CN113563251A

CN113563251A - Separation and extraction method of 5-hydroxytryptophan

Info

Publication number: CN113563251A
Application number: CN202110677155.9A
Authority: CN
Inventors: 徐庆阳; 陈志超; 余子辰; 张震
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2021-06-18
Filing date: 2021-06-18
Publication date: 2021-10-29

Abstract

The invention provides a method for separating and extracting 5-hydroxytryptophan, wherein fermentation engineering bacteria are Escherichia coli E.coli HTP10, and the extraction steps comprise fermentation to obtain fermentation liquor, thallus separation, pigment separation, byproduct separation, protein separation and drying to obtain a product; the method solves the problem of difficult extraction of 5-HTP produced by microbial fermentation, has simple operation of extraction process and few steps, and improves the extraction yield; the extraction equipment requirement is low, the extraction cost is reduced, and the extraction efficiency is improved; the product has high extraction purity and considerable extraction yield, and the product competitiveness and the production profit are improved; the extraction process is clean and environment-friendly, the environment-friendly property is realized, the subsequent waste gas and liquid treatment cost is reduced, and the economic benefit is further improved.

Description

Separation and extraction method of 5-hydroxytryptophan

Technical Field

The invention relates to the field of amino acid production, in particular to a method for separating and extracting 5-hydroxytryptophan.

Background

5-hydroxytryptophan (5-HTP) is a precursor for synthesizing serotonin and melatonin in animals, has important regulation effects on the control of emotion and behavior and physiological functions such as sleep, pain sensation and body temperature, and has been successfully used for treating diseases such as depression, insomnia and migraine.

Due to the high medicinal health care and market value of 5-HTP, the production research of 5-HTP is rapidly developed in recent years. The existing method for producing 5-HTP mainly comprises the process technologies of natural product extraction, chemical synthesis, microbial fermentation and the like, and the 5-HTP is extracted from seeds of African plants Ganacard by the procedures of oil pressing, extraction, coarse filtration, fine filtration, vacuum concentration, extraction impurity removal, solid-liquid separation, refining, water washing solvent removal, drying, crushing and the like (CN 111978238A). Huwenhui et al (CN102351775B) use L-tryptophan methyl ester/ethyl ester to obtain L-tryptophan methyl ester/ethyl ester hydrochloride, desalt acid to obtain L-tryptophan methyl ester/ethyl ester under alkaline condition, acetylate to obtain N-acetyl-L-tryptophan methyl ester/ethyl ester, reduce indole ring under triethylsilane-trifluoroacetic acid reduction system, oxidize indole ring 1-site nitrogen under sodium tungstate-30% hydrogen peroxide system, and finally deacetylate protecting group to obtain 5-hydroxytryptophan under acidic condition. The two methods have the defects that the yield of the extraction method is low, the extraction method is easily influenced by seasons, the raw materials are insufficient, and the region limitation becomes the main bottleneck limiting large-scale production, and the chemical method has the disadvantages of complex synthesis reaction system, complicated steps, high cost and difficult control, so that the microbial fermentation method attracts great attention in the industry in recent years. Although the microbial fermentation method has low cost and simple operation, the separation and extraction of 5-HTP is always a difficult problem in the industry, and because of the immaturity of fermentation, the yield of 5-HTP is lower, the extraction yield of the conventional extraction method is lower, and the product loss is serious; the increase of soluble protein caused by the decomposition of the thalli further improves the difficulty of separation and extraction; fermentation byproducts such as tryptophan are difficult to control, and the physicochemical property of the tryptophan is extremely similar to that of 5-HTP, so that the tryptophan becomes the biggest difficulty in the separation and extraction of the 5-HTP.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for separating and extracting 5-hydroxytryptophan.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a method for separating and extracting 5-hydroxytryptophan comprises fermenting engineering bacteria such as Escherichia coli HTP10 to obtain fermentation broth, separating thallus, separating pigment, separating by-products, separating protein, and drying to obtain the final product.

