CN101812488A - Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method - Google Patents
Method for preparing D-cystine and L-tryptophane by using DL-cysteine split by microbial enzyme method Download PDFInfo
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Abstract
The invention discloses a method for preparing D-cystine and L-tryptophane by using DL-cysteine split by a microbial enzyme method. The method comprises the following steps of: splitting the DL-cysteine to produce D-cysteine and L-tryptophane through an enzymatic reaction by using fermentation liquor of recombined Bacillus subtillis which efficiently express tryptophanase as an enzyme source and using the DL-cysteine and indole as a substrate; and oxidizing the enzymatic reaction liquor to ensure that the D-cysteine is oxidized into D-cystine, then regulating pH to be 5 and performing isoelectric point crystallization to separate the D-cystine out, and collecting the precipitation to obtain the D-cystine. A method for purifying the L-tryptophane comprises the following steps of: removing the residual indole from the supernatant which is subjected to the isoelectric point crystallization by adopting an S-8 type macroporous resin, then absorbing the L-tryptophane in the supernatant by adopting an NKA-II type macroporous resin, eluting by using 50 percent ethanol, reducing pressure, concentrating and drying to obtain the L-tryptophane. The invention provides a novel process route of green production of the D-cystine and the L-tryptophane.
Description
[technical field]:
The invention belongs to biotechnology and produce amino acid whose technical field, relate to the method that splits DL-cysteine production D-Gelucystine and L-tryptophane by microbial enzyme method.
[background technology]:
Gelucystine is important medicine intermediate, also is usually used in preparing amino acid oral preparations or transfusion, dying agent, makeup, dairy products additive, grease oxidation inhibitor etc.; Also can be used for medicines such as heavy metal detoxification agent, hepatitis.Gelucystine has the body cell of promotion redox ability, strengthen white cell to prevent pathogenic bacterial infection, can be used for treating leukopenia, coronary heart disease, prevent and treat fatty liver, liver cirrhosis, short hair growth, anti-skin aging also can be used for the treatment of illness such as typhoid fever dysentery, influenza, asthma, neurodynia, eczema and various poisonings.
The D-Gelucystine is the dimer of D-halfcystine, can effectively obtain important pharmaceutical intermediate D-halfcystine by the reduction means.The D-halfcystine is a kind of alpha-non-natural amino acid, can be widely used in industries such as medicine, food, makeup, feed, it is the important intermediate of chiral drug, as the D-halfcystine is synthetic third generation cephalosporin---the important source material of Cefminox sodium salt, introduced the D-halfcystine in 7 β position side chain terminal, have very strong germicidal action, can be used for the treatment of respiratory tract infection; The Beracilline that is obtained by the D-cysteine derivatives can be used as toxicide, is used for the antagonist of heavy metal, also is used for immunological diseases such as rheumatoid arthritis and chronic active hepatitis; In addition; 2-(2; 4-two phenolic hydroxyl groups)-4, medicine series such as 5-thiazoline-4 (S)-carboxylic acid, N-acetyl-D-halfcystine, FK-228, N-(S)-3-alkyl oenanthyl-D-R-glutamyl-glycyl-D-L-Ala and Azythromycin are material medicine with the D-halfcystine all.In addition, the D-cysteine salt still is colibacillary potent inhibitor, also can be used as the alleviating agent of acute alcoholism, also is the inhibitor of some proteolytic enzyme simultaneously, also have certain curative effect at aspects such as inflammation and HIV treatments, so purposes is very extensive.
Preparation technology's more complicated of D-cysteine hydrochloride adopts the method for asymmetric synthesis preparation usually, and reactions steps is many, and the production control difficulty is big, needs the chiral reagent of use value costliness; And enzyme process resolution reaction mild condition, selectivity is strong, and side reaction is few, the yield height, the optical purity height is the effective ways of the production of D-halfcystine.
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 production method of L-tryptophane 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 the precursor of chemosynthesis to be raw material, has both given full play to the advantage of organic synthesis technology, has production concentration height, yield height, purity height again, an advantage such as by product is few and purification operations is easy.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.And be in the approach of the synthetic L-tryptophane of raw material with L-Serine and indoles, because the price of substrate L-Serine is almost suitable with the L-tryptophane, so practicality is not strong.Therefore 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.
