CN101525641A - Method for producing L-tryptophan by microbial enzyme method - Google Patents
Method for producing L-tryptophan by microbial enzyme method Download PDFInfo
- Publication number
- CN101525641A CN101525641A CN200810053749A CN200810053749A CN101525641A CN 101525641 A CN101525641 A CN 101525641A CN 200810053749 A CN200810053749 A CN 200810053749A CN 200810053749 A CN200810053749 A CN 200810053749A CN 101525641 A CN101525641 A CN 101525641A
- Authority
- CN
- China
- Prior art keywords
- consumption
- tryptophan
- substrate
- tryptophane
- tryptophanase
- 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.)
- Pending
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for producing L-tryptophan by a microbial enzyme method. The method comprises that: full cells of Pseudomonas putida Ts1138 and bacillus coli thalli expressing tryptophanase in high efficiency, a thallus cracking solution or immobilized enzyme are used as enzyme sources; and from a substrate DL-2-amino-delta <2>-thiazoline-4-carboxylic acid (DL-ATC), the L-tryptophan is synthesized by a two-step continuous or one-step mixing enzyme catalytic reaction. The purification method comprises that: an S8 type macroporous resin is utilized to remove the DL-2-amino- delta <2>-thiazoline-4-carboxylic acid remained in a reaction solution containing the L-tryptophan and indole; under the condition of pH of 5, an NKA-II type macroporous resin is utilized to adsorb the L-tryptophan; the interference of L-cysteine is eliminated; and finally, the obtained L-tryptophan is subjected to ethanol elution, decompression, condensation and drying to obtain the L-tryptophan. Therefore, the method provides a novel process route for the green production of the L-tryptophan. Because the production cost of the substrate DL-ATC is relatively low, the method has a wide application prospect in the field of industrialized production of the L-tryptophan.
Description
[technical field]:
The invention belongs to amino acid whose production technical field, relate to the method for producing the L-tryptophane by microbial enzyme method conversion of substrate precursor.
[background technology]:
The L-tryptophane is the intravital important indispensable amino acid of humans and animals, all has purposes very widely in fields such as medicine, food and feeds.In recent years, because feed industrial development is rapid, and the L-tryptophane constantly enlarges in the purposes of pharmaceutical industries, and the demand for the L-tryptophane increases day by day both at home and abroad.But since for a long time the production difficulty height of L-tryptophane, cost an arm and a leg, except that medicinal use, the L-tryptophane is all failed large-scale popularization and is used in other field.
The L-tryptophane is direct fermentation one of the amino acid of difficult production, and its production method mainly contains four kinds of proteolysis extraction method, chemical synthesis, microbe fermentation method and enzyme process synthesis methods.Wherein, the enzyme process synthesis method utilizes chemical industry synthetic precursor to be raw material, both given full play to the advantage of organic synthesis technology, advantage such as have production concentration height, yield height, purity height again, by product is few and purification operations is easy is the good method that current cheapness of being praised highly is produced the L-tryptophane.
The approach of the synthetic L-tryptophane of enzyme process relates generally to tryptophan synthetase (EC 4.2.1.20) and tryptophanase (EC 4.1.99.1), but the two equal catalysis L-Serine and the synthetic L-tryptophane of indoles.Because indoles has had strong inhibitory effects to tryptophan synthetase, tryptophanase then has satisfactory stability to indoles, so people more pay close attention to the application of tryptophanase in the biosynthesizing of L-tryptophane in recent years.
Decompose the L-tryptophane under the tryptophanase normal circumstances and generate pyruvic acid, indoles and ammonia, but under the condition of high density pyruvic acid and ammonia also effectively catalysis pyruvic acid, indoles and ammonia synthesis L-tryptophane.This enzyme can also catalysis L-Serine or L-halfcystine and the synthetic L-tryptophane of indoles.In the approach of tryptophanase catalysis pyruvic acid, indoles and ammonia synthesis L-tryptophane, since the substrate indoles to tryptophane enzyme inhibition a little less than, and the pyruvic acid price is not high, thereby has a certain practicality, but this approach is the reversed reaction of tryptophane hydrolysis, the requirement concentration of substrate is higher, and molecular balance is difficult for holding.With L-Serine and indoles is in the approach of the synthetic L-tryptophane of raw material, because the price of substrate L-Serine is almost suitable with the L-tryptophane, so practicality is not strong.Utilize in the approach of the synthetic L-tryptophane of tryptophanase catalysis L-halfcystine and indoles, because the production cost of substrate L-halfcystine is comparatively cheap, so this approach has important industrial applications value.