Preferably, the method for separating and extracting 5-hydroxytryptophan comprises the following steps:

(1) carrying out amplification culture on Escherichia coli E.coli HTP10 to obtain fermentation liquor containing 5-HTP, adjusting the pH of the fermentation liquor to 4.0-4.2, heating to 70 ℃, and then carrying out ultrafiltration to remove thalli and part of macromolecular protein;

(2) performing nanofiltration on the clarified fermentation liquor after ultrafiltration through a nanofiltration membrane to remove insoluble small molecular proteins and residual insoluble substances;

(3) concentrating the nanofiltration liquid to 35-50% of the original volume, and decolorizing with anion adsorption resin;

(4) decoloring the fermentation liquor obtained in the step (3) again by using activated carbon;

(5) concentrating the fermentation liquor obtained in the step (4) to 30-40% of the original volume, performing primary separation and purification by using cation exchange resin, and selecting a low-concentration ammonia water solution as an eluent;

(6) concentrating the clear liquid obtained in the step (5) to 40-60% of the original volume and drying;

(7) grinding the solid obtained in the step (6) into fine powder, redissolving the powder by using low-concentration formic acid, and quickly filtering the redissolved solution after slight stirring to obtain pure 5-HTP;

(8) and (4) drying the solid obtained by filtering in the step (7).

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the pH adjusting reagent used in step (1) is sulfuric acid, the heating mode is microwave oven or plate heat exchanger according to the total amount of fermentation broth, the ultrafiltration is performed by inorganic ceramic membrane filtration, the cut-off molecular weight is 25000-30000, and bacteria, macromolecular proteins and other insoluble substances in the fermentation broth are removed by circular filtration.

Preferably, in the 5-hydroxytryptophan separation and extraction method, the molecular weight cut-off of the nanofiltration membrane in the step (2) is 300-400, so that most substances with the molecular weight of more than 400 can be removed.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the concentration in step (3) is reverse osmosis concentration or evaporation concentration, the anion adsorption resin is weak base type anion adsorption resin with model number SQD301-FD, and the small molecule soluble protein is primarily decolorized and removed.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the amount of activated carbon used in step (4) is 0.2-0.3% by mass/volume, i.e. 2-3g of activated carbon, preferably 3g/L, is used per liter of fermentation broth for further decolorization.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the cation exchange resin in step (5) is strong acid type cation exchange resin with model number SD001 × 7H, and the eluent is 0.3moL/L ammonia water, and the step is used for separating other amino acid by-products in the fermentation broth, such as glutamic acid, and primarily separating tryptophan and 5-HTP, but the elution time of the two is similar, so that the separation of tryptophan and 5-HTP is not good.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the drying method in step (6) is spray drying, evaporation drying, freeze drying or flash drying.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the low-concentration formic acid in step (7) is 8-10% formic acid solution, tryptophan is very soluble in the formic acid solution, and 5-HTP is relatively stable in formic acid, and by using this characteristic, tryptophan and 5-HTP can be further separated, wherein if the concentration of formic acid is too low, 5-HTP is dissolved, the solubility of tryptophan is also reduced, the yield and purity are reduced, and if the cost is too high, waste is caused; and (4) grinding the solid obtained in the step (6) into fluffy powder, adding a formic acid solution, slightly stirring, and quickly filtering, wherein a suction filtration mode, a plate-frame filtration mode or a rotary vacuum filtration mode is selected according to the volume of the solution.

Preferably, in the above 5-hydroxytryptophan separation and extraction method, the drying in step (8) is oven drying, tray drying or air drying at 65-85 ℃.

Has the advantages that:

the method for separating and extracting 5-hydroxytryptophan solves the problem of difficult extraction of 5-HTP produced by microbial fermentation, has simple operation in the extraction process and few steps, and improves the extraction yield; the extraction equipment requirement is low, the extraction cost is reduced, and the extraction efficiency is improved; the product has high extraction purity and considerable extraction yield, and the product competitiveness and the production profit are improved; the extraction process is clean and environment-friendly, the environment-friendly property is realized, the subsequent waste gas and liquid treatment cost is reduced, and the economic benefit is further improved.

