CN115521953A - Method for synthesizing p-acetamidophenol through enzyme catalysis - Google Patents
Method for synthesizing p-acetamidophenol through enzyme catalysis Download PDFInfo
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- CN115521953A CN115521953A CN202110706349.7A CN202110706349A CN115521953A CN 115521953 A CN115521953 A CN 115521953A CN 202110706349 A CN202110706349 A CN 202110706349A CN 115521953 A CN115521953 A CN 115521953A
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- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 32
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- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 11
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 10
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims abstract description 52
- 229960004050 aminobenzoic acid Drugs 0.000 claims abstract description 25
- 108020005224 Arylamine N-acetyltransferase Proteins 0.000 claims abstract description 20
- 108010074633 Mixed Function Oxygenases Proteins 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 102000006809 Arylamine N-Acetyltransferase Human genes 0.000 claims abstract description 15
- 102000008109 Mixed Function Oxygenases Human genes 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 230000035484 reaction time Effects 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 23
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 17
- 239000007995 HEPES buffer Substances 0.000 claims description 17
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- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 claims description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 6
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- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 3
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- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 2
- VWWQXMAJTJZDQX-UHFFFAOYSA-N Flavine adenine dinucleotide Natural products C1=NC2=C(N)N=CN=C2N1C(C(O)C1O)OC1COP(O)(=O)OP(O)(=O)OCC(O)C(O)C(O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UHFFFAOYSA-N 0.000 claims description 2
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- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 description 2
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- 108020004705 Codon Proteins 0.000 description 2
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- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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Abstract
A method for synthesizing acetaminophen through enzyme catalysis, which belongs to the technical field of biological engineering. In order to solve the problems of high toxicity, serious environmental pollution caused by waste liquid, high catalyst price, more reaction process steps, complicated post-treatment and the like in the synthesis process of the p-acetaminophenol, the p-aminobenzoic acid is used as a substrate, the p-aminobenzoic acid hydroxylase and the arylamine-N acetyltransferase are added to catalyze and synthesize the p-acetaminophenol, a way for enzymatically synthesizing the p-acetaminophenol by using the p-aminobenzoic acid as the substrate is established for the first time, and a new thought is provided for the synthesis of the p-acetaminophenol. Compared with chemical synthesis, the synthesis method has the advantages of neutral reaction conditions, no need of any metal catalyst, capability of reducing the pollution of three-waste discharge to the environment, safe and simple operation, short reaction time, high efficiency and yield of more than 98 percent, and the reaction is carried out at normal temperature and normal pressure.
Description
Technical Field
The invention relates to a method for synthesizing acetaminophen through enzyme catalysis, and belongs to the technical field of biological engineering.
Background
Paracetamol is a commonly used antipyretic analgesic, has slow and lasting antipyretic action and is listed in the basic medicine list of the world health organization in the 20 th century. At present, acetaminophen is one of the most widely used medicines in the world, and is one of the varieties with the largest yield in raw material medicines in China, and in addition, acetaminophen can also be used for synthesis of medicine paracetamol, organic medicine intermediates, photographic chemicals, stabilizers of hydrogen peroxide and the like. In recent years, demand for acetaminophen is increasing at home and abroad, and the original production process is synthesized by a chemical method, so that the following problems exist: on one hand, the precursors or intermediates in the synthesis process all relate to benzene derivative compounds, such as nitrobenzene and the like, and are flammable and explosive, high in toxicity and serious in environmental pollution caused by waste liquid; on the other hand, the catalyst is expensive, the reaction process has many steps, and the post-treatment is complicated. Therefore, the development of a safe and efficient acetaminophen synthesis process with less environmental pollution is of great significance. The enzyme catalysis reaction has the characteristics of environmental protection, mild reaction conditions, high efficiency and the like, so people hope to develop an enzyme catalysis synthesis method of p-aminophenol.
