CN 110499343B CN 201910856791 A CN201910856791 A CN 201910856791A CN 110499343 B CN110499343 B CN 110499343B
- Authority
- CN
- China
- Prior art keywords
- producing
- primary amine
- amine oxidase
- hydroxyphenylacetaldehyde
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03021—Primary-amine oxidase (1.4.3.21), i.e. VAP-1
Abstract
The invention discloses a method for producing 4-hydroxybenzaldehyde by an enzyme method, belonging to the technical field of biology. The method for producing the 4-hydroxyphenylacetaldehyde by the enzyme method has the advantages of mild reaction conditions, environmental friendliness, strong specificity of the biological enzyme catalytic reaction, reduction of the inhibition effect of the product on the enzyme by adopting a two-phase catalytic system, high reaction yield, coupling of catalysis and separation, contribution to product extraction, cheap and easily-obtained raw materials, simple and easy production process and low production cost.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method.
Background
Aromatic aldehyde is a very important fine chemical, and the molecule of the aromatic aldehyde contains active aldehyde group, so that the aromatic aldehyde can be extended to synthesize various products and can be widely applied to the fields of medicine, food and agriculture. 4-hydroxybenzaldehyde is an important aromatic aldehyde, can be used as an important precursor for synthesizing benzyl isoquinoline alkaloid, tyrosol, 4-hydroxyphenylacetic acid and other substances, and has wide application value and very wide market prospect.
Primary amine oxidases (PrAOs, EC 1.4.3.21) are important copper-containing amine oxidases that play an important role in biogenic amine metabolism, converting primary amines to the corresponding aldehydes and producing equimolar amounts of ammonium and hydrogen peroxide. Primary amine oxidase is widely present in prokaryotic and eukaryotic microorganisms such as escherichia coli, arthrobacter, enterobacter aerogenes, hansenula polymorpha and the like, most of the primary amine oxidase has a wider substrate spectrum, but microorganisms of different species still have different substrate spectra. The Escherichia coli primary amine oxidase has higher substrate preference on aromatic amine, and the method for producing 4-hydroxybenzaldehyde by oxidizing tyramine with the Escherichia coli primary amine oxidase is a very promising method and has the advantages of environmental friendliness, high conversion efficiency and the like.
The 4-hydroxyphenylacetaldehyde can be prepared by a chemical synthesis method, is obtained by using phenylephrine as a raw material and performing hot acid treatment, and has the advantages of severe reaction conditions, long reaction time, low product yield, environmental friendliness, high cost and difficulty in scale-up production. New bioconversion production processes are considered to be the most promising alternative. However, the catalytic reaction of the biological enzyme in the water phase is limited by poor solubility of the substrate and toxicity of the product to the biological enzyme, and the scale production is difficult.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method. The method utilizes the recombinant bacteria for producing the primary amine oxidase to produce the primary amine oxidase, and uses the primary amine oxidase to produce the 4-hydroxybenzaldehyde.
The technical scheme adopted by the invention is as follows:
a method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method comprises the following specific steps:
(1) constructing a recombinant bacterium for producing the primary amine oxidase, and fermenting to obtain a wet bacterium for producing the recombinant bacterium for producing the primary amine oxidase;
(2) single water phase conversion: preparing tyramine aqueous solution with the pH value of 7.0 and the concentration of 0.5-1.5g/100ml, adding catalase with the final concentration of 200-400U/ml and recombinant bacteria wet bacteria with the final concentration of 1-3% (g/100ml) for producing primary amine oxidase for catalysis, controlling the temperature to be 28-40 ℃, ventilating to control the dissolved oxygen to be 5-15% of air saturation, converting and producing 4-hydroxyphenylacetaldehyde in an automatic control fermentation tank, continuously sampling and measuring the concentration of the 4-hydroxyphenylacetaldehyde in the reaction process until the concentration of the 4-hydroxyphenylacetaldehyde is not increased any more, and finishing the reaction.