In the approach of the synthetic L-tryptophane of enzymatic conversion method L-halfcystine and indoles, the main difficult point of separation and purification product L-tryptophane is, the moiety of reaction solution is comparatively complicated, material such as residual indoles and L-halfcystine and product L-tryptophane are not easily separated, and the method report of mature and feasible is not arranged at present as yet.
[summary of the invention]:
The objective of the invention is to solve the above-mentioned problems in the prior art, a kind of novel method of producing L-tryptophan by microbial enzyme method is provided.
The full cell of coli somatic, cellular lysate liquid or the immobilized enzyme that the present invention relates to utilize pseudomonas putida TS1138 (Pseudomonas putida TS1138) cell and efficiently express tryptophanase are the enzyme source, from substrate DL-2-amino-Δ
2-thiazoline-4-carboxylic acid (DL-2-amino-Δ
2-thiazoline-4-carboxylic acid DL-ATC) sets out, and through two step successive or the synthetic L-tryptophane of step blended enzymatic reaction, and final separation and purification obtains the operational path of product L-tryptophane.
The present invention separates acquisition one strain pseudomonas putida TS1138 bacterial strain (Pseudomonas putida TS-1138) voluntarily, this bacterial strain is preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; preserving number is CGMCC No.1920, L-cysteine synthase that this bacterial strain comprised system can the synthetic L-halfcystine of catalytic substrate DL-ATC.In addition, the present invention has also made up the bacillus coli gene engineering strain that efficiently expresses tryptophanase voluntarily, be template promptly with the e. coli jm109 strain gene group DNA, pcr amplification obtains tryptophan gene, and it is expressed in e. coli bl21 (DE3), recombinant expressed tryptophanase vigor reaches 110 times of host bacterium.
The full cell of thalline, cellular lysate liquid or immobilized enzyme with above-mentioned two kinds of bacterial strains are the enzyme source, and the present invention has set up the novel method of or step blended enzymatic reaction biosynthesizing L-tryptophane continuous through two steps of L-halfcystine respectively, and synthetic route is seen Fig. 1.
The method of producing L-tryptophan by microbial enzyme method provided by the invention specifically comprises:
1st, two steps successive enzymatic reaction
1.1st, be the enzyme source at first with the full cell or the cellular lysate liquid of pseudomonas putida TS1138 thalline, enzymatic conversion method substrate DL-2-amino-Δ
2-thiazoline-4-carboxylic acid obtains containing the reaction solution of L-halfcystine;
1.2nd, being the enzyme source with full cell, cellular lysate liquid or the immobilized enzyme of the coli somatic that efficiently expresses tryptophanase again, is substrate with above-mentioned L-halfcystine reaction solution and indoles, through the synthetic L-tryptophane of enzymatic conversion method;
Perhaps,
2nd, step blended enzymatic reaction
Full cell or cellular lysate liquid with pseudomonas putida TS1138 thalline are the mixed enzyme source with the full cell, cellular lysate liquid or the immobilized enzyme that efficiently express the coli somatic of tryptophanase, enzymatic conversion method substrate DL-2-amino-Δ
2-thiazoline-4-carboxylic acid and indoles synthesize the L-tryptophane.
In the described two steps successive enzymatic reaction process, add the coenzyme pyridoxal phosphate simultaneously, and azanol is as the inhibitor of L-cysteine desulfhydrase, and the consumption of described coenzyme pyridoxal phosphate is 0.05g/L to 1g/L, and the final concentration of described azanol is 0.5mmol/L to 3.0mmol/L; TS1138 thalline consumption is generally 50g/L to 500g/L, and cellular lysate liquid consumption is generally the lysate of 10g to 100g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is generally 1g/L to 100g/L, and cellular lysate liquid consumption is generally the lysate of 0.5g to 30g somatic cells, and the immobilized enzyme consumption is generally 1g/L to 50g/L; Substrate DL-2-amino-Δ
2The concentration of-thiazoline-4-carboxylic acid is 1g/L to 15g/L; Substrate indoles consumption is 1g/L to 20g/L.