Disclosure of Invention
The invention provides a method for synthesizing p-acetaminophenol under enzyme catalysis, aiming at solving the problems of high toxicity, serious environmental pollution caused by waste liquid, high catalyst price, multiple reaction process steps, complex post-treatment and the like in the synthesis process of p-acetaminophenol.
In one embodiment of the present invention, the reaction system further comprises NADH, FAD, acetyl-CoA.
In one embodiment of the present invention, the nucleotide sequence of the p-aminobenzoic acid hydroxylase gene is shown as SEQ ID No. 1; the nucleotide sequence of the arylamine-N acetyltransferase gene is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the method for preparing the p-aminobenzoic acid hydroxylase and the arylamine-N acetyltransferase comprises the steps of:
s1, cloning a p-aminobenzoic acid hydroxylase gene and an arylamine-N acetyltransferase gene to a vector pET-Duet-1 and a vector pET-28a respectively to obtain recombinant plasmids respectively;
s2, introducing the recombinant plasmid obtained in the step S1 into Escherichia coli BL21 (DE 3) to obtain a recombinant bacterium;
s3, culturing the recombinant bacteria obtained in the step S2 by using an LB liquid culture medium, inducing by using IPTG, centrifuging fermentation liquor to obtain thalli, redissolving the thalli by using HEPES lysate, crushing by using a high-pressure crusher, and centrifuging and collecting supernatant to obtain crude enzyme liquid;
and S4, purifying the crude enzyme solution obtained in the step S3 by adopting a nickel column to obtain the purified enzyme.
In one embodiment of the invention, the induction of S3 is induced by adding 20. Mu.L of 1M IPTG at 16 ℃ for 16h.
In one embodiment of the present invention, the elution method for the nickel column purification described in S4 is as follows: and (4) fully incubating the crude enzyme solution obtained in the step (S3) and nickel beads for 1h at 4 ℃, transferring the incubated solution into a gravity column, washing to remove protein by using a washing solution, eluting the target protein by using an eluent, and storing the purified enzyme in a protein storage solution for cryopreservation at-80 ℃.
In one embodiment of the invention, the composition of the wash solution is 25mM HEPES,0.5M NaCl,20mM imidazole, pH7.5; the composition of the eluent is 25mM HEPES,0.3M NaCl,150mM imidazole, pH7.5; the protein stock solution had a composition of 25mM HEPES, pH7.5,0.3M NaCl.
In one embodiment of the invention, the temperature of the enzymatic reaction of the process is 25-35 ℃.
In one embodiment of the invention, the enzymatic reaction of the process has a pH of 6.5 to 8.0.
In one embodiment of the invention, the enzymatic reaction time of the method is 2 to 3.5 hours.
The invention has the beneficial effects that:
the invention establishes a path (shown in figure 2) for synthesizing the paracetamol by taking the para aminobenzoic acid as a substrate through enzyme catalysis for the first time, and provides a new idea for the synthesis of the paracetamol. Compared with chemical synthesis: 1) The reaction condition is neutral, and the pollution of three-waste discharge to the environment can be reduced without any metal catalyst; 2) The reaction is carried out at normal temperature and normal pressure, and the operation is safe and simple; 3) Short reaction time, high efficiency and high yield up to more than 98%.
Drawings
FIG. 1 is a HPLC check of an enzyme catalyzed reaction to acetaminophen, wherein A is the reaction product; b is a acetaminophen standard substance;
FIG. 2 is a diagram showing the pathway for enzymatically synthesizing acetaminophen.
Detailed Description
The present invention will be described in further detail with reference to specific examples and drawings, and procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for those specifically mentioned below, and the present invention is not particularly limited thereto.
The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available. The enzyme reagent is purchased from NEW ENGLAND BioLabs, the kit for extracting plasmid and the kit for recovering DNA fragment are purchased from American OMEGA company, and the corresponding operation steps are carried out according to the product instruction; all media were prepared with deionized water unless otherwise specified.