Further, preparing a tyramine two-phase reaction system with the pH of 7.0 and the concentration of 0.5-1.5g/100mL, wherein the two-phase reaction system is an organic solvent phase positioned at the lower layer and the upper layer of the container, the organic solvent is 20-60% of the volume of the water phase, and then adding 400U/mL catalase with the final concentration of 200-.
Further, the organic solvent is one of ethyl acetate and butyl acetate.
Further, the construction method of the recombinant bacterium for producing the primary amine oxidase comprises the following steps:
(1) cloning a primary amine oxidase gene tynA of escherichia coli K12, wherein the nucleotide sequence of the primary amine oxidase gene tynA is shown as SEQ ID No. 1;
(2) carrying out double enzyme digestion and connection on the nucleotide sequence shown in SEQ ID No.1 and a vector pET28a, and screening positive clones to obtain a recombinant vector pET28 a-tynA;
(3) the recombinant vector pET28a-tynA is transformed into a host bacterium E.coli BL21(DE3) to obtain a recombinant bacterium E.coli BL21(DE3)/pET28a-tynA for producing the primary amine oxidase.
Further, the method for obtaining the recombinant bacterium wet thallus for producing the primary amine oxidase comprises the following steps: (1) inoculating the constructed recombinant bacteria producing the primary amine oxidase into an LB slant culture medium to be cultured for 14-24 h at 37 ℃;
(2) inoculating a recombinant strain which produces primary amine oxidase on a 1-ring inclined plane into an LB liquid seed culture medium, and carrying out shake culture at 37 ℃ for 6-12 h at 200 r/min;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid cultured in the step (2) is inoculated into the fermentation culture medium by an inoculum size of 5-10% of the volume ratio, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value above 20% of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25% to be stabilized at 7.0, and the temperature is reduced to 22-28 ℃ for expression after the seed liquid is cultured for 6-10 h at 37 ℃; adding 500g/L of glycerol solution in a flowing manner in the fermentation process to maintain the concentration of the glycerol at 8000r/min and 4 ℃ for 15min after the fermentation is finished, collecting thalli by centrifugation, and washing the thalli twice by using sterile normal saline;
further, the LB slant culture medium: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 20g/L of agar, pH 7.0-7.2, and performing high-pressure steam sterilization at 121 ℃ for 20 min.
Further, the LB liquid seed culture medium: 5g/L yeast extract powder, 10g/L peptone, 5g/L NaCl, 50mg/L kanamycin, 7.0-7.2 pH, and high-pressure steam sterilization at 121 ℃ for 20 min.
Further, the fermentation medium: 20g/L of glycerol, 10g/L of peptone, 8g/L of yeast extract powder, 1 percent of corn steep liquor and MgSO4 5g/L，KH2PO4 10g/L，(NH4)2SO43g/L, citric acid 2g/L, CuSO450mg/L, pH7.2-7.4, and sterilizing with high pressure steam at 121 deg.C for 20 min.
Compared with the prior art, the invention has the beneficial effects that:
the method adopts the enzyme method to produce the 4-hydroxyphenylacetaldehyde, the reaction condition is mild, the environment is friendly, the specificity of the biological enzyme catalysis reaction is strong, when the concentration of tyramine in a reaction system is lower, the conversion rate of the 4-hydroxyphenylacetaldehyde produced by the enzyme method is over 90 percent, when the concentration of a reaction substrate tyramine aqueous solution is improved, the conversion rate of the 4-hydroxyphenylacetaldehyde produced by the enzyme method in a single-phase reaction system is reduced to about 35 percent, and when the concentration of the reaction substrate tyramine aqueous solution is improved, the conversion rate of the 4-hydroxyphenylacetaldehyde produced by the enzyme method in an organic solvent and aqueous phase two-phase reaction system is improved. Therefore, the method adopts a two-phase catalytic system to reduce the inhibition effect of the product on the enzyme, has high reaction yield, is beneficial to product extraction by coupling catalysis and separation, and has the advantages of cheap and easily-obtained raw materials, simple and easy production process and lower production cost.
Detailed Description
The technical solution of the present invention is further and timely provided by the following embodiments, but the scope of the present invention is not limited in any way by the embodiments.
