CN102140483A - Method for synthesizing L-tryptophan by immobilized enzyme - Google Patents
Method for synthesizing L-tryptophan by immobilized enzyme Download PDFInfo
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- CN102140483A CN102140483A CN2011100078905A CN201110007890A CN102140483A CN 102140483 A CN102140483 A CN 102140483A CN 2011100078905 A CN2011100078905 A CN 2011100078905A CN 201110007890 A CN201110007890 A CN 201110007890A CN 102140483 A CN102140483 A CN 102140483A
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Abstract
The invention discloses a method for synthesizing L-tryptophan by immobilized enzyme. In the method, immobilized tryptophanase is taken as an enzyme source, L-cysteine and indole are taken as substrates, the L-tryptophan is produced through enzymatic conversion, the immobilized enzyme is removed through filtration, and the L-cysteine is obtained through further separation. In the method, the tryptophanase is immobilized through GE resin, and the optimized catalysis reaction conditions are that: the pH is 8.5, the reaction time is 3 hours, the concentration of the substrates is 10g/L of L-cysteine and 10g/L of indole, the cell concentration of the enzyme source is 20mL/L, and the concentration of phosphopyridoxal coenzyme is 0.15g/L. Under the optimum reaction conditions, the conversion rate of the cysteine substrate is 81.5 percent. Because the immobilized enzyme is adopted for direct enzymatic synthesis, the method is easy and convenient to implement, high in catalysis efficiency and low in fermentation cost, and the products are easy to separate; therefore, the method has good industrial prospect in the field of industrial production of L-tryptophan.
Description
Technical field
The invention belongs to biotechnology and produce amino acid whose technical field, relate to by the synthetic L-tryptophane of immobilized enzyme method.
Background technology
The L-tryptophane is one of eight kinds of indispensable amino acids in human body and the animal life activity, and the growing of humans and animals, metabolism are played important effect, is widely used in all many-sides such as medicine, food and feed.The production of L-tryptophane mainly relies on chemical synthesis and proteolysis method the earliest, but along with microbial method being produced deepening continuously of tryptophane research, this method has been moved towards practical and has been in dominant position.Microbial method can be divided into direct fermentation, microbe transformation method and enzyme process synthesis method substantially.Wherein the enzyme process synthesis method is to utilize the catalysis of tryptophane biosynthetic enzyme system in the microorganism to produce tryptophane.Enzyme process can utilize chemical industry synthetic precursor to be raw material, both given full play to the advantage of organic synthesis technology, have production concentration height, yield height, purity height again, by product is few and purification operations is easy to advantage, is a kind of method of the comparatively effectively cheap L-of production tryptophane.Path for transformation 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.Although the former obviously is better than the latter by dynamic characteristic, indoles has had strong inhibitory effects to it, and tryptophanase then has satisfactory stability to indoles, so people more pay close attention to tryptophanase in recent years, and uses it for the biosynthesizing of L-tryptophane.
Degraded L-tryptophane generates pyruvic acid, indoles and ammonia under the tryptophanase normal circumstances, 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 enzyme process 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 the enzyme process approach of the synthetic L-tryptophane of raw material, and the price of substrate L-Serine is almost suitable with the L-tryptophane, so practicality is not strong.
2000, Wei Pinghe etc. reported the technology of utilizing tryptophan gene engineering bacteria catalysis L-halfcystine and the synthetic L-tryptophane of indoles, and the product total yield reaches 70%.This technology is the enzyme source with the engineering bacteria, is unfavorable for follow-up separation and purification, and can not reuse, and cost is higher relatively.It is the enzyme source that the present invention adopts immobilized enzyme, can recycle at least 10 times, and be easy to separate.Because substrate L-halfcystine price is comparatively cheap, so this enzyme process approach has important industrial applications value.
Summary of the invention
The objective of the invention is to provide the method for the synthetic L-tryptophane of a kind of immobilized enzyme, solve prior art cost height, shortcomings such as difficult separation for the green production of L-tryptophane.
The present invention relates to genetic engineering bacterium e. coli bl21 (DE3)-pET21a (+)-tnaA, set out with substrate L-halfcystine, through L-tryptophanase direct catalysis L-halfcystine and the indole reaction that genetic engineering bacterium is expressed, preparation L-tryptophane.
The present invention has made up engineering strain e. coli bl21 (DE3)-pET21a (+)-tnaA that efficiently expresses the L-tryptophanase voluntarily, be that template is carried out pcr amplification L-tryptophan gene promptly with the e. coli jm109 strain gene group DNA, and in e. coli bl21 (DE3), express, enzyme activity can reach 3912.6 U/g, and wherein e. coli bl21 (DE3) and corresponding expression vector pET21a (+) are bibliographical information and the available expression system of commercialization.
