CN102808008A - Method for synthesizing 5-hydroxytryptophan by enzymic method - Google Patents
Method for synthesizing 5-hydroxytryptophan by enzymic method Download PDFInfo
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- CN102808008A CN102808008A CN2012103034246A CN201210303424A CN102808008A CN 102808008 A CN102808008 A CN 102808008A CN 2012103034246 A CN2012103034246 A CN 2012103034246A CN 201210303424 A CN201210303424 A CN 201210303424A CN 102808008 A CN102808008 A CN 102808008A
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- oxyindole
- tryptophanase
- htp
- halfcystine
- hydroxytryptophan
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Abstract
The invention discloses a new method for synthesizing L-5-hydroxytryptophan by catalyzing 5-hydroxy-indole and L-cysteine by using tryptophanase. According to the method, the 5-hydroxytryptophan is generated through enzymatic reaction by innovatively using tryptophanase as an enzyme source, and using the 5-hydroxy-indole and the L-cysteine as substrates. The tryptophanase is derived from fermentation bacterium cells or lysate of escherichia coli genetic engineering recombinant bacteria BL21(DE3)-pET21a(+)-tnaA, or fermentation broth of bacillus subtilis genetic engineering bacteria WB600-pWB980-tnaA. The invention provides a new green production process route for the L-5-hydroxytryptophan.
Description
Technical field
The invention belongs to biotechnology and produce amino acid whose technical field, relate to method through tryptophanase catalysis 5-oxyindole and the synthetic L-5-hydroxytryptophan of L-halfcystine.
Background technology
The 5-hydroxytryptophan (has another name called the L-5-hydroxytryptophan; Be called for short 5-HTP) be a kind of natural aromatic amino acid; It is the precursor of neurotransmitter serotonin important in the human body; Serotonin is a kind of hormone of brain excretory, can influence our mood, sleep and appetite, needs 5-HTP to keep health when causing the thrombotonin imbalance in the brain and is in standard state.Modern medicine has proved that 5-HTP has the various remarkable pharmacological action, comprising: control the emotion, reduce anxiety, treatment dysthymia disorders; Improve sleep; Weaken appetite, treatment is fat; Treatment fibromyalgia syndrome; Alleviate symptoms such as the headache of going slowly.5-HTP has obtained using widely at medicine and protective foods industry.Most important antidepressant tonic and nonprescription drugs have been become at American-European 5-HTP.
At present, the 5-HTP working method mainly comprises:
(1) extraction method is from leguminous plants such as Ghana's seed (patent CN101648900), to extract.This technology is the main flow technology that present 5-HTP produces, conditional stability, and product purity is high.But, the output of raw material Ghana seed and determined the production cost of this technology collection period, and, cause holding at high price of 5-HTP in recent years along with the regional raw material in Africa the high year by year of cost of gathering.
(2) chemical synthesis is to be raw material with tryptophane and oxyacetic acid, in autoclave, carries out chemical hydroxylation reaction and obtains the poly-hydroxy tryptophane, and under the effect of catalyzer tryptophanase, hydrolysis finally obtains 5-HTP under alkaline condition then.This technological reaction is complicated, reaction conditions is strict, energy consumption is huge, and the output of product 5-HTP is unstable, therefore fails to enlarge always and produces.
(3) biotransformation method, be with microorganism cells or the enzyme that from organism, extracts as biological catalyst, utilize the specificity of enzyme to prepare 5-HTP, have the reaction conditions gentleness, an advantage such as speed of response is fast and specificity is strong.Traditional enzyme process be with TPH as catalyzer, be substrate with the tryptophane, generate 5-HTP specifically.Existing both at home and abroad report is to TPH gene clone in the animal body and measure dna sequence dna and protein sequence.Then, the corresponding gene order of screening and utilize sophisticated plasmid method for transformation that this gene is imported engineering strain and carry out large scale fermentation production in the CDNA library is used for 5-HTP production with the TPH of external acquisition.Domestic patent CN101864466 utilizes the gene constructed recombinant expression vector of rabbit Oryctolagus cuniculus TPH of existing report; Transform BL21 through plasmid and obtain engineering strain; Utilize this bacterial strain further to ferment, add the substrate tryptophane in the fermenting-ripening phase and obtain end product 5-HTP.
Tryptophanase, claim also that the tryptophane indoles splits and enzyme (tryptophanase, EC4.1.99.1), by the tetramer that the same subunit that contains a Vitazechs binding site is formed, this tetramer is at NH
4+, K
+Or Rb
+Have catalytic activity under existing, but activity receives Na
+And Li
+Suppress.This enzyme particularly has a large amount of existence in the intestinal bacteria in mikrobe, main catalysis tryptophane anaerobic digestion produces indole,pyruvic acid and ammonia, to halfcystine and the existing certain effect of Serine.Chinese patent (application number CN201010031351.0) adopts recombined bacillus subtilis to efficiently express tryptophanase, is used for enzyme process fractionation DL-cysteine and prepares D-Gelucystine and L-tryptophane.At present, do not see that also this enzyme is used to prepare the report of 5-HTP.