The pharmaceutical agent kit and the like used in the examples are commercially available unless otherwise specified.
Example 1: construction of engineering bacteria
Designing an upstream primer F-tynA containing a BamH I enzyme cutting site according to an escherichia coli K12 tynA gene sequence (SEQ ID No. 1): 5'-AAGGATCCATGGGAAGCCCCTCTCTGTATTC-3', and a downstream primer R-tynA containing an EcoR I cleavage site: 5'-AACTCGAGTCACTTATCTTTCTTCAGCGCC-3' are provided. Extracting Escherichia coli K12 genome DNA by using a bacterial genome DNA extraction kit, and performing PCR amplification by using the genome DNA as a template, wherein a PCR reaction system comprises 10 XExTaq buffer (Mg)2+Plus) 5. mu.L, dNTPs mix 4.0. mu.L, TaKaRa ExTaq 0.5. mu.L, template 1.0. mu.L, upstream and downstream primers 1.0. mu.L each, with dd H2O make up to 50. mu.L. The PCR reaction parameters are as follows: 94 ℃ for 30s30s at 60 ℃ and 1min at 72 ℃, and after 30 cycles, the extension is carried out for 10min at 72 ℃. The PCR amplification products were detected on a 1% agarose gel and the target DNA fragments were purified using AxyPrep DNA gel recovery kit. Vector pET28a and the tynA fragment were digested with BamH I and EcoR I, respectively, and the tynA fragment was ligated to the vector with DNA ligase and transformed into E.coli DH5 α. The positive clone is verified to be correct by colony PCR and enzyme digestion identification to obtain a pET28a-tynA recombinant plasmid, and the recombinant plasmid is used for transforming E.coli BL21(DE3) competent cells to obtain recombinant bacteria containing primary amine oxidase.
Example 2 production of Primary amine oxidase by fermentation of recombinant bacteria and production of 4-hydroxyphenylacetaldehyde by enzymatic conversion of tyramine
(1) Inoculating the constructed genetic engineering bacteria into an LB slant culture medium to be cultured for 16h at 37 ℃;
(2) inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shake culture at 37 deg.C and 200r/min for 6 h;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by 8 percent of seed amount, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value to be more than 20 percent, the pH value is adjusted by 25 percent ammonia water to be stabilized at 7.0, and the culture is carried out for 6 hours at 37 ℃ and is cooled to 30 ℃ for expression. 500g/L of glycerol solution is fed during the fermentation process to maintain the glucose concentration at 5 g/L. After the fermentation is finished, the thalli is collected by centrifugation at 8000r/min and 4 ℃ for 10min, and the thalli is washed twice by sterile normal saline.
(4) Weighing 2.5g of tyramine, dissolving in 200mL of water, adjusting the pH value to 7.0 by 2mol/L of hydrochloric acid, fixing the volume to 500mL, adding 100000U of catalase and 10g of wet bacteria for catalysis, controlling the temperature to be 30 ℃, ventilating to control the dissolved oxygen to be 5% of air saturation, converting and producing 4-hydroxyphenylacetaldehyde on a 1L automatic control fermentation tank, continuously sampling and determining the concentration of the 4-hydroxyphenylacetaldehyde in the process, determining the concentration of the 4-hydroxyphenylacetaldehyde not to increase after reacting for 1h, determining the concentration of the 4-hydroxyphenylacetaldehyde to be 4.8g/L by liquid chromatography, and determining the mass conversion rate to be 96%.
The 4-hydroxyphenylacetaldehyde concentration was determined by liquid chromatography under conditions of Diamonsil C18 column (250 mm. times.4.6 mm,5 μm, DIKMA), column temperature: 30 ℃; detection wavelength: 276 nm; mobile phase A: 5% methanol, 0.1% trifluoroacetic acid, mobile phase B: 90% methanol, 0.1% trifluoroacetic acid. And (3) carrying out gradient elution according to the mobile phase B according to the flow rate of 0-35%, 10min, 35%, 4min, 35-100% and 4min, wherein the flow rate of the mobile phase is 1 mL/min.