At present to split mainly be to adopt chemical process to DL-cysteine, and chemical resolution method is to make racemic modification and chiral reagent effect generation diastereomer, utilizes the difference of diastereomer physics, chemical property and they are separated.With the DL-cysteine is raw material, and adopt route as follows respectively: (1) is resolving agent with L-tartrate, via 2,2-dimethyl-thiazolidine--asymmetric the conversion of 4-carboxylic acid generates D-2,2-dimethyl-thiazolidine--4-carboxylic acid-L-tartrate, make its hydrolysis in the aqueous solution, obtain the D-halfcystine.(2) with the DL-cysteine be raw material, prepare the D-4-tetrahydro-thiazoles-4-carboxylic acid earlier, and then make itself and azanol reaction obtain halfcystine, utilize the method for preferential crystallization to obtain optically active isomer afterwards.(3) be resolving agent with L-thiazoline carboxylic acid, generate DL-cysteine-thiazoline carboxylate salt, utilize the method for preferential crystallization to obtain D-, L-halfcystine-thiazoline carboxylate salt, adopt triethylamine to handle the D-and the L-halfcystine that can obtain optical purity afterwards.And that chemical method for splitting has step is long, and yield is low, is unfavorable for the characteristics of environmental protection.
Enzyme is that optically active substance is arranged, and because the specificity of its chemical reaction, so can select the resolution reagent of suitable enzyme as racemic modification.But often under the effect of enzyme, one of enantiomorph is consumed, and this is the shortcoming of this method.2009, burnt celebrating just wait (200910032018.9) with DL-cysteine as raw material, the somatic cells that will have the gene engineering colibacillus of high vigor L-tryptophanase mixes with the conversion fluid of kilnitamin that contains DL-cysteine or DL-cysteine hydrochloride, carry out enzymatic reaction under 30～45 ℃, then with isoelectric point crystallizing method or isoelectric point crystallizing with from the method that combines of exchange resin separate converted product, obtain high purity D-halfcystine and L-tryptophane.And efficiently expressing the fermented liquid of the recombinant bacillus bacillus of tryptophanase, utilization of the present invention is the enzyme source, when enzyme process fractionation DL-cysteine prepares the D-Gelucystine L-halfcystine is converted into the L-tryptophane, it is low to have overcome employing somatic cells temperature tolerance, catalytic efficiency is low, shortcomings such as mixing in the product has somatic cells, and separation and purification is loaded down with trivial details.The inventive method not only can realize the fractionation of DL-cysteine and the preparation of L-tryptophane, and fermentation costs is low, the transformation efficiency height, and good separating effect has good industrialization value.
[summary of the invention]:
The objective of the invention is to solve the problem that exists in the existing DL-cysteine disassemble technique, provide a kind of microbial enzyme method to split the green method for transformation that DL-cysteine is produced D-Gelucystine and L-tryptophane.
The present invention relates to genetic engineering bacterium Bacillus subtillis WB600-pWB980-tnaA fermented liquid is the enzyme source, set out with substrate DL-cysteine and indoles, split through the direct catalysis of the tryptophanase of genetically engineered secreting, expressing and to obtain D-halfcystine and by product L-tryptophane, and finally obtain the operational path of product D-Gelucystine and L-tryptophane through oxidation and separation and purification.
The present invention has made up the Bacillus subtillis engineering strain WB600-pWB980-tnaA of efficient secretion type expression tryptophanase voluntarily, be template promptly with the e. coli jm109 strain gene group DNA, pcr amplification obtains tryptophan gene (Genebank No.X15974.1), carry out recombinant expressedly in Bacillus subtillis WB600, the recombinant expressed tryptophanase vigor of the fermented liquid after the optimization can reach more than the 1000U/mL.Wherein Bacillus subtillis F-strain WB600 and corresponding expression vector pWB980 are bibliographical information and the available expression system of commercialization.
Fermented liquid with above-mentioned recombinant bacterial strain is the enzyme source, and it is substrate that the present invention has set up with DL-cysteine and indoles, through the biological method that splits DL-cysteine production D-Gelucystine and L-tryptophane of enzymatic reaction.