Detailed Description

Example 1

Escherichia coli HTP10 (obtained by artificial modification of e.coli W3110(ATCC 27325), purchased from tianjin science and technology university) was used as a producer, and activated on a slant medium for two generations: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast powder, 2.5g/L of NaCl, 25g/L of agar powder and pH 6.8-7.0; inoculating all the second-generation activated strains to a seed tank, and performing seed tank culture, wherein a seed culture medium is as follows: glucose 20g/L, MgSO₄ 0.5g/L，KH₂PO₄ 1.2g/L，(NH₄)₂SO₄ 5g/L，FeSO₄.7H₂O 10mg/L，MnSO₄.H₂O5 mg/L, yeast extract powder 10g/L, V_B10.3mg/L，V_H0.2mg/L, 1g/L of defoaming agent, adjusting the culture medium with ammonia water and maintaining the pH value to 6.7-7.0 when the OD of the seeding tank is₆₀₀Fermenting in a fermentation tank when the fermentation temperature is more than or equal to 25 ℃; inoculating 15% of seed liquid to a fermentation tank, continuously culturing, adding 100ml of composite auxiliary materials in a flowing manner for 2h, adding 2g of tryptophan, 4g of glutamine and 2g of alpha-ketoglutaric acid in each liter of fermentation liquid, and fermenting for 32h to obtain the fermentation liquid. The adopted fermentation medium is as follows: glucose 20g/L, yeast powder 4g/L, MgSO₄ 1.2g/L，KH₂PO₄ 2g/L，(NH₄)₂SO₄5g/L，FeSO₄.7H₂O 10mg/L，MnSO₄.H₂O10 mg/L, corn steep liquor 20ml/L, VB_Mix2mg/L, adjusting the culture of the fermentation tank by ammonia water and maintaining the pH value to be between 6.7 and 7.0.

The specific method for obtaining the E.coli HTP10 by artificially modifying E.coli W3110(ATCC 27325) comprises the following steps: the wild type Escherichia coli is obtained by modifying the wild type Escherichia coli by the following method: knocking out tnaA gene to prevent decomposition of tryptophan and 5-hydroxytryptophanMetabolizing; the lacIZ site of its genome integrates the xylose promoter P_xylFThe controlled T7RNAP gene can make the cells to generate RNA polymerase T7RNAP by utilizing xylose induction while inactivating lacI protein; with feedback-inhibiting releasing mutant trpE^fbrReplacing the original trpE gene of Escherichia coli with a gene, and using P_trcThe promoter directs expression of the tryptophan operon to enhance the chorismate pathway; mutant aroG that will relieve feedback inhibition^fbrGene serA^fbrThe gene is integrated into the yjiV site of the Escherichia coli genome in tandem and is expressed by P_trcThe promoter guides expression to strengthen shikimic acid pathway and serine synthesis pathway; knocking out tyrR gene and trpR gene to realize deletion of negative transcription regulatory protein TyrR and TrpR; the TPH150 gene which is guided by a T7 promoter to express and codes the human type 2 tryptophan hydroxylase truncation mutant is integrated at the genome mbhA site so as to construct an intracellular tryptophan hydroxylation path; the mtrA gene from bacillus subtilis and the PTPS gene, SPR gene, PCD gene and DHPR gene from human being are serially integrated to the yghX site of colibacillus genome to introduce the synthesis path and regeneration path of coenzyme tetrahydropterin, wherein the mtrA gene, PTPS gene and SPR gene are composed of the same P_trcThe promoter directs the expression, the PCD gene and the DHPR gene are from the same P_trcThe promoter directs the expression; wherein the content of the first and second substances,

coli W3110, accession number ATCC 27325;

the xylose promoter P_xylFHas a nucleotide sequence shown in a sequence table SEQ ID NO. 1;

the RNA polymerase T7RNAP has a nucleotide sequence shown in a sequence table SEQ ID NO. 2;

the trpE^fbrThe gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 3;

the P is_trcThe promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 4;

the aroG^fbrThe gene has a nucleotide sequence shown in a sequence table SEQ ID NO. 5;

the serA^fbrThe gene has a sequenceThe nucleotide sequence shown in Table SEQ ID NO. 6;

the strong promoter PT7 promoter has a nucleotide sequence shown in a sequence table SEQ ID NO. 7;

the TPH150 gene for coding the human 2-type tryptophan hydroxylase truncation mutant has a nucleotide sequence shown in a sequence table SEQ ID NO. 8;