The consumption of described coenzyme pyridoxal phosphate is preferably 0.15g/L, and the final concentration of used azanol is preferably 1.0mmol/L; TS1138 thalline consumption is preferably 150g/L, and cellular lysate liquid consumption is preferably the lysate of 30g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is preferably 10g/L, and cellular lysate liquid consumption is preferably the lysate of 3g somatic cells, and the immobilized enzyme consumption is preferably 15g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is preferably 6g/L; Substrate indoles consumption is preferably 4.5g/L.
In the described step blended enzymatic reaction process, can add the coenzyme pyridoxal phosphate simultaneously, its consumption is 0.05g/L to 1g/L; TS1138 thalline consumption is generally 50g/L to 500g/L, and cellular lysate liquid consumption is generally the lysate of 10g to 100g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is generally 1g/L to 100g/L, and cellular lysate liquid consumption is generally the lysate of 0.5g to 30g somatic cells, and the immobilized enzyme consumption is generally 1g/L to 50g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is 1g/L to 15g/L, and the indoles consumption is 1g/L to 20g/L.
Coenzyme pyridoxal phosphate consumption is preferably 0.15g/L, and TS1138 thalline consumption is preferably 200g/L, and cellular lysate liquid consumption is preferably the lysate of 40g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is preferably 15g/L, and cellular lysate liquid consumption is preferably the lysate of 5g somatic cells, and the immobilized enzyme consumption is preferably 20g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is preferably 6g/L, and the indoles consumption is preferably 5g/L.
The separation purification method of the synthetic L-of institute tryptophane of the present invention is to adopt S8 type macroporous resin to remove and contain DL-2-amino-Δ residual in the L-tryptophan reaction liquid
2-thiazoline-4-carboxylic acid and indoles, again pH be under 5 the condition with NKA-II type macroporous resin adsorption L-tryptophane, get rid of the interference of L-halfcystine, after ethanol elution, concentrating under reduced pressure drying obtain the L-tryptophane.
In embodiments of the present invention, adopt the Gaitonde method to measure the content of L-halfcystine, concrete grammar is as follows: accurately take by weighing a certain amount of L-halfcystine standard substance, be dissolved among a certain amount of 0.05mol/L HCl, the preparation final concentration is the standard mother liquor of 500mg/L, and further with distilled water above-mentioned mother liquor being diluted respectively is 10,20, ..., the standardized solution of 100mg/L.Get the diluent 0.2mL of a certain amount of standardized solution or testing sample solution, add the 0.2mL Glacial acetic acid, add 0.2mL acid ninhydrine reagent again, in boiling water bath, react 10min, cool off in cold water immediately then, add 2.4mL alcohol at last, making cumulative volume is 3mL.After placing 10min, measure its absorbancy down in 560nm, with concentration and absorbancy drawing standard curve.Calculate L-semicystinol concentration in the unknown solution by this typical curve.
In embodiments of the present invention, adopt high performance liquid chromatography to measure the content of L-tryptophane, concrete grammar is as follows: get 50～1 respectively, the L-tryptophane reference liquid 20 μ L sample introductions of 500mg/L concentration, continuous sample introduction 5 times, the calculating peak height is also averaged, with L-tryptophane concentration (X, mg/L) be X-coordinate, peak height (Y) is an ordinate zou drawing standard curve.The L-tryptophane that enzymatic reaction produced is compared with typical curve after high performance liquid chromatography detects can carry out accurate quantification.
Beneficial effect of the present invention: the present invention is from the substrate DL-ATC of chemosynthesis, utilize pseudomonas putida TS1138 and the full cell, cellular lysate liquid or the immobilized enzyme that efficiently express the coli somatic of tryptophanase to be the enzyme source, produce the L-tryptophane through two step successive and step blended enzymatic reaction respectively.Because the production cost of substrate DL-ATC is comparatively cheap, so the present invention has broad application prospects in the field of industrialized production of L-tryptophane.