Microbial enzyme method provided by the invention splits the method that DL-cysteine is produced D-Gelucystine and L-tryptophane, specifically comprises:
Fermented liquid with the gene engineering recombinant bacterium of expressing tryptophanase is the enzyme source, and in the enzymatic reaction process, adding substrate DL-cysteine consumption in reaction system is 5g/L to 50g/L; Substrate indoles consumption is 3g/L to 30g/L; The consumption of coenzyme pyridoxal phosphate is 0.05g/L to 0.25g/L.Under 40 to 60 ℃, the pH value is 7 to 9, to 6h, produces D-halfcystine and L-tryptophane through enzymatic conversion method reaction 2, further obtains the D-Gelucystine after the bubbling air oxidation.
It is that above-mentioned reaction solution is regulated pH to 5.0 that the present invention separates the purification process that splits the D-Gelucystine that obtains after the oxidation, and the D-Gelucystine is carried out isoelectric precipitation.The separation purification method of the L-tryptophane that the present invention produced is that the supernatant liquor behind the isoelectric precipitation adopts S-8 type macroporous resin to remove wherein residual indoles, use NKA-II type macroporous resin adsorption L-tryptophane again, through 50% ethanol elution, the concentrating under reduced pressure drying obtains the L-tryptophane.
In embodiments of the present invention, adopt pre-column derivatization HPLC method to measure the content of D/L-halfcystine, derivatization reagent is a Marfey reagent, it is 1-fluoro-2,4-dinitrophenyl-5-L-alanimamides (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA).It contains a chiral carbon atom, when reacting with the amino acid that contains chiral carbon atom, because sterically hindered influence, can suppress amino acid generation racemization reaction effectively, therefore can with two kinds not the same amino acid derived of isomorphism type be diastereomer, thereby reach the purpose of isolation identification.The pre-column derivatization method is as follows: with 100 μ L 10mmol/L Na
2CO
3Solution (pH9) dissolving halfcystine, making its final concentration is 10～40mmol/L, join subsequently in 1% the FDAA 200 μ L acetone solns, 40 ℃ hatch 1h after, add 2N HCl 20 μ L termination reactions, 20 μ L sample introductions are used for HPLC and analyze.Chromatographic condition: C
18Chromatographic column (Phenomenex Luna 5 μ, 100A, 250 * 4.6mm), be moving phase with A phase water (containing 0.1%TFA), B phase acetonitrile (containing 0.1%TFA), gradient elution: 0.0min, 55%A phase; 11.0min, the 47%A phase; Room temperature detects wavelength 340nm, and flow velocity is 1mL/min.Continuous sample introduction 5 times, the calculating peak area is also averaged, and (X mmol/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with the D/L semicystinol concentration.The D-halfcystine that enzymatic reaction produced is got 100 μ L after diluting 10 times, after deriving as stated above, measure with HPLC, and the establishing criteria curve carries out accurate quantification.
In embodiments of the present invention, adopt high performance liquid chromatography to measure the content of L-tryptophane, concrete grammar is as follows: get the L-tryptophane reference liquid 20 μ L sample introductions of 50～200mg/L concentration respectively, chromatographic condition: C
18Chromatographic column (Phenomenex Luna 5 μ, 100A, 250 * 4.6mm), moving phase: methyl alcohol: 1mmol/L potassium primary phosphate (30: 70), room temperature detects wavelength 225nm, and flow velocity is 1mL/min.Continuous sample introduction 5 times, the calculating peak area is also averaged, and (X mg/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with L-tryptophane concentration.The L-tryptophane that enzymatic reaction produced and refining after the L-tryptophane, after high performance liquid chromatography detects, compare and can carry out accurate quantification with typical curve.
Beneficial effect of the present invention: the present invention sets out with substrate indoles and DL-cysteine, the fermented liquid that utilization efficiently expresses the Bacillus subtillis WB600-pWB980-tnaA of tryptophanase is the enzyme source, and enzyme process splits DL-cysteine and produces D-Gelucystine and L-tryptophane.Split owing to adopt fermented liquid directly to carry out enzyme process, temperature tolerance is good, the catalytic efficiency height, do not contain somatic cells in the product, separation and purification is simple, and fermentation costs is low, so the present invention has good industrialization value in the field of industrialized production of D-Gelucystine and L-tryptophane.