the mtrA gene is derived from bacillus subtilis, is responsible for encoding GTP cyclohydrolase I and has a nucleotide sequence shown in a sequence table SEQ ID NO. 9;

the humanized PTPS gene is responsible for encoding 6-pyruvoyl tetrahydrobiopterin synthetase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 10;

the human SPR gene is responsible for coding the guanine reductase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 11;

the human PCD gene is responsible for encoding pterin-4 alpha-methanol ammonia dehydratase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 12;

the humanized DHPR gene is responsible for encoding dihydropterin reductase and has a nucleotide sequence shown in a sequence table SEQ ID NO. 13.

Example 2

Fermentation in a 5L fermenter for 32h with the cultivation method described in example 1 gave 4L of fermentation broth with a 5-HTP yield of 2.7g/L, a tryptophan yield of 1.5g/L and a biomass of 66 (OD)₆₀₀) And 3.7g/L of soluble protein, dividing the fermentation liquor into 4 parts, and carrying out the study on the using amount of the activated carbon, wherein each part is 1L. The decolorizing time is 30min, the decolorizing temperature is 50 ℃, and an ultrasonic device is arranged for assisting decolorization. The amounts of activated carbon were 0.15%, 0.2%, 0.3% and 0.4%, which were A, B, C, D groups, and the absorbance before decolorization was A₀When the absorbance after decolorization is A, the decolorization rate is

The results are shown in table 1, using the decolorization ratio, extraction yield and product purity as indexes:

TABLE 1 Effect of activated carbon dosage on decolorization ratio

As shown in Table 1, the optimum decoloring effect is achieved with an addition amount of 0.3%, and an addition amount higher than 0.3% only increases the cost and fails to achieve a better decoloring effect.

Example 3

16L of fermentation broth with a 5-HTP yield of 2.6g/L, a tryptophan yield of 1.4g/L and a microbial biomass of 68 (OD) were obtained by fermentation in a 30L fermenter for 32h according to the cultivation method in example 1₆₀₀) The concentration of formic acid was measured by dividing the fermentation broth into 4 portions of 3.7g/L soluble protein, each 4L. Concentration gradients were set at 5%, 8%, 10%, 20%, 40%, corresponding to A, B, C, D, E groups, each group using 1: 60 (mass to volume ratio, namely 60mL of formic acid solution is used for 1g of dry powder), and the extraction yield and the product purity are taken as indexes, and the results are shown in Table 2:

TABLE 2 Effect of reconstituted formic acid concentration on extraction

As shown in Table 2, when the concentration of formic acid is too low, tryptophan and 5-HTP can be well dissolved, so that the extraction yield and the product purity are low, when the concentration is increased to 8%, the solubility of 5-HTP is reduced, and the solubility of tryptophan is increased, so that after slight stirring, tryptophan is almost completely dissolved, and after rapid filtration, the purity of 5-HTP is good, and it is noted that the stirring process must be slight, and when the stirring amplitude is too large, 5-HTP is also dissolved to a certain extent, so that slight stirring and rapid filtration are important in the step.

Example 4

Using the cultivation method of example 1, and combining the optimum conditions of examples 2 and 3, 60L of fermentation broth with 5-HTP yield of 2.7g/L, tryptophan yield of 1.4g/L, and microbial biomass of 67 (OD) was obtained after fermentation in a 100L fermentor for 32h₆₀₀) And the soluble protein is 3.2 g/L. The extraction process of the embodiment of the part is as follows:adjusting the pH value of the fermentation liquor to 4.0 by using sulfuric acid, heating to 75 ℃, performing circulating filtration by using a ceramic membrane filter to obtain clear fermentation liquor, performing secondary filtration on the obtained clear liquor by using a nanofiltration membrane to remove small molecular proteins and partial pigments, then concentrating the fermentation liquor to 40% of the original volume by using an evaporation concentration method, performing secondary decolorization on the obtained concentrated clear liquor by using weak-base anion adsorption resin, removing soluble proteins, performing secondary decolorization on the decolorized clear liquor by using 0.3% of active carbon, then concentrating to 30% of the original volume, separating other byproducts except tryptophan by using strong acid type cation exchange resin, performing secondary evaporation concentration, performing air-flow drying, fully crushing the dried solid into fluffy powder, re-dissolving by using 8% formic acid (volume/volume) after filtering, and performing drying by using an air-flow dryer after filtering to obtain the final extraction yield of 56%, the purity of the product is 98%.