Thalline with above-mentioned recombinant bacterial strain is the enzyme source, and it is substrate that the present invention has set up with L-halfcystine and indoles, through the method for enzymatic reaction biosynthesizing L-tryptophane.
The method of the synthetic L-tryptophane of immobilized enzyme provided by the invention specifically comprises:
Tryptophanase being carried out immobilization, obtain immobilized enzyme, is the enzyme source with the immobilized enzyme, with L-halfcystine and indoles is substrate, and under 35 to 55 ℃, the pH value is 6 to 11, to 5h, produce the L-tryptophane through enzymatic conversion method reaction 1, obtained the L-tryptophane further the separation after immobilized enzyme was removed in filtration.
Preferred invert point is 45 ℃; Preferred conversion pH value is 8.5; Preferred transformation time is 3 h.
The purification process of L-tryptophane is, regulate catalyzed reaction supernatant liquor pH to 5.0, adopt S-8 type macroporous resin to remove residual indoles, adopt NKA-II type macroporous resin adsorption L-tryptophane again, through 50% (v/v) ethanol elution, concentrating under reduced pressure drying, obtain the L-tryptophane.
In the above enzymatic reaction process, add immobilized enzyme 5 mL/L to 50 mL/L in reaction system, substrate L-halfcystine and indoles are pressed mass ratio 1:1 and are added, and are respectively 2 g/L to 25 g/L, and the amount of coenzyme pyridoxal phosphate is 0.05 g/L ~ 0.25 g/L.
The consumption of substrate L-halfcystine and indoles preferably is respectively 10 g/L; The preferable amount of coenzyme pyridoxal phosphate is 0.15 g/L.
The carrier that immobilized enzyme uses is GE, ES-2,0404-2 resin.Be preferably the GE resin.
Reusable 10 times of described immobilized enzyme, and under The optimum reaction conditions and system, preceding 5 uses, its catalytic substrate transformation efficiency all can reach more than 80%.
In embodiments of the present invention, adopt the HPLC method to measure the content of L-tryptophane, chromatographic condition:
Chromatographic column: Nucleosil SA(250 * 4.6 mmol/L); Sampling volume: 20 mL.
Moving phase: glacial acetic acid-triethylamine-water (0.1:0.1:99.8); Flow velocity: 1.0 mL/min; Column temperature: 25 ℃.Ultraviolet detection wavelength: 280 nm.
ELSD detector parameters: 40 ℃ of drift tube temperatures, nitrogen pressure: 2.8 Bar.
Beneficial effect of the present invention: the present invention is a substrate with L-halfcystine and indoles, utilizes the immobilized L-of efficiently expressing tryptophanase to be the enzyme source, and enzyme process synthesizes the L-tryptophane.Synthesize owing to adopt immobilized enzyme directly to carry out enzyme process, convenient and easy, the catalytic efficiency height, fermentation costs is low, the product separate easily, so the present invention has good industrial prospect in the field of industrialized production of L-tryptophane.
Description of drawings
Fig. 1 is the abduction delivering of tryptophanase,
1 ~ 3, comprise pET-tnaA's
E. coliBL21 (DE3) cellular lysate liquid; 4, comprise pET-21a's (+)
E.coliBL21 (DE3) cellular lysate liquid; 5, the molecular weight of albumen standard.
Fig. 2 is the optimization of catalytic condition,
A: the relation of temperature of reaction and L-halfcystine transformation efficiency; The relation of B:pH and L-halfcystine transformation efficiency; C: the relation of reaction times and L-halfcystine transformation efficiency; D: substrate indoles and L-halfcystine are to the relation of L-halfcystine transformation efficiency; E: the relation of immobilized enzyme and L-halfcystine transformation efficiency; F: the relation of coenzyme PLP and L-halfcystine transformation efficiency.
Fig. 3 is the immobilized enzyme study on the stability.
Fig. 4 is in the HPLC method 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, and L-tryptophane (retention time, 4.732 min); 2, indoles (retention time, 5.965 min); 3, L-halfcystine (retention time, 3.223 min).