[description of drawings]
Fig. 1: D-halfcystine typical curve
A:FDAA deutero-D-cysteine standard substance color atlas; B:D-halfcystine quantitative analysis typical curve; 1: derivative reagent FDAA; The 2:FDAA-D-halfcystine
Fig. 2: L-tryptophane typical curve
A:L-tryptophane standard substance color atlas; B:L-tryptophane quantitative analysis typical curve
Fig. 3: the tryptophanase specificity is analyzed
The accurate product induced color of A:DL-halfcystine standard spectrogram; B:L-tryptophane induced color spectrogram; C: reaction solution induced color spectrogram;
1: derivative reagent FDAA; The 2:FDAA-D-halfcystine; The 3:FDAA-L-halfcystine; The 4:FDAA-L-tryptophane
Fig. 4: the recombined bacillus subtilis fermented liquid is the conversion condition analysis in enzyme source
A: the relation of reaction times and L-halfcystine transformation efficiency; B pH: with the relation of L-halfcystine transformation efficiency; C: temperature of reaction with the relation of L-halfcystine transformation efficiency; D: the relation of coenzyme PLP and L-halfcystine transformation efficiency; E: the relation of substrate indoles and L-halfcystine transformation efficiency; F: the relation of substrate DL-cysteine and L-halfcystine transformation efficiency
Fig. 5: the purity check of D-Gelucystine
1: derivative reagent FDAA; The 2:FDAA-D-Gelucystine
Fig. 6: L-tryptophane purifying and purity check
A: elution curve; B: purity check figure.
[embodiment]
Efficiently expressing of structure of genetic engineering bacterium Bacillus subtillis and tryptophanase
Tryptophan gene sequence according to e. coli k12 strain, design tryptophan gene upstream primer P1:5 '-CCG AAGCTT ATG GAA AAC TTT AAA CAT CTC C-3 ' and downstream primer P2:5 '-CCC GGA TCC TFA AAC TFCTTT 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 pWB980-tnaA respectively.
Recombinant plasmid transformed to subtilis WB600, is made up and obtains the tryptophan gene engineering strain.
Positive recombinant bacterial strain is inoculated in 4mL contains incubated overnight in the LB substratum of kantlex 20 μ g/mL, inserting 50mL with 2% switching amount subsequently contains in the fresh LB substratum of kantlex 20 μ g/mL, 37 ℃ of shaking culture 24h, centrifugal removal thalline, get supernatant liquor, measure corresponding tryptophanase vigor, the enzyme activity of every milliliter of fermented liquid can reach 510U.
Column front derivation HPLC method is measured the content of D/L-halfcystine
The pre-column derivatization method is as follows: with 100 μ L 10mmol/L Na
2CO
3Solution (pH9) dissolving halfcystine, making its final concentration is 10～40mmol/L, join subsequently in 1% the FDAA 200 μ L acetone solns, 40 ℃ hatch 1h after, add 2NHCl 20 μ L termination reactions, 20 μ L sample introductions are used for HPLC and analyze.Chromatographic condition: C
18Chromatographic column (Phenomenex Luna 5 μ, 100A, 250 * 4.6mm), be moving phase with A phase water (containing 0.1%TFA), B phase acetonitrile (containing 0.1%TFA), gradient elution: 0.0min, 55%A phase; 11.0min, the 47%A phase; Room temperature detects wavelength 340nm, and flow velocity is 1mL/min.Continuous sample introduction 5 times, the calculating peak area is also averaged, and (X mmol/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with the D-semicystinol concentration.The D-halfcystine that enzymatic reaction produced is got 100 μ L after diluting 10 times, after deriving as stated above, measure with HPLC, and the establishing criteria curve carries out accurate quantification.As Fig. 1.
The HPLC method is measured the content of L-tryptophane
Accurately the L-tryptophane reference liquid of configuration 50～200mg/L concentration is got 20 μ L sample introductions respectively, adopts the HPLC method to measure the content of L-tryptophane.Chromatographic condition: the C18 chromatographic column (Phenomenex Luna 5 μ, 100A, 250 * 4.6mm), moving phase: methyl alcohol: 0.001mol/L potassium primary phosphate (30: 70), room temperature detects wavelength 225nm, and flow velocity is 1mL/min.Continuous sample introduction 5 times, the calculating peak area is also averaged, and (X mg/L) is X-coordinate, and peak area (Y) is an ordinate zou drawing standard curve with L-tryptophane concentration.
The L-tryptophane that enzymatic reaction produced and refining after the L-tryptophane, after high performance liquid chromatography detects, compare and can carry out accurate quantification with typical curve.As Fig. 2.