Example 5

Using the cultivation method of example 1, and combining the optimum conditions of examples 2 and 3, 300L of fermentation broth with 5-HTP yield of 2.6g/L, tryptophan yield of 1.2g/L, and bacterial biomass of 69 (OD)₆₀₀) And the soluble protein is 3.7 g/L. The extraction process of the embodiment of the part is as follows: adjusting the pH value of fermentation liquor to 4.0 by using sulfuric acid, heating to 75 ℃ by using a plate heat exchanger, circularly filtering by using a ceramic membrane filter to obtain clear fermentation liquor, filtering the obtained clear liquor again by using a nanofiltration membrane to remove small molecular proteins and partial pigments, then concentrating the fermentation liquor to 40% of the original volume by using an evaporation concentration method, decoloring the obtained concentrated clear liquor again by using weak alkaline anion adsorption resin to remove soluble proteins, decoloring the decolored clear liquor again by using 0.3% of activated carbon, then concentrating to 30% of the original volume, adsorbing and separating other byproducts except tryptophan by using strong acid type cation exchange resin, evaporating and concentrating again, fully crushing the dried solid into fluffy powder by using a spray dryer, redissolving by using 8% formic acid (volume/volume), filtering, and then volatilizing and drying by using an oven at 85 ℃, the final extraction yield is 55%, and the product purity is 99%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Tianjin science and technology university

<120> method for separating and extracting 5-hydroxytryptophan

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> promoter

<220>

<221> promoter

<222> (1)..(268)

<400> 1

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aaatcaagaa ataaaccaaa aatcgtaatc gaaagataaa aatctgtaat tgttttcccc 180

tgtttagttg ctaaaaattg gttacgttta tcgcggtgat tgttacttat taaaactgtc 240

ctctaactac agaaggccct acaccatg 268

<210> 2

<211> 2652

<212> DNA

<213> Bacteriophage T7

<220>

<221> tRNA

<222> (1)..(2652)

<400> 2

atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60

ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120

catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180

gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag 240

atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300

acagccttcc agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag 360

accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420

atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480

cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa 540

gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600

tggtcttcgt ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc 660

attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720

tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780

ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc 840

attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900

agtaagaaag cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960

aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020

atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080

ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct 1140

gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200

atgcttgagc aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg 1260

gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320

aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380

aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag 1440

ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact ggagaacact 1500

tggtgggctg agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560

gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620

tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680

ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag 1740

attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt gaccgatgag 1800

aacactggtg aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860

ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggcttacggg 1920

tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat 1980

tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg 2040

atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100

tctgctgcta agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc 2160

aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220

aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280

attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct 2340

aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400

aagtacggaa tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac 2460

gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520

gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa 2580

atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc 2640

gcgttcgcgt aa 2652

<210> 3

<211> 1563

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(1563)

<400> 3

atgcaaacac aaaaaccgac tctcgaactg ctaacctgcg aaggcgctta tcgcgacaat 60

cccaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaatcc 120

gcagatatcg acagcaaaga tgatttaaaa agcctgctgc tggtagacag tgcgctgcgc 180

attacagttt taggtgacac tgtcacaatc caggcacttt ccggcaacgg cgaagccctc 240

ctggcactac tggataacgc cctgcctgcg ggtgtggaaa gtgaacaatc accaaactgc 300

cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgctcg cttatgctcc 360

ctttcggttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420

cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480

ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540

ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600

aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660

gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720

agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780

attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840

tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900

ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960

attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020

gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080

gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140

agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200

tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260

atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320

gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380

gatctcgaca cctacattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440

caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500

aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560

tga 1563

<210> 4

<211> 74

<212> DNA

<213> promoter

<220>

<221> promoter

<222> (1)..(74)

<400> 4

ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac 60

acaggaaaca gacc 74

<210> 5

<211> 1053

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(1053)