Example 3 production of Primary amine oxidase by fermentation of recombinant bacteria and production of 4-hydroxyphenylacetaldehyde by enzymatic conversion of tyramine
(1) Inoculating the genetically engineered bacteria constructed in the embodiment 1 into an LB slant culture medium to be cultured for 15h at 37 ℃;
(2) inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shake culture at 37 deg.C and 200r/min for 8 h;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by the amount of 7 percent of the volume ratio, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value above 20 percent of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25 percent to be stabilized at 7.0, and the culture is carried out at 37 ℃ for 7h and then is cooled to 28 ℃ for expression. 500g/L of glycerol solution is fed during the fermentation process to maintain the concentration of glycerol at 3-5 g/L. After the fermentation is finished, the thalli is collected by centrifugation at 8000r/min and 4 ℃ for 10min, and the thalli is washed twice by sterile normal saline.
(4) Weighing 7.5g of tyramine, dissolving in 200mL of water, adjusting the pH value to 7.0 by 2mol/L of hydrochloric acid, fixing the volume to 500mL, adding 100000U of catalase and 15g of wet bacteria for catalysis, controlling the temperature to be 30 ℃, ventilating to control the dissolved oxygen to be 5% of air saturation, converting and producing 4-hydroxyphenylacetaldehyde on a 1L automatic control fermentation tank, continuously sampling and determining the concentration of the 4-hydroxyphenylacetaldehyde in the process, determining the concentration of the 4-hydroxyphenylacetaldehyde not to increase after reacting for 2 hours, determining the concentration of the 4-hydroxyphenylacetaldehyde to be 5.3g/L by liquid chromatography, and determining the mass conversion rate to be 35.3%. 4-Hydroxyphenylacetaldehyde was determined as in example 2.
Example 4 production of Primary amine oxidase by fermentation of recombinant bacteria and production of 4-hydroxyphenylacetaldehyde by enzymatic conversion of tyramine
(1) Inoculating the constructed genetic engineering bacteria into an LB slant culture medium to be cultured for 16h at 37 ℃;
(2) inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shake culture at 37 deg.C and 200r/min for 8 h;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by 7 percent of seed amount, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value to be more than 20 percent, the pH value is adjusted by 25 percent ammonia water to be stabilized at 7.0, and the culture is carried out for 6 hours at 37 ℃ and is cooled to 26 ℃ for expression. 500g/L of glycerol solution is fed during the fermentation process to maintain the concentration of glycerol at 3 g/L. After the fermentation is finished, the thalli is collected by centrifugation at 8000r/min and 4 ℃ for 10min, and the thalli is washed twice by sterile normal saline.
(4) Weighing 7.5g of tyramine, dissolving in 200mL of water, adjusting the pH value to 7.0 by 2mol/L of hydrochloric acid, fixing the volume to 300mL, adding 200mL of ethyl acetate to make the final volume reach 500mL, uniformly mixing, adding 150000U of catalase and 10g of wet bacteria, catalyzing, controlling the temperature to be 30 ℃, introducing air to control the dissolved oxygen to be 5%, converting and producing 4-hydroxyphenylacetaldehyde on a 1L automatic control fermentation tank, continuously sampling and determining the concentration of the 4-hydroxyphenylacetaldehyde in the process, ensuring that the concentration of the 4-hydroxyphenylacetaldehyde does not increase after reacting for 3.5h, determining the concentration of the 4-hydroxyphenylacetaldehyde to be 12.1g/L by liquid chromatography, and ensuring the mass conversion rate to be 80.6%. 4-Hydroxyphenylacetaldehyde was determined as in example 2.