[description of drawings]
The route synoptic diagram of the synthetic L-tryptophane of Fig. 1: enzymatic conversion method DL-ATC,
Fig. 2: in the high performance liquid chromatography detection reaction liquid and the L-tryptophane behind the purifying,
(A), reaction solution (ultraviolet detection); (B), reaction solution (the diffusing look device of evaporation light detects); (C), the L-tryptophane (ultraviolet detection) of purifying; (D), the L-tryptophane of purifying (the diffusing look device of evaporation light detects); Chromatographic peak is respectively: 1, the L-tryptophane (retention time, 4.732min); 2, and indoles (retention time, 5.965min); 3, and the L-halfcystine (retention time, 3.223min).
Pseudomonas putida TS1138 of the present invention, classification name: Pseudomonas putida, be preserved on January 17th, 2007 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; address during preservation: in the institute of microbiology of the Chinese Academy of Sciences of Zhongguangcun, Haidian District, Beijing City, preserving number is CGMCC No.1920.Simultaneously, this bacterial strain has been documented in disclosed No. 200710056754.9 patent application documents on October 17th, 2007.
[embodiment]
Embodiment 1
The cloning and expression of intestinal bacteria tryptophan gene
According to the tryptophan gene sequence of e. coli k12 strain, design tryptophan gene upstream primer 5 '-CCGGAATTCATGGAAAACTTTAAACATCTCC-3 ' and downstream primer 5 '-CCCAAGCTTTTAAACTTCTTTCAGTTTTGCGG-3 '.Genomic dna with bacillus coli DH 5 alpha, JM109 and BL21 bacterial strain is a template respectively, and pcr amplification obtains tryptophan gene, and makes up recombinant expression plasmid pET-tnaA (D), pET-tnaA (J) and pET-tnaA (B) respectively.
Three kinds of recombinant plasmids are converted into e. coli bl21 (DE3) respectively, make up the tryptophan gene engineering strain that obtains separately.The inoculation recombinant bacterial strain contains in the LB substratum of penbritin 100 μ g/mL in 4mL, behind 37 ℃ of shaking culture 12～16h, is forwarded to the fresh LB substratum of 50mL with 1% inoculum size, continues to be cultured to OD
600Reach at 0.5 o'clock, adding final concentration was the IPTG of 0.1mmol/L, in 37 ℃ of abduction deliverings 2 hours.Collect thalline and adopt potassium phosphate buffer (pH 8.0) washing back resuspended, ultrasonic disruption was handled 5 minutes, made the abundant cracking of thalline, and supernatant liquor is collected in centrifugal back, measures the vigor of recombinant expressed tryptophanase.The result shows that the enzyme activity that derives from the different colibacillary tryptophan gene recombination expression products of three strains is close, all reaches more than 100 times (table 1) of host bacterium self tryptophanase vigor.
The tryptophanase vigor of table 1 different strains
All numerical value are mean+SD (the Mean value ± S.D.) of three parallel laboratory tests.
The preparation in pseudomonas putida TS1138 bacterial strain enzyme source
Picking one ring pseudomonas putida TS1138 inserts (glucose 20g, ATC3g, corn steep liquor 5g, urea 3g, NaCl 1.5g, MnSO the 4mL seed culture medium from flat board
4H
2O 0.1g, K
2HPO
43g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g is settled to 1L, and pH 7.5), after 28 ℃ of 160r/min cultivate 16h, be transferred to 50mL with 1% inoculum size and produce (glucose 30g, ATC 4g, corn steep liquor 1g, urea 3g, NaCl 1.5g, MnSO in the enzyme substratum
4H
2O 0.1g, K
2HPO
43g, MgSO
47H
2O0.5g, FeSO47H
2O 0.01g is settled to 1L, and pH 7.5), after 28 ℃ of 160r/min cultivate 16h, centrifugal, collect thalline, wash thalline 1 to 2 time with glycine-sodium hydrate buffer solution (pH 8.0), take by weighing a certain amount of above-mentioned thalline, add glycine-sodium hydrate buffer solution, promptly make enzyme source cell suspension.
The enzyme source cell is carried out ultrasonic disruption, and supernatant liquor is collected in centrifugal back, promptly makes cellular lysate liquid.