<400> 5

atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60

gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120

aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180

tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240

gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300

acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360

gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420

gcaggtgagt ttctcgatat gatcacccca caatatctcg ctgacctgat gagctggggc 480

gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540

tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600

aatgccgccg gtgcgccgca ctgcttcctg ttcgtaacga aatgggggca ttcggcgatt 660

gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720

tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780

caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840

gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900

gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960

ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020

ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053

<210> 6

<211> 1233

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(1233)

<400> 6

atggcaaagg tatcgctgga gaaagacaag attaagtttc tgctggtaga aggcgtgcac 60

caaaaggcgc tggaaagcct tcgtgcagct ggttacacca acatcgaatt tcacaaaggc 120

gcgctggatg atgaacaatt aaaagaatcc atccgcgatg cccacttcat cggcctgcga 180

tcccgtaccc atctgactga agacgtgatc aacgccgcag aaaaactggt cgctattggc 240

tgtttctgta tcggaacaaa ccaggttgat ctggatgcgg cggcaaagcg cgggatcccg 300

gtatttaacg caccgttctc aaatacgcgc tctgttgcgg agctggtgat tggcgaactg 360

ctgctgctat tgcgcggcgt gccggaagcc aatgctaaag cgcaccgtgg cgtgtggaac 420

aaactggcgg cgggttcttt tgaagcgcgc ggcaaaaagc tgggtatcat cggctacggt 480

catattggta cgcaattggg cattctggct gaatcgctgg gaatgtatgt ttacttttat 540

gatattgaaa ataaactgcc gctgggcaac gccactcagg tacagcatct ttctgacctg 600

ctgaatatga gcgatgtggt gagtctgcat gtaccagaga atccgtccac caaaaatatg 660

atgggcgcga aagaaatttc actaatgaag cccggctcgc tgctgattaa tgcttcgcgc 720

ggtactgtgg tggatattcc ggcgctgtgt gatgcgctgg cgagcaaaca tctggcgggg 780

gcggcaatcg acgtattccc gacggaaccg gcgaccaata gcgatccatt tacctctccg 840

ctgtgtgaat tcgacaacgt ccttctgacg ccacacattg gcggttcgac tcaggaagcg 900

caggagaata tcggcctgga agttgcgggt aaattgatca agtattctga caatggctca 960

acgctctctg cggtgaactt cccggaagtc tcgctgccac tgcacggtgg gcgtcgtctg 1020

atgcacatcg ccgaaaaccg tccgggcgtg ctaactgcgc tgaacaaaat cttcgccgag 1080

cagggcgtca acatcgccgc gcaatatctg caaacttccg cccagatggg ttatgtggtt 1140

attgatattg aagccgacga agacgttgcc gaaaaagcgc tgcaggcaat gaaagctatt 1200

ccgggtacca ttcgcgcccg tctgctgtac taa 1233

<210> 7

<211> 61

<212> DNA

<213> promoter

<220>

<221> promoter

<222> (1)..(61)

<400> 7

taatacgact cactataggg tctagaaata attttgttta actttaagaa ggagatatac 60

c 61

<210> 8

<211> 951

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(951)