Example 5 production of Primary amine oxidase by fermentation of recombinant bacteria and production of 4-hydroxyphenylacetaldehyde by enzymatic conversion of tyramine
(1) Inoculating the constructed genetic engineering bacteria into an LB slant culture medium to be cultured for 16h at 37 ℃;
(2) inoculating 1-loop slant strain to LB liquid seed culture medium, and performing shake culture at 37 deg.C and 200r/min for 8 h;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid is inoculated into the fermentation culture medium by 7 percent of seed amount, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value to be more than 20 percent, the pH value is adjusted by 25 percent ammonia water to be stabilized at 7.0, and the culture is carried out for 7 hours at 37 ℃ and is cooled to 26 ℃ for expression. 500g/L of glycerol solution is fed during the fermentation process to maintain the concentration of the glycerol at 5 g/L. After the fermentation is finished, the thalli is collected by centrifugation at 8000r/min and 4 ℃ for 10min, and the thalli is washed twice by sterile normal saline.
(4) Weighing 7.5g of tyramine, dissolving in 150mL of water, adjusting the pH value to 7.0 by 2mol/L of hydrochloric acid, fixing the volume to 250mL, adding 250mL of butyl acetate to make the final volume reach 500mL, uniformly mixing, adding 100000U of catalase and 15g of wet bacteria, catalyzing, controlling the temperature to be 30 ℃ and introducing air to control the dissolved oxygen to be 5%, converting and producing 4-hydroxyphenylacetaldehyde on a 1L automatic control fermentation tank, continuously sampling and determining the concentration of the 4-hydroxyphenylacetaldehyde in the process, ensuring that the concentration of the 4-hydroxyphenylacetaldehyde does not increase after reacting for 3.5h, determining the concentration of the 4-hydroxyphenylacetaldehyde to be 12.5g/L by liquid chromatography, and ensuring the mass conversion rate to be 83.3%. 4-Hydroxyphenylacetaldehyde was determined as in example 2.
Sequence listing
<110> university of Ludong
<120> method for preparing 4-hydroxyphenylacetaldehyde by enzyme method
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2274
<212> DNA
<213> Escherichia coli K12(Escherichia coli K-12)
<400> 1
atgggaagcc cctctctgta ttctgcccgt aaaacaaccc tggcgttggc agtcgcctta 60
agtttcgcct ggcaagcgcc ggtatttgcc cacggtggtg aagcgcatat ggtgccaatg 120
gatgaaacgc ttaaagaatt tggtgccgat gtgcagtggg acgactacgc ccagatcttt 180
accctgatta aagatggcgc gtacgtgaaa gtgaagcctg gtgcgcaaac agcaattgtt 240
aatggtcaac ctctggcact gcaagtgccg gtagtgatga aagacaataa ggcctgggtt 300
tctgacacct ttattaacga tgttttccag tccggtctgg atcaaacctt tcaggtagaa 360
aagcgccctc acccacttaa tgcgctaact gcggacgaaa ttaaacaggc cgttgaaatt 420
gttaaagctt ccgcggactt caaacccaat acccgtttta ctgagatctc cctgctaccg 480
ccagataaag aagctgtctg ggcgtttgcg ctggaaaaca aaccggttga ccagccacgc 540
aaagccgacg tcattatgct cgacggcaaa catatcatcg aagcggtggt ggatctgcaa 600
aacaacaaac tgctctcctg gcaacccatt aaagacgccc acggtatggt gttgctggat 660
gatttcgcca gtgtgcagaa cattattaac aacagtgaag aatttgccgc tgccgtgaag 720
aagcgcggta ttactgatgc gcagaaagtg attaccacgc cgctgaccgt aggttatttc 780
gatggtaaag atggcctgaa acaagatgcc cggttgctca aagtcatcag ctatcttgat 840
gtcggtgatg gcaactactg ggcacatccc atcgaaaacc tggtggcggt cgttgattta 900
gaacagaaaa aaatcgttaa gattgaagaa ggtccggtag ttccggtgcc aatgaccgca 960
cgcccatttg atggccgtga ccgcgttgct ccggcagtta agcctatgca aatcattgag 1020
cctgaaggta aaaattacac cattactggc gatatgattc actggcggaa ctgggatttt 1080