Embodiment
The present invention will make up BL21 (DE3)-pET21a (+)-tnaA voluntarily and carry out abduction delivering, collect enzyme liquid and carry out immobilization, with the immobilized enzyme is the enzyme source, with L-halfcystine and indoles is substrate, produce the L-tryptophane through the enzymatic conversion method reaction, obtained the L-tryptophane further the separation after immobilized enzyme was removed in filtration.This research has been carried out immobilization research to the tryptophanase of expressing, and immobilized tryptophanase reaction conditions has been carried out preliminary study, produces the production technique of L-tryptophane for microbial enzyme method and lays a good foundation.Specific examples is as follows:
The structure of genetic engineering bacterium, tryptophanase abduction delivering and crude enzyme liquid preparation
Tryptophan gene sequence according to e. coli k12 strain, design tryptophan gene upstream primer P1:5 '-CCG AAG CTT ATG GAA AAC TTT AAA CAT CTC C-3 ' and downstream primer P2:5 '-CCC GGA TCC TTA AAC TTC TTT CAG TTT TGC GG-3 ', genomic dna with the e. coli jm109 bacterial strain is a template, pcr amplification obtains tryptophan gene, and makes up recombinant expression plasmid pET21a (+)-tnaA respectively.
Recombinant plasmid transformed to e. coli bl21 (DE3), is made up and obtains tryptophan gene engineering strain BL21 (DE3)-pET21a (+)-tnaA.
Get a tryptophane engineering bacteria list colony inoculation in the LB substratum, 37 ℃ of overnight incubation are inoculated in saturated culture in the LB substratum that contains Amp (100 μ g/ml) with 1% to obtain saturated culture, and 37 ℃ are continued to cultivate OD
600Reach at 0.5 o'clock, add IPTG, induce 2h abduction delivering tryptophanase for 37 ℃ to final concentration 0.1mmol/L.
Medium centrifugal is collected thalline, add 0.1mol/L potassium phosphate buffer (pH 8.0) suspension washing, collect thalline and take by weighing weight in wet base, and adding potassium phosphate buffer, to make final concentration be 20%(W/V) enzyme source cell bacteria suspension, ultrasonic wave (35kHz, 120W) broken wall 30min, centrifugal collection supernatant is crude enzyme liquid.
Crude enzyme liquid is carried out SDS-PAGE analyze, the results are shown in Figure 1, have amounts of protein to express at 53 kD places, consistent with the tryptophanase size, the Recombinant Protein Expression amount accounts for 68.2% of somatic cells soluble proteins.
The mensuration of tryptophanase vigor
In 10 mL triangular flasks, add respectively in order: pyridoxal phosphate (PLP) solution of 20 μ L, 0.2 mg/mL, reduced glutathion (GSH) solution of 10 μ L, 0.005 M, the tryptophanase liquid that obtains among the 270 μ L embodiment 1; Above-mentioned 0.3 mL solution covers with 1 mL toluene, add the L-tryptophane solution of 100 μ L, 5 mg/mL behind 37 ℃ of insulation 5 min, place 37 ℃ of light and slow oscillatory reaction 10 min of shaking table, add 3 mL indoles colour developing liquid stopped reaction then, mixing leaves standstill behind 30 min and measures absorbancy in 570 nm.
The enzyme activity definition: under above-mentioned reaction conditions, in 37 ℃, it is an enzyme activity unit that 10 min catalysis form the needed enzyme amount of 0.001 μ mol indoles.
Measure the tryptophanase vigor of expressing through aforesaid method and reach 3912.6 U/g, be about 110 times of original host bacterium self tryptophanase vigor.
The high performance liquid chromatography of L-tryptophane detects in the reaction solution
Tryptophanase being carried out immobilization, obtain immobilized enzyme, is the enzyme source with the immobilized enzyme, is substrate with L-halfcystine and indoles, and under 35 to 55 ℃, the pH value is 6 to 11, to 5h, produces the L-tryptophane through enzymatic conversion method reaction 1.Reaction solution after the catalysis is removed immobilized enzyme, and the clear liquid after the filtration with the tryptophane concentration that high effective liquid chromatography for measuring generates, with the comparison of tryptophane typical curve, calculates the molar yield of L-halfcystine in the substrate solution.
Chromatographic column: Nucleosil SA(250 * 4.6 mmol/L); Sampling volume: 20 mL.
Moving phase: glacial acetic acid-triethylamine-water (0.1:0.1:99.8); Flow velocity: 1.0 mL/min; Column temperature: 25 ℃.Ultraviolet detection wavelength: 280 nm.
ELSD detector parameters: 40 ℃ of drift tube temperatures, nitrogen pressure: 2.8 Bar.