The formula of the culture medium is as follows:
LB liquid medium: 5g/L yeast powder, 10g/L NaCl,10g/L peptone and the balance water, sterilizing at 121 ℃ for 20min. During the actual culture process, antibiotics with a certain concentration, such as 100mg/L ampicillin and 50mg/L chloramphenicol, can be added to the above culture medium to maintain the stability of plasmids.
Example 1: preparation of p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase
1) Codon optimization
Because the host bacteria selected by the invention are escherichia coli, the nucleotide sequence of the escherichia coli is synthesized after the escherichia coli codon optimization is carried out on the p-aminobenzoic hydroxylase and arylamine-N acetyltransferase sequences, the obtained p-aminobenzoic hydroxylase gene is derived from Agaricus bisporus (Agaricus bisporus), and the nucleotide sequence is shown as SEQ ID NO. 1; the arylamine-N acetyltransferase gene is derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa), and the nucleotide sequence is shown as SEQ ID NO. 2. The gene sequences of the synthesized and optimized p-aminobenzoic acid hydroxylase and the arylamine-N acetyltransferase are respectively cloned to pET-Duet-1 (the sequence of the used forward primer is shown as SEQ ID NO.3, and the sequence of the used reverse primer is shown as SEQ ID NO. 4) and pET-28a (the sequence of the used forward primer is shown as SEQ ID NO.5, and the sequence of the used reverse primer is shown as SEQ ID NO. 6) vectors to obtain the recombinant plasmid containing the target gene.
2) Competent preparation of E.coli BL21 (DE 3)
(1) Streaking the preserved strain BL21 (DE 3) on an LB (Langmuir-Blodgett) plate, culturing overnight, picking a single clone in a test tube containing 3mL of LB culture medium, and culturing overnight;
(2) Inoculating into 100mL LB medium at 0.5%, and culturing at 37 deg.C to OD 600 About 0.4 (about 2h to about 3 h);
(3) Precooling the cultured bacterial liquid on ice for 20min, subpackaging in precooled sterile test tubes, and centrifuging at 6000rpm/min at 4 ℃ for 10min;
(4) The supernatant was discarded and 20mL of pre-cooled 0.1mol/L CaCl were added 2 Suspending thallus in the solution, standing on ice for 30min, and centrifuging at 6000rpm/min at 4 deg.C for 10min. Repeating twice;
(5) The supernatant was discarded and 1mL of pre-cooled 0.1mol/L CaCl was added 2 The solution (containing 15% glycerol), the cells were gently suspended and distributed into pre-cooled 1.5mL test tubes, each containing 100. Mu.L of the cell suspension, and stored in a freezer at-80 ℃.
3) Recombinant plasmid transformation BL21 (DE 3)
(1) Adding 1 μ L recombinant plasmid into the prepared BL21 (DE 3) competence, mixing lightly, and ice-cooling for 30min;
(2) Immediately carrying out ice bath for 2min after the heat shock treatment for 45s in a preheated 42 ℃ water bath;
(3) Adding 700 μ L LB culture solution without antibiotic, culturing at 37 deg.C for 1h to recover thallus;
(4) Uniformly coating 200 mu L of bacterial liquid on an LB flat plate containing the aminobenzyl antibiotic;
(5) Cultured overnight at 37 ℃ and stored for later use.
4) Induction expression, separation and purification of p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase
(1) Add 500. Mu.L of recombinant bacteria to 200mL of LB medium. Culturing at 37 deg.C to OD 600 And (4) cooling on ice for 20min at 0.8. 20 μ L of 1M IPTG was added and induced at 16 ℃ for 16h. The fermentation broth was centrifuged (6000 rpm/min, 10 min) to obtain cells, the cells were redissolved with HEPES lysate (25mM HEPES, pH7.5,0.5M NaCl,5mM imidazole), disrupted with a high pressure disrupter, and the supernatant was collected by centrifugation (13000 rpm/min,30 min) to obtain a crude enzyme solution.