The tryptophanase fermentation
Recombined bacillus subtilis among the embodiment 1 is inoculated in 4mL contains incubated overnight in the LB substratum of kantlex 20 μ g/mL, insert 120mL with 2% switching amount subsequently and contain in the fresh LB substratum of kantlex 20 μ g/mL, cultivate 12h for 37 ℃; At last this seed all is inoculated in 6 liters of fermention mediums, 37 ℃ of fermentation 12h, fermentative medium formula is: 0.5% Zulkovsky starch, 1% yeast powder, 0.4% corn steep liquor.Collect filtering fermentation liquor and remove thalline, get the recombined bacillus subtilis fermented liquid.Measure corresponding tryptophanase vigor, the enzyme activity of every milliliter of fermented liquid can reach 1020U.
The tryptophanase specificity is analyzed
With recombined bacillus subtilis fermented liquid in the above-mentioned example 4 is the enzyme source, and the add-on that adds the substrate DL-cysteine in every liter of fermented liquid is 10g; Substrate indoles consumption is 5g; The amount of coenzyme pyridoxal phosphate is 0.1g; Invert point is 45 ℃; Transforming the pH value is 8; Transformation time is 3h.Adopt the above-mentioned HPLC method detection D-halfcystine and the content of L-tryptophane, to determine the Substratspezifitaet of tryptophanase.
The result does not almost have L-halfcystine chromatographic peak as shown in Figure 3 in the reaction solution after the catalyzed reaction, the substitute is corresponding L-tryptophane peak, and almost not loss of D-halfcystine simultaneously proves that this tryptophanase has specificity preferably.
With fermentation of bacillus subtilis liquid is the enzyme source, investigates the transformation efficiency of L-halfcystine
With recombined bacillus subtilis fermented liquid in the above-mentioned example 4 is the enzyme source, and the add-on that adds the substrate DL-cysteine respectively in every liter of fermented liquid is 5～50g; Substrate indoles consumption is 3～30g; The amount of coenzyme pyridoxal phosphate is 0.05g～0.25g; Invert point is 20～60 ℃; Transforming the pH value is 6～10; Transformation time is 1～6h.(the concrete value of above each parameter is omited shown in each point among Fig. 4 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.
Fig. 4 A: the relation of investigating temperature of reaction and L-halfcystine transformation efficiency; Substrate DL-cysteine consumption is 25g; Substrate indoles consumption is 10g; The amount of coenzyme pyridoxal phosphate is 0.1g; Transforming the pH value is 7; Transformation time is 2h.
Fig. 4 B: the relation of investigating pH and L-halfcystine transformation efficiency; Substrate DL-cysteine consumption is 25g; Substrate indoles consumption is 10g; The amount of coenzyme pyridoxal phosphate is 0.1g; Invert point is 50 ℃; Transformation time is 2h.
Fig. 4 C: the relation of investigating reaction times and L-halfcystine transformation efficiency; Substrate DL-cysteine consumption is 25g; Substrate indoles consumption is 10g; The amount of coenzyme pyridoxal phosphate is 0.1g; Invert point is 50 ℃; Transforming the pH value is 8.
Fig. 4 D: the relation of investigating coenzyme PLP and L-halfcystine transformation efficiency; Substrate DL-cysteine consumption is 25g; Substrate indoles consumption is 10g; Invert point is 50 ℃; Transforming the pH value is 8; Transformation time is 3h.
Fig. 4 E: the relation of investigating substrate indoles and L-halfcystine transformation efficiency; Substrate DL-cysteine consumption is 25g; The amount of coenzyme pyridoxal phosphate is 0.15g; Invert point is 50 ℃; Transforming the pH value is 8; Transformation time is 3h.
Fig. 4 F: the relation of investigating substrate DL-cysteine and L-halfcystine transformation efficiency; Substrate indoles consumption is 15g; The amount of coenzyme pyridoxal phosphate is 0.15g; Invert point is 50 ℃; Transforming the pH value is 8; Transformation time is 3h.