Summary of the invention
The purpose of this invention is to provide the method that a kind of new enzyme process synthesizes 5-hydroxytryptophan (5-HTP), this method is catalyzer with the tryptophanase, is substrate with 5-oxyindole and L-halfcystine, and technology is simple, environment friendly and pollution-free.
The method of the synthetic 5-hydroxytryptophan (5-HTP) of enzyme process provided by the invention comprising: with the zymophyte somatocyte of bacillus coli gene engineering reorganization bacterium BL21 (DE3)-pET21a (+)-tnaA of expressing tryptophanase or the fermented liquid of lysate or subtilis gene engineering recombinant bacterium WB600-pWB980-tnaA is the enzyme source; With 5-oxyindole and L-halfcystine is substrate, and in the enzymatic reaction liquid, the add-on of substrate 5-oxyindole is 5 to 20g/L; The add-on of L-halfcystine is 6 to 24g/L; The addition in enzyme source is 2000 to 8000U/L, under 40 to 60 ℃, and pH7 to 9; To 12h, produce 5-HTP through enzymatic conversion method reaction 3.Also comprise the coenzyme Vitazechs in the enzymatic reaction liquid, the add-on of Vitazechs is 0.005 to 0.02g/L.
The consumption of substrate 5-oxyindole is preferably 15g/L, and the consumption of substrate L-halfcystine is preferably 18g/L, and the addition in enzyme source is preferably 6000U/L; The consumption of Vitazechs is preferably 0.015g/L; Preferred invert point is 45 ℃, preferably transforms pH8, preferred transformation time 12h.
The subtilis gene engineering recombinant bacterium WB600-pWB980-tnaA of described expression tryptophanase is from patent CN201010011351.0.
In embodiments of the present invention, adopt HPLC to measure the content of 5-HTP, concrete grammar is following: performance liquid chromatography adopts Agilent ZORBASB-C18 post (250 * 4.6mm; 5 μ m) content of 5-HTP is measured; This liquid-phase condition is: mobile phase A is the 1mM/L potassium dihydrogen phosphate aqueous solution mutually, and B is 100% methyl alcohol mutually, 20%B-40%B during 0-10min; 40%B-20%B during 10-20min stops during 23min; Sample size 10 μ l; Overall flow rate is 1.0mL/min; Detect wavelength 225nm; Column temperature is 25 ℃.
Advantage of the present invention and beneficial effect:
Novelty of the present invention ground is substrate with 5-oxyindole and L-halfcystine; Zymophyte somatocyte/lysate or the fermented liquid of subtilis genetic engineering bacterium that utilization efficiently expresses the bacillus coli gene engineering bacteria of tryptophanase are the enzyme source; Produce 5-HTP; Have that mild condition, cycle are short, few, advantages such as the product separation and Extraction convenient, process step simple, operational safety of impurity in the enzymatic system, for the green production of 5-HTP provides a kind of novel method.
Description of drawings
Fig. 1 is a reaction mechanism;
Fig. 2 is the 5-HTP typical curve;
A: standard substance 5-HTP color atlas; B:5-HTP quantitative analysis typical curve;
Fig. 3 is the optimization of catalytic condition;
A: the relation of temperature of reaction and transformation efficiency; The relation of B:pH and transformation efficiency; C: the relation of reaction times and transformation efficiency; D: the relation of substrate 5-oxyindole and transformation efficiency; The relation of E:L-halfcystine and transformation efficiency; F: the relation of immobilized enzyme and transformation efficiency; G: the relation of Vitazechs and transformation efficiency;
Fig. 4 is that the HPLC of 5-HTP and 5-oxyindole in the reaction solution detects.
Embodiment
Embodiment 1 subtilis genetic engineering bacterium is expressed the preparation of tryptophanase
The fermentation of tryptophan gene engineering strain is referring to Chinese patent (application number CN201010011351.0).
Following jar of fermented liquid descends 3 for 4 ℃, and the centrifugal 15min of 000rpm removes, and obtains expressing the supernatant of tryptophanase, and the mensuration enzyme activity is 550U/mL.