(2) Purifying the crude enzyme liquid obtained in the step (1) by adopting a nickel column, wherein the elution method comprises the following steps: and (3) fully incubating the crude enzyme solution and the nickel beads for 1h at 4 ℃, and transferring the incubated solution into a gravity column. The foreign proteins were washed with a washing solution (25mM HEPES, pH7.5,0.5M NaCl,20mM imidazole), and the objective protein was eluted with an eluent (25mM HEPES, pH7.5,0.3M NaCl,150mM imidazole). The purified enzyme was stored in a protein stock (25mM HEPES, pH7.5,0.3M NaCl), frozen at-80 ℃.
Example 2: enzymatic synthesis of acetaminophen
(1) Reaction temperature testing of Acetaminophenase-catalyzed pathway
Taking p-aminobenzoic acid as a substrate, adding the amounts of p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase of 20 mu g/mL, carrying out water bath on the substrate and 50mM HEPES buffer solution with the pH value of 7.0 for 30min at different temperatures of 20-60 ℃, and measuring the enzyme activities of the p-aminobenzoic acid decarboxylation hydroxylase and the arylamine-N acetyltransferase, wherein the reaction temperature of the enzyme is preferably 25-35 ℃ and the optimal reaction temperature is 30 ℃.
(2) Reaction optimum pH test for Acetaminophenase catalytic pathway
Taking p-aminobenzoic acid as a substrate, wherein the addition amount of p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase is 20 mu g/mL, performing water bath on the substrate and 50mM HEPES buffer solution with the pH value of 4.0-10.0 for 30min at different temperatures of 30 ℃, and measuring the enzyme activity of the p-aminobenzoic acid decarboxylation hydroxylase and the arylamine-N acetyltransferase, wherein the reaction pH value of the enzyme is preferably 6.5-8.0, and the optimal reaction pH value is 7.5.
(3) Optimum time for reaction test of Acetaminophenase catalytic pathway
Taking p-aminobenzoic acid as a substrate, adding the amounts of p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase of 20 mu g/mL, carrying out water bath on the substrate and 50mM HEPES buffer solution with the pH value of 7.5 at different temperatures of 30 ℃ for 0.5-6 h, and measuring the enzyme activities of the p-aminobenzoic acid decarboxylation hydroxylase and the arylamine-N acetyltransferase, wherein the reaction time of the enzyme is preferably 2-3.5 h, and the optimal reaction time is 3h.
(4) Enzymatic synthesis of acetaminophen
To a 50mM HEPES buffer system pH7.5, 2mM p-aminobenzoic acid, 12U/mL p-aminobenzoic acid hydroxylase, 15U/mL arylamine-N acetyltransferase, 0.1mM FAD,0.2mM NADH and 400. Mu.M acetyl-CoA were added and reacted at 30 ℃ for 30 minutes.