Embodiment 3
Efficiently express the preparation in the intestinal bacteria enzyme source of tryptophanase
E.coli BL21 (DE3) recombinant bacterial strain that comprises pET-tnaA (J) of preparation contains in the LB substratum of penbritin 100 μ g/mL in 4mL among the inoculation embodiment 1, behind 37 ℃ of shaking culture 12～16h, be forwarded to the fresh LB substratum of 50mL with 1% inoculum size, continue to be cultured to OD
600Reach at 0.5 o'clock, adding final concentration was the IPTG of 0.1mmol/L, in 37 ℃ of abduction deliverings 2 hours.Collect thalline and adopt potassium phosphate buffer (pH 8.0) washing thalline 1 to 2 time, take by weighing a certain amount of above-mentioned thalline, add potassium phosphate buffer, promptly make enzyme source cell suspension.Adopt ultrasonic disruption enzyme source cell, supernatant liquor is collected in centrifugal back, promptly makes cellular lysate liquid.
Take by weighing 1g DEAE-22 Mierocrystalline cellulose, add 3.5% glutaraldehyde 40mL, stir 5h under the room temperature, standing over night, centrifugal removal supernatant liquor washes the gained carrier to remove remaining glutaraldehyde repeatedly with distilled water, promptly gets crosslinked carrier behind the suction filtration.Adding soluble protein content in the carrier after crosslinked is the cellular lysate liquid 30mL of 15g/L, and 4 ℃ leave standstill 12h, stirs once every 0.5h.Centrifugal, abandoning supernatant, throw out washes repeatedly with distilled water and removes free lysate, promptly makes immobilized enzyme behind the suction filtration.
With the full cell of thalline is the enzyme source, through the synthetic L-tryptophane of two continuous enzymatic reactions of step
Be 500mL and be equipped with in the container of agitator that preparation 300mL enzymatic reaction liquid comprises 6g/L DL-ATC, 6g/L K at an internal volume
2HPO
4, 1.0mmol/L azanol and 150g/L embodiment 2 in the TS1138 bacterial strain enzyme source cell of preparation, the potassium hydroxide aqueous solution adjustment pH value to 8.0 with 5% places 42 ℃ of waters bath with thermostatic control, stirring reaction 2.5h.
After reaction is finished, under 4 ℃ of conditions 12, the centrifugal 5min of 000r/min, with solid-liquid separation, supernatant liquor is the mixed solution that contains the L-halfcystine, detects wherein that the L-cysteine content is 4.8g/L, the molar yield of substrate DL-ATC is 96.5%.
In the reaction solution of the above-mentioned L-of containing halfcystine, add the indoles that final concentration is 4.5g/L, the coenzyme pyridoxal phosphate of 0.15g/L and the intestinal bacteria enzyme source cell that efficiently expresses tryptophanase of 10g/L respectively, potassium hydroxide aqueous solution with 5% is adjusted pH value to 8.0, in 45 ℃ of waters bath with thermostatic control, stirring reaction 3h.
After reaction is finished, under 4 ℃ of conditions 12, the centrifugal 5min of 000r/min, with solid-liquid separation, supernatant liquor is the mixed solution that contains the L-tryptophane, detects wherein that the L-tryptophane is 6.2g/L, and the molar yield of substrate L-halfcystine is 76.6%, the molar yield of indoles is 79.0%, and the total molar yield from substrate DL-ATC to product L-tryptophane is 73.9%.
The mensuration of above-mentioned L-cysteine content adopts the Gaitonde method, concrete grammar is as follows: accurately take by weighing a certain amount of L-halfcystine standard substance, be dissolved among a certain amount of 0.05mol/L HCl, the preparation final concentration is the standard mother liquor of 500mg/L, further with distilled water above-mentioned mother liquor being diluted respectively is 10,20 ..., the standardized solution of 100mg/L.Get the diluent 0.2mL of a certain amount of standardized solution or testing sample solution, add the 0.2mL Glacial acetic acid, add 0.2mL acid ninhydrine reagent again, in boiling water bath, react 10min, cool off in cold water immediately then, add 2.4mL alcohol at last, making cumulative volume is 3mL.After placing 10min, measure its absorbancy down in 560nm, with concentration and absorbancy drawing standard curve.Calculate L-semicystinol concentration in the unknown solution by this typical curve.
The mensuration of L-tryptophane adopts high performance liquid chromatography, concrete grammar is as follows: get 50～1 respectively, the L-tryptophane reference liquid 20 μ L sample introductions of 500mg/L concentration, continuous sample introduction 5 times, the calculating peak height is also averaged, (X mg/L) is X-coordinate, and peak height (Y) is an ordinate zou drawing standard curve with L-tryptophane concentration.The L-tryptophane that enzymatic reaction produced is compared with typical curve after high performance liquid chromatography detects can carry out accurate quantification.