cacctcagca tgaactcgcg cgtcggacct atgatctcca ccgtgactta taacgacaat 1140
ggcaccaaac gcaaagtcat gtacgaaggt tctctcggcg gcatgattgt gccttacggt 1200
gatcctgata ttggctggta ctttaaagcg tatctggatt ctggtgacta cggtatgggc 1260
acgctaacct caccaattgc tcgtggtaaa gatgccccgt ctaacgcagt gctccttaat 1320
gaaaccatcg ccgactacac tggcgtgccg atggagatcc ctcgcgctat cgcggtattt 1380
gaacgttatg ccgggccgga gtataagcat caggaaatgg gccagcccaa cgtcagtacc 1440
gaacgccggg agttagtggt gcgctggatc agtacagtgg gtaactatga ctacattttt 1500
gactggatct tccatgaaaa cggcactatt ggcattgatg ccggtgctac gggcatcgaa 1560
gcggtgaaag gtgttaaagc gaaaaccatg cacgatgaga cggcgaaaga tgacacgcgc 1620
tacggcacgc ttatcgatca caatatcgtg ggtactacac accaacatat ttataatttc 1680
cgcctcgatc tggatgtaga tggcgagaat aacagcctgg tggcgatgga cccagtggta 1740
aaaccgaata ctgccggtgg cccacgcacc agtaccatgc aagttaatca gtacaacatc 1800
ggcaatgaac aggatgccgc gcagaagttt gatccgggca cgattcgtct gttgagtaac 1860
ccgaacaaag agaaccgcat gggcaatcca gtttcctatc aaattattcc ttatgcaggt 1920
ggtactcacc cggtagcaaa aggtgcccag ttcgcgccgg acgagtggat ctatcatcgt 1980
ttaagcttta tggacaagca gctttgggta acgcgttatc atcctggcga gcgtttcccg 2040
gaaggcaaat atccgaaccg ttctactcat gacaccggtc ttggacaata cagtaaagat 2100
aacgagtcgc tggacaacac cgacgccgtt gtctggatga ccaccggcac cacacatgtg 2160
gcccgcgccg aagagtggcc gattatgccg accgaatggg tacatactct gcttaaacca 2220
tggaacttct ttgacgaaac gccaacgcta ggggcgctga agaaagataa gtga 2274
<210> 2
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaggatccat gggaagcccc tctctgtatt c 31
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aactcgagtc acttatcttt cttcagcgcc 30
Claims (7)
1. A method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method is characterized by comprising the following specific steps:
(1) constructing a recombinant bacterium for producing the primary amine oxidase, and fermenting to obtain a wet bacterium for producing the recombinant bacterium for producing the primary amine oxidase; the primary amine oxidase is derived from a primary amine oxidase gene tynA of Escherichia coli K12, and the nucleotide sequence of the primary amine oxidase gene tynA is shown as SEQ ID No. 1;
(2) single water phase conversion: preparing tyramine aqueous solution with the pH value of 7.0 and the concentration of 0.5-1.5g/100mL, adding catalase with the final concentration of 200-400U/mL and recombinant bacteria wet bacteria with the final concentration of 1-3% g/100mL for producing primary amine oxidase for catalysis, controlling the temperature to be 28-40 ℃, ventilating to control the dissolved oxygen to be 5-15% of air saturation, converting and producing 4-hydroxyphenylacetaldehyde in an automatic control fermentation tank, continuously sampling and measuring the concentration of the 4-hydroxyphenylacetaldehyde in the reaction process until the concentration of the 4-hydroxyphenylacetaldehyde is not increased any more, and finishing the reaction.
2. The method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method as claimed in claim 1, wherein the method comprises preparing a tyramine two-phase reaction system with a pH of 7.0 of 0.5-1.5g/100mL, wherein the two-phase reaction system comprises an organic solvent phase located at the lower aqueous phase and the upper aqueous phase of a container, the organic solvent is 20-60% of the volume of the aqueous phase, and then adding a recombinant wet bacterial strain with a final concentration of 200-400U/mL catalase and a final concentration of 1-3% g/100mL primary amine oxidase to perform a catalytic reaction.