Detecting the generation amount of the product acetaminophen by using high performance liquid chromatography, wherein the detection conditions of the product high performance liquid chromatography are as follows: 0-5min 15-30% acetonitrile (0.1% TFA), 85-70% water (0.1% TFA); 5-10min 30-60% acetonitrile (0.1% TFA), 70-40% water (0.1% TFA); 10-11min 60-80% acetonitrile (0.1% TFA), 40-20% water (0.1% TFA); 11-15min 80% acetonitrile (0.1% TFA), 20% water (0.1% TFA); 15-1695in 80-15% acetonitrile (0.1% TFA), 20-85% water (0.1% TFA); 16-20min 15% acetonitrile (0.1%; TFA), 85% water (0.1%; TFA), and an SPD-20A UV-visible light detector with detection wavelengths of 250nm, 270nm. The HPLC detection of acetaminophen is shown in FIG. 1.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> institute for bioenergy and Process in Qingdao of Chinese academy of sciences
<120> method for synthesizing acetaminophen by enzyme catalysis
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1401
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<213> Artificial Synthesis
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atggtgcagg gcgaacgcag ccatattgcg attattggcg cgggcattgt gggcctggcg 60
tttgcggttg cattaaatgc gttggataaa gaacataagt tcgcgatcga tctgtacgag 120
gcgaccccgg aattagcgga aattggcgca ggtattaatg tgtggccgct gaccttaagc 180
attctgaaag aaatgggcct gcatcagacc ctgattccgt tttttgatca ttatccggat 240
ctggagcgcc gcgtgatttt tggcttgcgc aaagcggatg aaaaaaatgg ctttcatgtg 300
tacgacgtga tgaacgaagg cggcgcgtta cgtattcatc gcgcggattt acagcgtggc 360
ctgattcagc atctgccgct gagcaaaagc aataaagtgc atattaatac cccgtgcacc 420
tttcatctga atcatcgcct gaaagattat acccgcgatg cgagcgaaga ttttggccgc 480
attaaactgc attttgatgg caaaccgagc cgcgaatgcg atgtgctgat tggcgcagat 540
ggcattcata gcaccgtgcg ccaattattt ctgagccgct tgccgagccc agaacgttat 600
gataaatatc gcaaaccggt gtggagcggc ctggtggcat atcgtggttt agttagccgt 660
gaagatttgg aagaaaccta tccgggccat cgcgcgctga cccatccagg tttaatttat 720
accggcaaaa cccgctatgt gaccatttat ccggtgagcg gcggcaaatt tattaatgtg 780
gtggcgattg cgcgcgatac cagcaatgat accggcgtgt ggaaaggccc gtggaaagtg 840
gaagtgaccc aggaagaatt ttttcacacc tatcagggct ttgacgagga ggtgctggcg 900
ctgattcatt gcattaaaaa accgaccaaa tgggcgctgc atgtgctgga tcatctggat 960
atttttagca aacagcaggt gtttctgatg ggcgatgcgg cgcatgcgat gctgccacat 1020
ttaggtgcgg gtgcgaccgt tggcattgaa gatgcgtata ttctggcgag catgctgacc 1080
catcagagca ccagccgtcc attgaatagc gaaaaaatta aactgatcag caccatctac 1140
aacaccgtgc tggtgccgca tgcgacccgt atgagcaaat tgaccaatga taccggccat 1200
ctgctggatc tgaccgcgcc aggttttgat ctggaacgct ataccctggg cgatcgcatt 1260
ccgctggaaa ccttaattaa tgcgtttcgc caggtggaac gcaattggat ttggagcagc 1320
agcgatccgg aagaagatcg ccgcaaagtg gaagatctgc tggaaagctg gagcggcccg 1380
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cctggtcctc cgagcctggc gaatttagat cgcctgatcg atgcgcattt acgccgcgtt 180
gcgtttgaaa acctggatgt gctgctggat cgcccgattg aaattgatgc ggacaaagtg 240
tttgcgaaag tggtggaagg tagccgtggc ggctattgct ttgaactgaa cagcctgttt 300
gcgcgtctgt tactggcgtt aggctatgaa ctggaactgc tggtggcacg tgttcgttgg 360
ggtttacctg atgatgcgcc gttaacccag cagagccatt taatgctgcg cctgtatctg 420
gcggaaggcg aatttctggt ggatgtgggc tttggttcag cgaacccgcc tcgtgcatta 480
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catgcgggct tatatgaaag cgcggtgcgt ggtcgtagcg gttggttacc tctgtatcgc 600
tttgatctgc gcccgcagct gtggattgat tatattccgc gcaactggta taccagcacc 660
catccgcata gcgtgtttcg ccagggtctg aaagcggcga ttaccgaagg tgatctgcgt 720
ctgaccttag cggatggtct gtttggtcag cgtgcgggta acggtgaaac cctgcagcgt 780
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Claims (10)
1. The method for synthesizing the p-acetamidophenol through enzyme catalysis is characterized in that p-aminobenzoic acid is taken as a substrate, and the p-aminobenzoic acid hydroxylase and arylamine-N acetyltransferase are added to catalyze and synthesize the p-acetamidophenol.