Embodiment 5
With cellular lysate liquid is the enzyme source, mixes the synthetic L-tryptophane of enzymatic reaction through a step
Be 500mL and be equipped with in the container of agitator that preparation 300mL enzymatic reaction liquid comprises 6g/L DL-ATC, 0.6%K at an internal volume
2HPO
4, 1.0mmol/L azanol, 5g/L indoles, 0.15g/L pyridoxal phosphate, the lysate of 25g TS1138 bacterial strain thalline and the colibacillary cellular lysate liquid that 5g efficiently expresses tryptophanase, potassium hydroxide aqueous solution with 5% is adjusted pH value to 8.0, place 42 ℃ of waters bath with thermostatic control, stirring reaction 3h.
After reaction is finished, under 4 ℃ of conditions 12, the centrifugal 5min of 000r/min, with solid-liquid separation, supernatant liquor is the mixed solution that contains the L-tryptophane, detect wherein that the L-tryptophane is 6.7g/L (method is with example 4), the molar yield of substrate DL-ATC is 79.9%, and the molar yield of indoles is 76.9%.
With the immobilized enzyme is the enzyme source, through the synthetic L-tryptophane of two continuous enzymatic reactions of step
Be 500mL and be equipped with in the container of agitator that preparation 300mL enzymatic reaction liquid comprises 6g/L DL-ATC, 6g/L K at an internal volume
2HPO
4, 1.0mmol/L azanol and 150g/L TS1138 bacterial strain enzyme source cell, the potassium hydroxide aqueous solution with 5% is adjusted pH value to 8.0, places 42 ℃ of waters bath with thermostatic control, stirring reaction 2.5h.
After reaction is finished, under 4 ℃ of conditions 12, the centrifugal 5min of 000r/min, with solid-liquid separation, supernatant liquor is the mixed solution that contains the L-halfcystine, detects wherein that the L-cysteine content is 4.8g/L (method is with example 4), the molar yield of substrate DL-ATC is 96.5%.
In the reaction solution of the above-mentioned L-of containing halfcystine, add the indoles that final concentration is 4.5g/L, the coenzyme pyridoxal phosphate of 0.15g/L and the immobilization tryptophanase of 15g/L respectively, potassium hydroxide aqueous solution with 5% is adjusted pH value to 8.0, in 50 ℃ of waters bath with thermostatic control, stirring reaction 3h.
After reaction is finished, under 4 ℃ of conditions 12, the centrifugal 5min of 000r/min, with solid-liquid separation, supernatant liquor is the mixed solution that contains the L-tryptophane, detects wherein that the L-tryptophane is 6.8g/L (method is with example 4), and the molar yield of substrate L-halfcystine is 84.0%, the molar yield of indoles is 86.7%, and the total molar yield from substrate DL-ATC to product L-tryptophane is 81.1%.
Embodiment 7
The separation and purification of L-tryptophane
The enzymatic reaction liquid that will comprise the L-tryptophane is in 4 ℃ following 12, the centrifugal 10min of 000r/min removes thalline and undissolved indoles, supernatant liquor is regulated pH value to 5.0 with HCl, getting the above-mentioned solution of 300mL is splined on S8 type macroporous resin column (40 * 2.6cm), flow velocity 1.5mL/min is with DL-ATC residual in the adsorbent solution and indoles, effluent liquid is splined on NKA-II macroporous resin column (40 * 2.6cm) again, flow velocity 1.0mL/min, behind the water thorough washing with 50% ethanol elution, elution speed 0.7mL/min.Collect elutriant, and detect the content of L-tryptophane in the elutriant.Merge active wash-out part, through obtaining L-tryptophane white powder 1.652g after the concentrating under reduced pressure drying treatment.Detect (Fig. 2) through high performance liquid chromatography, product purity reaches 98.3% of standard substance.