The preparation of embodiment 2 bacillus coli gene engineering bacterium expression tryptophanases
Tryptophan gene sequence (the sequence number: NP418164) of the E.coli K12 that includes according to ncbi database; Design upstream primer tnaA1:5 '-CCG GAA TTC ATG GAAAAC TTT AAA CAT CTC C-3 ' (SEQ ID No.1), downstream primer tnaA2:5 '-CCC AAG CTT TTA AAC TTC TTT CAG TTT TGC GG-3 ' (SEQ ID No.2).Extract the genomic dna of E.coli JM109 bacterial strain, the pcr amplification tryptophan gene.Adopt gene engineering method, make up recombinant expression plasmid pET-21a (+)-tnaA.Recombinant expression vector pET-21a (+)-tnaA is converted into E.coli BL21 (DE3), obtains engineering strain BL21 (DE3)-pET21a (+)-tnaA.
Positive recombinant bacterial strain is inoculated in 4mL contains incubated overnight in the LB substratum of penbritin 100 μ g/mL; Inserting 100mL with 1% switching amount subsequently contains in the fresh LB substratum of penbritin 100 μ g/mL; 37 ℃ of shaking culture 3h; Then, adding final concentration is the IPTG induced liquid of 1mM, and specificity is induced the expression of tryptophanase.
The gained nutrient solution descends 6, the centrifugal 10min of 000rpm, collection thalline in 4 ℃; Obtain to express the gene engineering recombinant bacterium of tryptophanase; Centrifugal again after phosphoric acid buffer (pH8.0) the suspension washing with 50mmol/L, collect the fermentation thalline as the enzyme source, the mensuration enzyme activity is 1000U/g.
The thalline that will ferment carries out ultrasonic disruption, obtains the crude enzyme liquid of tryptophanase, and the mensuration enzyme activity is 200U/mL.
Embodiment 3HPLC method is measured 5-HTP
Performance liquid chromatography adopts Agilent ZORBA SB-C18 post (250 * 4.6mm; 5 μ m) content of 5-HTP is measured; This liquid-phase condition is: mobile phase A is the 1mM/L potassium dihydrogen phosphate aqueous solution mutually, and B is 100% methyl alcohol mutually, 20%B-40%B during 0-10min; 40%B-20%B during 10-20min stops during 23min; Sample size 10 μ l; Overall flow rate is 1.0mL/min; Detect wavelength 225nm; Column temperature is 25 ℃.
Accurately prepare the 5-HTP reference liquid of series concentration, continuous sample introduction 5 times calculates peak area and also averages, and (X mmol/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with 5-HTP concentration.The 5-HTP that enzymatic reaction produced is through suitably measuring with HPLC after the dilution, and the establishing criteria curve carries out accurate quantification, like Fig. 2.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 5000U/L respectively; Substrate 5-oxyindole 5g/L, L-halfcystine consumption 6g/L, coenzyme Vitazechs 0.005g/L; Transforming the pH value is 8, transformation time 6h.To the influence of 5-oxyindole transformation efficiency, adopt method among the embodiment 2 to detect the content of 5-HTP when investigating 40-60 ℃ of invert point.The result is shown in Fig. 3 A.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 5000U/L respectively; Substrate 5-oxyindole 5g/L, L-halfcystine consumption 6g/L, coenzyme Vitazechs 0.005g/L; Invert point is 45 ℃, transformation time 6h.To the influence of 5-oxyindole transformation efficiency, adopt the content of the method detection 5-HTP among the embodiment 2 when investigating conversion pH value 7-9.The result is shown in Fig. 3 B.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 5000U/L respectively; Substrate 5-oxyindole 5g/L, L-halfcystine consumption 6g/L, coenzyme Vitazechs 0.005g/L; Invert point is 45 ℃, and transforming the pH value is 8.To the influence of 5-oxyindole transformation efficiency, adopt the content of the method detection 5-HTP among the embodiment 2 when investigating transformation time 3-12h.The result is shown in Fig. 3 C.
Embodiment 7 investigates among the synthetic 5-HTP of tryptophane enzyme process, the relation of the input amount of substrate 5-oxyindole and the transformation efficiency of 5-oxyindole
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 5000U/L respectively; L-halfcystine consumption 24g/L, coenzyme Vitazechs 0.005g/L, invert point is 45 ℃; Transforming the pH value is 8, transformation time 12h.When being 5-20g/L, the input amount of investigation substrate 5-oxyindole, adopts the content of the method detection 5-HTP among the embodiment 2 to the influence of 5-oxyindole transformation efficiency.The result is shown in Fig. 3 D.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 5000U/L respectively; 5-oxyindole consumption 15g/L, coenzyme Vitazechs 0.005g/L, invert point is 45 ℃; Transforming the pH value is 8, transformation time 12h.When being 6-24g/L, the input amount of investigation L-halfcystine, adopts the content of the method detection 5-HTP among the embodiment 2 to the influence of 5-oxyindole transformation efficiency.The result is shown in Fig. 3 E.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, respectively in every liter transformation system, and 5-oxyindole consumption 20g/L; L-halfcystine consumption is 24g/L, coenzyme Vitazechs 0.02g/L, and invert point is 45 ℃; Transforming the pH value is 8, transformation time 12h.When being 2000-8000U/L, investigation enzyme amount, adopts the content of the method detection 5-HTP among the embodiment 2 to the influence of 5-oxyindole transformation efficiency.The result is shown in Fig. 3 F.