The immobilization of tryptophanase
Get 100 mL epoxy chloropropane activatory GE, ES-2,0404-2 resin respectively and mix 4 ℃ of shaken overnight with the cytoclasis crude enzyme liquid that 200 mL embodiment 1 obtain.Potassium phosphate buffer washing back 1M Gly seals 10 h, and potassium phosphate buffer (pH 8.0) washing obtains immobilized enzyme.In 100 mL reaction systems, add 1 mL immobilized enzyme, 0.5 the g indoles, the L-halfcystine of 0.5 g, 0.01 g pyridoxal phosphate, 37 ℃ of catalyzed reaction 2 h, L-halfcystine transformation efficiency relatively, it is the highest wherein to adopt the GE resin to carry out the transformation efficiency of catalysis L-halfcystine of the immobilized enzyme that immobilization obtains, and can reach 50.78%, the transformation efficiency of ES-2 resin immobilized enzyme is 40.12%, and the transformation efficiency of 0404-2 resin immobilized enzyme is 35.07%.Therefore select the highest GE resin immobilization tryptophanase of transformation efficiency for use, the immobilized enzyme of acquisition uses for subsequent embodiment.
The immobilized enzyme reaction condition optimization
With immobilized enzyme in the foregoing description 4 is the enzyme source, and adding substrate L-halfcystine and indoles consumption (1:1) are 2 ~ 25 g in every liter of reaction system respectively; The amount of coenzyme pyridoxal phosphate is 0.05 g ~ 0.25 g; Invert point is 35 ~ 55 ℃; Transforming the pH value is 6 ~ 11; Transformation time is 1 ~ 5 h, and immobilized enzyme is 5 ~ 50 mL/L.(the concrete value of above each parameter is omited shown in each point among Fig. 2 herein) investigates the influence of different conversion conditions to L-halfcystine transformation efficiency, and adopts the content of the method detection L-tryptophane among the embodiment 3, calculates the transformation efficiency of L-halfcystine.
As shown in Figure 2, wherein,
A: the relation of investigating temperature of reaction and L-halfcystine transformation efficiency; Immobilized enzyme 10 mL; Substrate L-halfcystine consumption is 5 g; Substrate indoles consumption is 5 g; The amount of coenzyme pyridoxal phosphate is 0.1 g; Transforming the pH value is 7; Transformation time is 2 h.
B: the relation of investigating pH and L-halfcystine transformation efficiency; Immobilized enzyme 10 mL; Substrate L-halfcystine consumption is 5 g; Substrate indoles consumption is 5 g; The amount of coenzyme pyridoxal phosphate is 0.1 g; Invert point is 45 ℃; Transformation time is 2 h.
C: the relation of investigating reaction times and L-halfcystine transformation efficiency; Immobilized enzyme 10 mL; Substrate L-halfcystine consumption is 5 g; Substrate indoles consumption is 5 g; The amount of coenzyme pyridoxal phosphate is 0.1 g; Invert point is 45 ℃; Transforming the pH value is 8.5.
D: the relation of investigating substrate and L-halfcystine transformation efficiency; Immobilized enzyme 10 mL; The amount of coenzyme pyridoxal phosphate is 0.1 g; Invert point is 45 ℃; Transforming the pH value is 8.5; Transformation time is 3 h.
E: the relation of investigating immobilized enzyme and L-halfcystine transformation efficiency; Substrate L-halfcystine consumption is 10 g; Substrate indoles consumption is 10 g; The amount of coenzyme pyridoxal phosphate is 0.1 g; 45 ℃ of invert points; Transforming the pH value is 8.5; Transformation time is 3 h.
F: the relation of investigating coenzyme PLP and L-halfcystine transformation efficiency; Immobilized enzyme 20 mL; Substrate DL-cysteine consumption is 10 g; Substrate indoles consumption is 10 g; Invert point is 45 ℃; Transforming the pH value is 8.5; Transformation time is 3 h.
The suitableeest enzymatic reaction condition of determining is: temperature of reaction, 45 ℃; Reaction system pH, 8.5; Reaction times, 3 h; Concentration of substrate, the L-halfcystine of 10 g/L+10 g/L indoles; The immobilized enzyme consumption, 20 mL/L; Coenzyme pyridoxal phosphate concentration, 0.15 g/L.Under this suitableeest enzymatic reaction condition, the transformation efficiency of L-halfcystine is about 81.5%.
The study on the stability of enzyme
The single catalyzed reaction finishes after-filtration and separates immobilized enzyme and product tryptophane, and the immobilized enzyme of recovery is recycled and reused for catalyzed reaction next time.Use repeatedly 10 times, the repeated experiments result of immobilized enzyme shows, 10 uses, and L-halfcystine transformation efficiency can reach more than 60%, and the transformation efficiency of preceding 5 substrate L-halfcystines all can be at (Fig. 3) more than 80%.