Claims (8)
1, a kind of method of producing L-tryptophan by microbial enzyme method is characterized in that this method comprises:
1st, two steps successive enzymatic reaction
1.1st, be the enzyme source at first with the full cell or the cellular lysate liquid of pseudomonas putida TS1138 thalline, enzymatic conversion method substrate DL-2-amino-Δ
2-thiazoline-4-carboxylic acid obtains containing the reaction solution of L-halfcystine;
1.2nd, being the enzyme source with full cell, cellular lysate liquid or the immobilized enzyme of the coli somatic that efficiently expresses tryptophanase again, is substrate with above-mentioned L-halfcystine reaction solution and indoles, through the synthetic L-tryptophane of enzymatic conversion method;
Perhaps,
2nd, step blended enzymatic reaction
Full cell or cellular lysate liquid with pseudomonas putida TS1138 thalline are the mixed enzyme source with the full cell, cellular lysate liquid or the immobilized enzyme that efficiently express the coli somatic of tryptophanase, enzymatic conversion method substrate DL-2-amino-Δ
2-thiazoline-4-carboxylic acid and indoles synthesize the L-tryptophane.
2, method according to claim 1, it is characterized in that, in the described two steps successive enzymatic reaction process, add the coenzyme pyridoxal phosphate simultaneously, and azanol is as the inhibitor of L-cysteine desulfhydrase, the consumption of described coenzyme pyridoxal phosphate is 0.05g/L to 1g/L, and the final concentration of described azanol is 0.5mmol/L to 3.0mmol/L; TS1138 thalline consumption is generally 50g/L to 500g/L, and cellular lysate liquid consumption is generally the lysate of 10g to 100g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is generally 1g/L to 100g/L, and cellular lysate liquid consumption is generally the lysate of 0.5g to 30g somatic cells, and the immobilized enzyme consumption is generally 1g/L to 50g/L zymoprotein; Substrate DL-2-amino-Δ
2The concentration of-thiazoline-4-carboxylic acid is 1g/L to 15g/L; Substrate indoles consumption is 1g/L to 20g/L.
3, method according to claim 2 is characterized in that, in the two steps successive enzymatic reaction process, described coenzyme pyridoxal phosphate consumption is preferably 0.15g/L, and the final concentration of used azanol is preferably 1.0mmol/L; TS1138 thalline consumption is preferably 150g/L, and cellular lysate liquid consumption is preferably the lysate of 30g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is preferably 10g/L, and cellular lysate liquid consumption is preferably the lysate of 3g somatic cells, and the immobilized enzyme consumption is preferably 15g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is preferably 6g/L; Substrate indoles consumption is preferably 4.5g/L.
4, method according to claim 1 is characterized in that, in the step blended enzymatic reaction process, adds the coenzyme pyridoxal phosphate simultaneously, and its consumption is 0.05g/L to 1g/L; TS1138 thalline consumption is generally 50g/L to 500g/L, and cellular lysate liquid consumption is generally the lysate of 10g to 100g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is generally 1g/L to 100g/L, and cellular lysate liquid consumption is generally the lysate of 0.5g to 30g somatic cells, and the immobilized enzyme consumption is generally 1g/L to 50g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is 1g/L to 15g/L, and the indoles consumption is 1g/L to 20g/L.
5, method according to claim 4 is characterized in that, in the described step blended enzymatic reaction process, coenzyme pyridoxal phosphate consumption is preferably 0.15g/L; TS1138 thalline consumption is preferably 200g/L, and cellular lysate liquid consumption is preferably the lysate of 40g somatic cells; The coli somatic consumption that efficiently expresses tryptophanase is preferably 15g/L, and cellular lysate liquid consumption is preferably the lysate of 5g somatic cells, and the immobilized enzyme consumption is preferably 20g/L; Substrate DL-2-amino-Δ
2The final concentration of-thiazoline-4-carboxylic acid is preferably 6g/L, and the indoles consumption is preferably 5g/L.
6, according to the described method of claim 1 to 5, it is characterized in that described pseudomonas TS1138 (Pseudomonas putidaTS-1138), be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preserving number are CGMCCNo.1920.
7,, it is characterized in that the described intestinal bacteria that efficiently express tryptophanase are by tryptophan gene of clone intestinal bacteria self and the engineering strain that efficiently expresses in intestinal bacteria according to the described method of claim 1 to 5.