Tryptophanase with preparation among the foregoing description 1 or the embodiment 2 is a catalyzer, in every liter transformation system, adds enzyme amount 8000U/L respectively; Substrate 5-oxyindole 20g/L, L-halfcystine consumption 24g/L, invert point is 45 ℃; Transforming the pH value is 8, transformation time 12h.When being 0.005-0.02g/L, the concentration of investigation Vitazechs, adopts the content of the method detection 5-HTP among the embodiment 2 to the influence of 5-oxyindole transformation efficiency.The result is shown in Fig. 3 G.
Visible from above result, with Vitazechs 0.005-0.02g/L, at 40-60 ℃, the pH value is 7-9, enzyme amount 2000-8000U/L, and reaction 3-12h, tryptophanase can catalytic substrate 5-oxyindole and L-halfcystine production 5-HTP.Simultaneously, preferred conversion condition is: 5-oxyindole 15g/L, L-halfcystine 18g/L, Vitazechs 0.015g/L, enzyme amount 6000U/L, 45 ℃ of temperature of reaction, pH value 8, reaction times 12h.Under this optimum condition, carry out confirmatory experiment, the transformation efficiency of 5-oxyindole is 73.4% (see figure 4).
Claims (5)
1. the method for the synthetic 5-hydroxytryptophan of tryptophanase catalysis 5-oxyindole and L-halfcystine is characterized in that this method is the enzyme source with the tryptophanase, is substrate with 5-oxyindole and L-halfcystine; In the enzymatic reaction liquid, the add-on of substrate 5-oxyindole is 5 to 20g/L, and the add-on of L-halfcystine is 6 to 24g/L; The addition in enzyme source is 2000 to 8000U/L, under 40 to 60 ℃, and pH7 to 9; Through enzymatic conversion method reaction 3h to 12h, produce the L-5-hydroxytryptophan.
2. method according to claim 1; It is characterized in that: described tryptophanase is from zymophyte somatocyte or the lysate of bacillus coli gene engineering reorganization bacterium BL21 (DE3)-pET21a (+)-tnaA, or the fermented liquid of subtilis genetic engineering bacterium WB600-pWB980-tnaA.
3. method according to claim 1 is characterized in that also comprising in the enzymatic reaction liquid coenzyme Vitazechs, and the add-on of Vitazechs is 0.005 to 0.02g/L.
4. method according to claim 3 is characterized in that the consumption of coenzyme Vitazechs is preferably 0.015g/L.
5. according to each described method in the claim 1 to 4, it is characterized in that: in the enzymatic reaction liquid, the consumption of substrate 5-oxyindole is preferably 15g/L; The consumption of substrate L-halfcystine is preferably 18g/L; The addition in enzyme source is preferably 6000U/L,, preferred invert point is 45 ℃; The preferred pH8 that transforms, preferred transformation time 12h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267417A (en) * | 2017-07-05 | 2017-10-20 | 保定保利瑞合生物科技有限公司 | A kind of method for producing 5 hydroxytryptophanes |
| WO2019006700A1 (en) * | 2017-07-05 | 2019-01-10 | 保定保利瑞合生物科技有限公司 | Method for producing 5-hydroxytryptophan |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525641A (en) * | 2008-07-04 | 2009-09-09 | 南开大学 | Method for producing L-tryptophan by microbial enzyme method |
| CN102140483A (en) * | 2011-01-14 | 2011-08-03 | 南开大学 | Method for synthesizing L-tryptophan by immobilized enzyme |
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- 2012-08-23 CN CN2012103034246A patent/CN102808008A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525641A (en) * | 2008-07-04 | 2009-09-09 | 南开大学 | Method for producing L-tryptophan by microbial enzyme method |
| CN102140483A (en) * | 2011-01-14 | 2011-08-03 | 南开大学 | Method for synthesizing L-tryptophan by immobilized enzyme |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267417A (en) * | 2017-07-05 | 2017-10-20 | 保定保利瑞合生物科技有限公司 | A kind of method for producing 5 hydroxytryptophanes |
| CN107267417B (en) * | 2017-07-05 | 2018-03-23 | 保定保利瑞合生物科技有限公司 | A kind of method for producing 5 hydroxytryptophanes |
| WO2019006700A1 (en) * | 2017-07-05 | 2019-01-10 | 保定保利瑞合生物科技有限公司 | Method for producing 5-hydroxytryptophan |
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Application publication date: 20121205 |