3. The method for producing 4-hydroxyphenylacetaldehyde by an enzymatic process according to claim 2, wherein the organic solvent is one of ethyl acetate and butyl acetate.
4. The method for producing 4-hydroxyphenylacetaldehyde by an enzymatic method according to claim 1, which is characterized in that the method for constructing the recombinant bacterium for producing the primary amine oxidase comprises the following steps:
(1) cloning a primary amine oxidase gene tynA of escherichia coli K12, wherein the nucleotide sequence of the primary amine oxidase gene tynA is shown as SEQ ID No. 1;
(2) carrying out double enzyme digestion and connection on the nucleotide sequence shown in SEQ ID No.1 and a vector pET28a, and screening positive clones to obtain a recombinant vector pET28 a-tynA;
(3) the recombinant vector pET28a-tynA is transformed into a host bacterium E.coli BL21(DE3) to obtain a recombinant bacterium E.coli BL21(DE3)/pET28a-tynA for producing the primary amine oxidase.
5. The method for obtaining the recombinant wet bacterial strain for producing the primary amine oxidase of claim 1, which comprises the following steps:
(1) inoculating the constructed recombinant bacteria producing the primary amine oxidase into an LB slant culture medium to be cultured for 14-24 h at 37 ℃;
(2) inoculating a recombinant strain which produces primary amine oxidase on a 1-ring inclined plane into an LB liquid seed culture medium, and carrying out shake culture at 37 ℃ for 6-12 h at 200 r/min;
(3) 3.0L of culture medium is filled in a 5L fermentation tank, the seed liquid cultured in the step (2) is inoculated into the fermentation culture medium by an inoculum size of 5-10% of the volume ratio, the initial rotating speed is 200r/min, the initial aeration flow is 1.5L/min, the rotating speed and the aeration flow are adjusted along with the increase of the thallus concentration so as to maintain the dissolved oxygen value above 20% of air saturation, the pH value is adjusted by ammonia water with the mass volume ratio of 25% to be stabilized at 7.0, and the temperature is reduced to 22-28 ℃ for expression after the seed liquid is cultured for 6-10 h at 37 ℃; adding 500g/L of glycerol solution in a flowing manner in the fermentation process to maintain the concentration of the glycerol at 8000r/min and 4 ℃ for 15min after the fermentation is finished, collecting thalli by centrifugation, and washing the thalli twice by using sterile normal saline;
the fermentation medium comprises: 20g/L of glycerol, 10g/L of peptone, 8g/L of yeast extract powder, 1 percent of corn steep liquor and MgSO4 5 g/L，KH2PO4 10 g/L，(NH4)2SO43g/L, citric acid 2g/L, CuSO450mg/L, pH7.2-7.4, and sterilizing with high pressure steam at 121 deg.C for 20 min.
6. The method according to claim 5, wherein the LB slant medium: 5g/L of yeast extract powder, 10g/L of peptone, 5g/L of NaCl, 100mg/L of ampicillin, 20g/L of agar, pH 7.0-7.2, and performing high-pressure steam sterilization at 121 ℃ for 20 min.