According to the described method of claim 1 to 5, it is characterized in that 8, the separation purification method of the synthetic L-of institute tryptophane is to adopt S8 type macroporous resin to remove and contain DL-2-amino-Δ residual in the L-tryptophan reaction liquid
2-thiazoline-4-carboxylic acid and indoles, again pH be under 5 the condition with NKA-II type macroporous resin adsorption L-tryptophane, get rid of the interference of L-halfcystine, after ethanol elution, concentrating under reduced pressure drying obtain the L-tryptophane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810053749A CN101525641A (en) | 2008-07-04 | 2008-07-04 | Method for producing L-tryptophan by microbial enzyme method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810053749A CN101525641A (en) | 2008-07-04 | 2008-07-04 | Method for producing L-tryptophan by microbial enzyme method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101525641A true CN101525641A (en) | 2009-09-09 |
Family
ID=41093711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810053749A Pending CN101525641A (en) | 2008-07-04 | 2008-07-04 | Method for producing L-tryptophan by microbial enzyme method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101525641A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140483A (en) * | 2011-01-14 | 2011-08-03 | 南开大学 | Method for synthesizing L-tryptophan by immobilized enzyme |
| CN102168118A (en) * | 2011-01-31 | 2011-08-31 | 安徽丰原发酵技术工程研究有限公司 | Method for increasing fermentation output of tryptophan |
| CN102808008A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for synthesizing 5-hydroxytryptophan by enzymic method |
-
2008
- 2008-07-04 CN CN200810053749A patent/CN101525641A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140483A (en) * | 2011-01-14 | 2011-08-03 | 南开大学 | Method for synthesizing L-tryptophan by immobilized enzyme |
| CN102168118A (en) * | 2011-01-31 | 2011-08-31 | 安徽丰原发酵技术工程研究有限公司 | Method for increasing fermentation output of tryptophan |
| CN102808008A (en) * | 2012-08-23 | 2012-12-05 | 天津启仁医药科技有限公司 | Method for synthesizing 5-hydroxytryptophan by enzymic method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106434782A (en) | Method for producing CIS-4-hydroxyproline | |
| CN100562581C (en) | A kind of method and special-purpose reaction column thereof of producing γ-An Jidingsuan | |
| CN102220400B (en) | Method for synthesis of glutathione in vitro | |
| CN103966276A (en) | Method for synthesizing DL-serine through enzyme catalysis method | |
| CN104178536A (en) | Biological preparation method for R-3-aminopiperidine | |
| CN101979646A (en) | Method for preparing guanosine by using immobilized cell | |
| CN101525641A (en) | Method for producing L-tryptophan by microbial enzyme method | |
| CN101985616A (en) | Method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine | |
| CN105316257A (en) | lysinibacillus xylanilyticus and method for preparing alpha-ketoacid with same | |
| CN102286602A (en) | Method for resolving DL-arginine by microbial enzyme method for preparing D-arginine hydrochloride and L-ornithine hydrochloride | |
| CN101358173A (en) | Aspergillus niger ZJUT712 and application thereof in arctium fruit preparation by solid-state fermentation | |
| CN108410918A (en) | A kind of tryptophan fermentation medium and tryptophan fermentation process | |
| CN102250970A (en) | Method for synthesizing 1,3-dioxyacetone by glycerol fermentation | |
| CN101709322B (en) | Method for synthesizing betulic acid by carrying out biocatalysis on betulin | |
| CN101701243B (en) | Method for producing R-mandelic acid and derivates thereof by biocatalysis | |
| CN108410749B (en) | Method for preparing (-) gamma-lactam by asymmetric hydrolysis of marine low-temperature (+) gamma-lactamase | |
| CN101134943A (en) | Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid | |
| CN101104861A (en) | Method for preparing S-ibuprofen and S-ibuprofen ester by biological catalysis | |
| CN105907742A (en) | Carboxyl magnetic bead immobilized glutamic acid decarboxylase and preparation method and application thereof | |
| CN104152384B (en) | A kind of method that marine bacteria strain catalytic asymmetric reduction prepares chirality phenylalanine | |
| CN101812488A (en) | Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method | |
| CN109136314A (en) | The method for synthesizing 2 '-deoxidation -2- amino adenosines using Michigan's Klebsiella | |
| CN109136313A (en) | Utilize the method for Michigan's Klebsiella synthesis 2'-deoxyadenosine | |
| CN102140483A (en) | Method for synthesizing L-tryptophan by immobilized enzyme | |
| CN101054597A (en) | Method for producing cystine by microorganism transformation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090909 |