2. The method of claim 1, wherein said reaction system further comprises NADH, FAD, acetyl-CoA.
3. The method of claim 1, wherein the nucleotide sequence of the p-aminobenzoic acid hydroxylase gene in the method is shown as SEQ ID No. 1; the nucleotide sequence of the arylamine-N acetyltransferase gene is shown in SEQ ID NO. 2.
4. The method according to claim 1, wherein the preparation method of the p-aminobenzoic acid hydroxylase and the arylamine-N acetyltransferase comprises the following steps:
s1, cloning a p-aminobenzoic acid hydroxylase gene and an arylamine-N acetyltransferase gene to a vector pET-Duet-1 and a vector pET-28a respectively to obtain recombinant plasmids respectively;
s2, introducing the recombinant plasmid obtained in the step S1 into Escherichia coli BL21 (DE 3) to obtain a recombinant bacterium;
s3, culturing the recombinant bacteria obtained in the step S2 by using an LB liquid culture medium, inducing by using IPTG (isopropyl-beta-D-thiogalactoside), centrifuging fermentation liquor to obtain bacteria, redissolving the bacteria by using HEPES lysate, crushing by using a high-pressure crusher, and centrifuging to collect supernatant to obtain crude enzyme solution;
and S4, purifying the crude enzyme solution obtained in the step S3 by adopting a nickel column to obtain the purified enzyme.
5. The method of claim 4, wherein the induction of S3 is induced by adding 20 μ L of 1M IPTG at 16 ℃ for 16h.
6. The method according to claim 4, wherein the elution method of the nickel column purification of S4 is as follows: and (4) fully incubating the crude enzyme solution obtained in the step (S3) and nickel beads for 1h at 4 ℃, transferring the incubated solution into a gravity column, washing to remove protein by using a washing solution, eluting the target protein by using an eluent, and storing the purified enzyme in a protein storage solution for cryopreservation at-80 ℃.
7. The method of claim 6, wherein the wash solution has a composition of 25mM HEPES,0.5M NaCl, 2mM imidazole, pH7.5; the composition of the eluent is 25mM HEPES,0.3M NaCl,150mM imidazole, pH7.5; the composition of the protein stock was 25mM HEPES, pH7.5,0.3MNaCl.
8. The method of claim 1, wherein the temperature of the enzymatic reaction is 25-35 ℃.
9. The method of claim 1, wherein the method is carried out at a pH of 6.5 to 8.0.
10. The method according to claim 1, wherein the enzymatic reaction time of the method is 2 to 3.5 hours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170211104A1 (en) * | 2016-01-21 | 2017-07-27 | 20n Labs, Inc. | Biosynthetic production of acetaminophen, p-aminophenol, and p-aminobenzoic acid |
| US20170260555A1 (en) * | 2014-09-29 | 2017-09-14 | The Regents Of The University Of California | Method for biosynthesis of acetaminophen |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170260555A1 (en) * | 2014-09-29 | 2017-09-14 | The Regents Of The University Of California | Method for biosynthesis of acetaminophen |
| US20170211104A1 (en) * | 2016-01-21 | 2017-07-27 | 20n Labs, Inc. | Biosynthetic production of acetaminophen, p-aminophenol, and p-aminobenzoic acid |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION：XP_006456258: "hypothetical protein AGABI2DRAFT_195451 [Agaricus bisporus var. Bisporus H97]", GENPEPT DATABASE, 14 January 2014 (2014-01-14), pages 1 - 2 * |
| WESTWOOD IM 等: "Chain A, Arylamine N-acetyltransferase", GENPEPT DATABASE, 1 December 2020 (2020-12-01), pages 1 - 2 * |
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