With fermentation of bacillus subtilis liquid is the enzyme source, and D-halfcystine and L-tryptophane are produced in enzymatic reaction
According to the splitting condition that embodiment 6 determines, be the enzyme source with recombined bacillus subtilis fermented liquid in the above-mentioned example 4, in 1 liter of fermented liquid, add pyridoxal phosphate 0.15g, indoles 15g, DL-cysteine 20g, 50 ℃ of temperature of reaction, pH8, transformation time 3h carries out the fractionation of DL-cysteine.After the filtration the conversion fluid after the enzymatic reaction, wherein the D-cysteine content is 10g; The L-tryptophane output that produces is 16.4g, and the molar yield of the L-halfcystine in the substrate DL-cysteine is 97.3%, and the molar yield of indoles is 62.7%.
The oxidation of D-Gelucystine with separate
With bubbling air in the conversion fluid of embodiment 7, stir oxidation 20h down, transfer the iso-electric point 5.0 of pH to D-Gelucystine, separate out the crystallization of D-Gelucystine with this understanding, filter collecting precipitation, obtain 9.27g D-Gelucystine crystal approximately, adopt the method identical with embodiment 2 to detect, product D-Gelucystine purity reaches 98.6%.As Fig. 5.
The separation and purification of L-tryptophane
Collect the conversion fluid of iso-electric point post precipitation among the embodiment 8, directly be splined on S-8 type macroporous resin column (40 * 2.6cm), flow velocity 1.5mL/min, with indoles residual in the adsorbent solution, 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 1mL/min.Collect elutriant, and detect the content of L-tryptophane in the elutriant, merge L-tryptophane elution peak, through obtaining L-tryptophane white powder 15.24g after the concentrating under reduced pressure drying treatment, adopt the high performance liquid chromatography of embodiment 3 to detect, product purity reaches 98.9%.As Fig. 6.
Claims (6)
1. a microbial enzyme method splits the method that DL-cysteine prepares D-Gelucystine and L-tryptophane, it is characterized in that this method comprises:
Fermented liquid with the gene engineering recombinant bacterium of expressing tryptophanase is the enzyme source, is substrate with DL-cysteine and indoles, under 40 to 60 ℃, the pH value is 7 to 9, to 6h, produce D-halfcystine and L-tryptophane through enzymatic conversion method reaction 2, further obtain the D-Gelucystine after the oxidation.
2. method according to claim 1, it is characterized in that: the host bacterium of the fermented liquid of described reorganization tryptophan gene engineering bacteria is Bacillus subtillis WB600, expression vector is a secretor type pWB980-tnaA recombinant expression vector, and expressing in the fermented liquid of reorganization tryptophanase somatic cells has highly active tryptophanase.
3. method according to claim 2 is characterized in that: with described recombined bacillus subtilis fermented liquid is the enzyme source, and the add-on of substrate DL-cysteine is 5 to 50g in every liter of fermented liquid; Substrate indoles consumption is 3 to 30g; The amount of coenzyme pyridoxal phosphate is 0.05g to 0.25g.
4. method according to claim 3 is characterized in that: in every liter of fermented liquid, the consumption of substrate DL-cysteine is preferably 20g; Substrate indoles consumption is preferably 15g; The preferable amount of coenzyme pyridoxal phosphate is 0.15g; Preferred invert point is 50 ℃; Preferred conversion pH value is 8; Preferred transformation time is 3h.
5. method according to claim 1, it is characterized in that: the purification process of D-Gelucystine is: the oxidation 5 of Enzymatic transformation liquid bubbling air is to 24h, make the D-halfcystine of generation be oxidized to the D-Gelucystine, re-adjustment pH 5 separates out D-Gelucystine isoelectric point crystallizing, and collecting precipitation gets the D-Gelucystine.
6. method according to claim 1, it is characterized in that: the purification process of L-tryptophane is, supernatant liquor behind claim 5 isoelectric point crystallizing, adopt S-8 type macroporous resin to remove wherein residual indoles, adopt NKA-II type macroporous resin adsorption L-tryptophane again, through 50% ethanol elution, concentrating under reduced pressure drying, obtain the L-tryptophane.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286602A (en) * | 2011-09-16 | 2011-12-21 | 天津启仁医药科技有限公司 | Method for resolving DL-arginine by microbial enzyme method for preparing D-arginine hydrochloride and L-ornithine hydrochloride |
| WO2015104464A1 (en) | 2014-01-07 | 2015-07-16 | Novasep Process | Process for purifying aromatic amino acids |
| 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 |
| CN112538092A (en) * | 2021-02-19 | 2021-03-23 | 江苏欣诺科催化剂有限公司 | Preparation method of chiral Trost ligand |
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