7. The method according to claim 5, wherein said LB liquid seed medium: 5g/L yeast extract powder, 10g/L peptone, 5g/L NaCl, 50mg/L kanamycin, 7.0-7.2 pH, and high-pressure steam sterilization at 121 ℃ for 20 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910856791.0A CN110499343B (en) | 2019-09-11 | 2019-09-11 | Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910856791.0A CN110499343B (en) | 2019-09-11 | 2019-09-11 | Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110499343A CN110499343A (en) | 2019-11-26 |
| CN110499343B true CN110499343B (en) | 2021-06-04 |
Family
ID=68591647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910856791.0A Active CN110499343B (en) | 2019-09-11 | 2019-09-11 | Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110499343B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2318514A4 (en) * | 2008-07-23 | 2013-08-21 | Opx Biotechnologies Inc | Methods, systems and compositions for increased microorganism tolerance to and production of 3-hydroxypropionic acid (3-hp) |
| BR112016029386A2 (en) * | 2014-06-16 | 2017-10-17 | Invista Tech Sarl | methods, reagents and cells to biosynthesize compounds |
| EP3314002A4 (en) * | 2015-06-23 | 2019-07-10 | Genomatica, Inc. | Microorganisms and methods for the production of biosynthesized target products having reduced levels of byproducts |
| CN105779522B (en) * | 2016-05-17 | 2016-12-28 | 河南巨龙生物工程股份有限公司 | A kind of microbial enzyme conversion method produces the method for L 4 hydroxyisoleucine |
-
2019
- 2019-09-11 CN CN201910856791.0A patent/CN110499343B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110499343A (en) | 2019-11-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107502599B (en) | Method for synthesizing 3-O-glucosyl glycyrrhetinic acid by enzyme method | |
| CN107502647A (en) | A kind of method that biological enzyme deracemization prepares L glufosinate-ammoniums | |
| CN112662638B (en) | Function of R-selective styrene monooxygenase | |
| CN112813013B (en) | Recombinant escherichia coli for producing hydroxytyrosol and application thereof | |
| CN114015669B (en) | Histidinol phosphate aminotransferase mutant 197, engineering bacteria and application | |
| CN102827853B (en) | Halogenohydrin dehalogenation enzyme gene mutant and application thereof | |
| US11098287B2 (en) | 17β-hydroxysteroid dehydrogenase mutants and application thereof | |
| CN110499343B (en) | Method for preparing 4-hydroxyphenylacetaldehyde by enzyme method | |
| CN101469318B (en) | Synthesis of (R)-styrene glycol by coupling acceleration of (R)-carbonyl reduction enzyme and formic dehydrogenase | |
| CN110257448B (en) | Method for converting arginine into agmatine by utilizing thallus whole cell catalysis | |
| CN110903993A (en) | Saccharomyces cerevisiae engineering bacterium for producing brassicasterol and construction method and application thereof | |
| CN107828752B (en) | Saccharopolyase, preparation method and application in production of alpha-arbutin | |
| CN106119272B (en) | Strategy for efficiently co-producing L-phenylglycine and gluconic acid | |
| CN104830744A (en) | Method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase | |
| CN116064435A (en) | Curcumin reductase Cfcura, coding gene and application thereof | |
| CN105820966B (en) | Efficient chitosanase producing strain and fermentation method thereof | |
| CN114457127A (en) | Recombinant engineering bacterium and construction method and application thereof | |
| CN109749948A (en) | A kind of recombinant pichia yeast strain and construction method sticking the expression of corn glutamine transaminage using TEF1 promoter regulation | |
| CN114958703B (en) | Recombinant bacterium for synthesizing succinic acid by utilizing grease, construction method and application thereof | |
| CN104673808B (en) | A kind of malic enzyme gene and its recombinant expression carrier | |
| CN102719463B (en) | A kind of method producing restructuring mixing L-arabinose isomerase | |
| CN116694585A (en) | Application of ketoamine reductase in synthesizing chiral pramipexole and synthetic method | |
| CN101104862A (en) | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin | |
| CN116694542A (en) | Double-enzyme co-expression strain for producing L-malic acid and construction method and application thereof | |
| CN106957854B (en) | Method for improving acetic acid tolerance of yeast cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for preparing 4-hydroxyphenylacetaldehyde by enzymatic method Effective date of registration: 20211216 Granted publication date: 20210604 Pledgee: Yantai financing guarantee Group Co.,Ltd. Pledgor: LUDONG University Registration number: Y2021980015152 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220317 Granted publication date: 20210604 Pledgee: Yantai financing guarantee Group Co.,Ltd. Pledgor: LUDONG University Registration number: Y2021980015152 |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221019 Address after: No. 1666, East Ring Road, Juancheng County, Heze City, Shandong Province 274699 Patentee after: SHANDONG YANGCHENG BIOTECH Co.,Ltd. Address before: 264025 No. 186 Hongqi Middle Road, Zhifu District, Shandong, Yantai Patentee before: LUDONG University |