<400> 8

atggttccgt ggtttcctcg caaaatcagc gagctggaca aatgcagcca ccgcgtcctg 60

atgtacggtt ccgaactgga cgccgatcac cctggtttca aagacaacgt ttaccgtcag 120

cgccgtaaat acttcgtaga cgtggccatg ggttacaaat acggtcagcc gatcccgcgc 180

gtcgaataca ctgaagaaga aaccaaaacg tggggcgtag tattccgtga actgtccaaa 240

ctgtacccga cccacgcttg ccgtgaatat ctgaaaaact ttccgctgct gaccaaatac 300

tgcggttacc gtgaagataa cgttccgcag ctggaagatg tttctatgtt cctgaaagag 360

cgttccggtt tcacggttcg tccagttgca ggttacctgt ctccgcgcga ttttctggcg 420

ggcctggctt accgtgtgtt ccactgtacc caatacatcc gtcacggcag cgatccgctg 480

tataccccgg aaccggacac ttgtcatgag ctgctgggcc acgttccact gctggctgac 540

ccaaaattcg cgcagttctc tcaggaaatt ggtctggcat ctctgggcgc gtctgacgaa 600

gacgtccaga aactggcaac ttgctacttc tttactatcg aatttggcct gtgcaagcaa 660

gaaggtcagc tgcgcgcgta tggtgcaggt ctgctgtcta gcatcggtga gctgaaacac 720

gcgctgtctg acaaggcctg cgtgaaggct tttgatccga aaaccacttg cctgcaggaa 780

tgcctgatca ccaccttcca ggaagcctac ttcgtaagcg agtccttcga agaagcgaaa 840

gagaaaatgc gtgatttcgc gaaaagcatt acccgtccgt tctctgtata cttcaacccg 900

tacacccagt ccatcgaaat cctgaaagat actcgttcca tcgaaaacgt t 951

<210> 9

<211> 573

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(573)

<400> 9

atgaaagaag ttaataaaga gcaaatcgaa caagctgttc gtcaaatttt agaagcgatc 60

ggagaagacc cgaatagaga agggcttctt gatactccga aaagagtcgc aaagatgtat 120

gccgaagtat tctccggctt gaatgaagat ccaaaagaac atttccagac tatcttcggt 180

gaaaaccatg aggagcttgt tcttgtaaaa gatatagcgt ttcattctat gtgtgagcat 240

caccttgttc ccttttatgg aaaagcacat gttgcatata tcccgcgagg cggaaaggtc 300

acaggactca gcaaactggc acgtgccgtt gaagccgttg caaagcgccc gcagcttcag 360

gaacgcatca cttctacaat tgcagaaagc atcgtagaaa cgcttgatcc gcatggcgta 420

atggtagtgg ttgaagcgga acacatgtgc atgacgatgc gcggtgtaag aaaaccgggt 480

gcgaaaactg tgacttcagc agtcagaggc gtttttaaag atgatgccgc tgcccgtgca 540

gaagtattgg aacatattaa acgccaggac taa 573

<210> 10

<211> 438

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(438)

<400> 10

atgagcacgg aaggtggtgg ccgtcgctgc caggcacaag tgtcccgccg catctccttc 60

agcgcgagcc accgattgta cagtaaattt ctaagtgatg aagaaaactt gaaactgttt 120

gggaaatgca acaatccaaa tggccatggg cacaattata aagttgtggt gacagtacat 180

ggagagattg accctgctac gggaatggtt atgaatctgg ctgatctcaa aaaatatatg 240

gaggaggcga ttatgcagcc ccttgatcat aagaatctgg atatggatgt gccatacttt 300

gcagatgtgg tgagcacgac tgaaaatgta gctgtttata tctgggacaa cctccagaaa 360

gttcttcctg taggagttct ttataaagta aaagtatacg aaactgacaa taatattgtg 420

gtttataaag gagaatag 438

<210> 11

<211> 783

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(783)

<400> 11

atggaaggtg gtctgggtcg tgctgtttgt ctgctgactg gtgctagccg tggtttcggc 60

cgtaccctgg cacctctgct ggcatctctg ctgtctccag gcagcgtgct ggtactgagc 120

gctcgtaacg atgaagcact gcgtcagctg gaagccgaac tgggtgctga acgttctggt 180

ctgcgtgtcg ttcgtgtacc tgcagatctg ggtgctgaag ctggcctgca acagctgctg 240

ggtgctctgc gtgaactgcc gcgtccaaaa ggcctgcaac gtctgctgct gatcaacaac 300

gctggttccc tgggtgacgt ttccaaaggt ttcgtagacc tgtccgattc cactcaggtt 360

aacaattact gggctctgaa cctgaccagc atgctgtgtc tgaccagctc cgttctgaaa 420

gcattcccag attctccggg cctgaaccgt accgttgtga acatttccag cctgtgcgcg 480

ctgcagccgt tcaagggttg ggcactgtat tgcgcgggta aagcagcccg tgacatgctg 540

ttccaggttc tggcgctgga agaaccaaac gttcgtgttc tgaactatgc tccgggtcct 600

ctggacaccg atatgcagca gctggcgcgt gaaacctccg ttgatccgga catgcgcaag 660

ggtctgcaag aactgaaagc taaaggtaaa ctggttgatt gcaaagtatc tgctcagaaa 720

ctgctgagcc tgctggaaaa agacgaattc aagagcggtg ctcacgtgga cttttacgac 780

aag 783

<210> 12

<211> 315

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(315)