The preparation of L-tryptophane and separation
The top condition of determining with embodiment 5, immobilized enzyme with embodiment 4 is the synthetic L-tryptophane of enzyme source enzyme process, subsequent filtration is collected supernatant liquor, is splined on S-8 type macroporous resin column (40 * 2.6 cm) behind the adjusting pH 5.0, flow velocity 1.5 mL/min, with indoles residual in the adsorbent solution, effluent liquid is splined on NKA-II macroporous resin column (40 * 2.6 cm), flow velocity 1.0 mL/min again, behind the water thorough washing with 50%(v/v) ethanol elution, elution speed 1 mL/min.Collect elutriant, and detect the content of L-tryptophane in the elutriant, merge L-tryptophane elution peak, through obtaining L-tryptophane white powder 15.24 g after the concentrating under reduced pressure drying treatment, adopt the high performance liquid chromatography of embodiment 3 to detect, product purity reaches 98.9%.
Claims (8)
1. the method for the synthetic L-tryptophane of an immobilized enzyme is characterized in that this method comprises:
Tryptophanase being carried out immobilization, obtain immobilized enzyme, is the enzyme source with the immobilized enzyme, is substrate with L-halfcystine and indoles, with the pyridoxal phosphate is coenzyme, and under 35 to 55 ℃, the pH value is 6 to 11, to 5h, produce the L-tryptophane through enzymatic conversion method reaction 1, further separate obtaining the L-tryptophane.
2. method according to claim 1 is characterized in that: fixed enzyme vector is GE, ES-2,0404-2 resin.
3. method according to claim 2 is characterized in that: described fixed enzyme vector is preferably the GE resin.
4. method according to claim 1, it is characterized in that: in every liter of catalyzed reaction liquid, the add-on of immobilized enzyme is 5 to 50 mL, and substrate L-halfcystine and indoles are pressed mass ratio 1:1 and added, be respectively 2 to 25 g, the amount of coenzyme pyridoxal phosphate is 0.05 g to 0.25 g.
5. method according to claim 1 is characterized in that: preferred invert point is 45 ℃; Preferred conversion pH value is 8.5; Preferred transformation time is 3 h.
6. method according to claim 4 is characterized in that: in every liter of reaction solution, the immobilized enzyme preferable amount is 20 mL; The consumption of substrate L-halfcystine and indoles preferably is respectively 10 g; The preferable amount of coenzyme pyridoxal phosphate is 0.15 g.
7. method according to claim 1 is characterized in that: reusable 10 times of immobilized enzyme, and under The optimum reaction conditions and system, preceding 5 uses, its catalytic substrate transformation efficiency all can reach more than 80%.
8. method according to claim 1, it is characterized in that: the purification process of L-tryptophane is, regulate catalyzed reaction supernatant liquor pH to 5.0, adopt S-8 type macroporous resin to remove wherein residual indoles, adopt NKA-II type macroporous resin adsorption L-tryptophane again, through 50 v/v % ethanol elutions, concentrating under reduced pressure drying, obtain the L-tryptophane.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110229807A (en) * | 2019-05-10 | 2019-09-13 | 河北工业大学 | The preparation method of organic phosphorus immobilized enzyme catalysis agent and organic phosphorus immobilized enzyme catalysis agent are applied with it |
| CN110982858A (en) * | 2019-11-13 | 2020-04-10 | 上海星酶生物科技有限公司 | Production process of D-tryptophan |
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| CN101525641A (en) * | 2008-07-04 | 2009-09-09 | 南开大学 | Method for producing L-tryptophan by microbial enzyme method |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS6349091A (en) * | 1986-08-15 | 1988-03-01 | Mitsui Toatsu Chem Inc | Production of tryptophan |
| CN101525641A (en) * | 2008-07-04 | 2009-09-09 | 南开大学 | Method for producing L-tryptophan by microbial enzyme method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110229807A (en) * | 2019-05-10 | 2019-09-13 | 河北工业大学 | The preparation method of organic phosphorus immobilized enzyme catalysis agent and organic phosphorus immobilized enzyme catalysis agent are applied with it |
| CN110229807B (en) * | 2019-05-10 | 2023-04-18 | 河北工业大学 | Preparation method of organophosphorus immobilized enzyme catalyst, organophosphorus immobilized enzyme catalyst and application thereof |
| CN110982858A (en) * | 2019-11-13 | 2020-04-10 | 上海星酶生物科技有限公司 | Production process of D-tryptophan |
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Application publication date: 20110803 |