<400> 12

atggctggta aagctcatcg tctgagcgcg gaagaacgtg atcagctgct gccaaacctg 60

cgtgcggttg gttggaacga actggaaggt cgtgatgcga tctttaaaca gtttcacttc 120

aaggatttta accgtgcttt cggtttcatg acccgtgtag cactgcaggc tgagaaactg 180

gaccaccacc cggaatggtt caacgtgtat aacaaagttc acatcactct gagcacccac 240

gaatgtgcag gcctgtctga acgtgacatc aacctggctt ctttcatcga acaggttgca 300

gtgtctatga cctag 315

<210> 13

<211> 735

<212> DNA

<213> gene

<220>

<221> gene

<222> (1)..(735)

<400> 13

atggcagcag ctgcagcagc aggtgaagct cgtcgtgttc tggtttacgg tggtcgtggt 60

gcgctgggtt ctcgttgtgt tcaggctttc cgcgctcgta actggtgggt agcttccgtg 120

gatgttgtag agaacgaaga ggcgtctgct tccatcatcg ttaaaatgac cgactctttc 180

acggaacaag cagatcaggt taccgcagaa gttggcaaac tgctgggcga agaaaaagtt 240

gacgctatcc tgtgtgttgc gggtggctgg gctggtggta acgcaaaatc taagtctctg 300

ttcaaaaact gcgatctgat gtggaaacag agcatctgga cttccacgat ctcctcccac 360

ctggcgacta aacacctgaa agaaggcggt ctgctgaccc tggctggtgc aaaagctgct 420

ctggacggca ctccgggtat gattggctat ggtatggcca aaggcgcagt acatcagctg 480

tgccaaagcc tggctggcaa aaactccggt atgccaccgg gtgcagccgc aattgcagtt 540

ctgccagtga ccctggatac cccgatgaac cgtaaaagca tgccggaagc tgatttctct 600

tcttggaccc cgctggaatt cctggttgaa actttccatg actggatcac cggcaaaaat 660

cgcccgtctt ccggttccct gattcaggtt gttactaccg aaggtcgtac tgaactgacc 720

ccggcatact tctag 735

Claims

1. A method for separating and extracting 5-hydroxytryptophan is characterized by comprising the following steps: the fermentation engineering bacteria is Escherichia coli HTP10, and the extraction steps comprise fermentation to obtain fermentation broth, thallus separation, pigment separation, byproduct separation, protein separation and drying to obtain the product.

2. The method for separating and extracting 5-hydroxytryptophan according to claim 1, wherein: the method comprises the following specific steps:

(8) and (4) drying the solid obtained by filtering in the step (7).

3. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the pH adjusting reagent used in the step (1) is sulfuric acid, the heating mode is a microwave oven or a plate heat exchanger, the ultrafiltration is performed by using an inorganic ceramic membrane for filtration, the cut-off molecular weight is 25000-30000, and thalli, macromolecular proteins and other insoluble substances in the fermentation liquor are removed by circulating filtration.

4. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the molecular weight cut-off of the nanofiltration membrane in the step (2) is 300-400.

5. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: and (3) concentrating in a reverse osmosis concentration or evaporation concentration manner, wherein the anion adsorption resin is weak base type anion adsorption resin.

6. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the active carbon in the step (4) is further decolorized with the use amount of 0.2-0.3% by mass volume ratio.

7. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: and (3) adopting strong acid type cation exchange resin as the cation exchange resin in the step (5), and adopting 0.3moL/L ammonia water as eluent.

8. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the drying method in the step (6) is spray drying, evaporation drying, freeze drying or flash drying.

9. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the low-concentration formic acid in the step (7) is 8-10% formic acid solution.

10. The method for separating and extracting 5-hydroxytryptophan according to claim 2, wherein: the drying in the step (8) is carried out in a mode of oven drying at 65-85 ℃, tray drying or airflow drying.