Method for converting L-tryptophan into tryptamine and D-tryptophan
1. A method for converting L-tryptophan to tryptamine and D-tryptophan comprising the steps of:
(1) adding L-tryptophan into a sodium hydroxide solution, stirring and dissolving, adding salicylaldehyde, carrying out racemization reaction in a water bath at 90-95 ℃ until the optical rotation of the solution is +1 to +1.5 degrees, stopping the reaction, cooling to room temperature, and adjusting the pH to 6.0 +/-0.5 to obtain a mother solution containing D-tryptophan and L-tryptophan;
(2) adding tryptophan decarboxylase and coenzyme PLP into the mother liquor, and specifically catalyzing the conversion of L-tryptophan into tryptamine under the conditions of 40-50 ℃ and pH of 6.0 +/-0.5 to obtain reaction liquid containing D-tryptophan and tryptamine;
(3) adjusting the pH value of the reaction liquid to 12.0 to separate out the tryptamine in the reaction liquid to form a precipitate, and separating to obtain the tryptamine;
(4) adjusting pH of the reaction solution after separating out tryptamine to 4.5-5 to separate out D-tryptophan, and separating to obtain D-tryptophan.
2. The method according to claim 1, wherein in the step (1), the concentration of the sodium hydroxide solution is 1-3 mol/L.
3. The method as claimed in claim 2, wherein in the step (1), the ratio of the addition amounts of the L-tryptophan, the sodium hydroxide solution and the salicylaldehyde is (95-105) g: (500- > 520) ml: (5-6) ml.
4. The method according to claim 1, wherein in the step (2), the amount of tryptophan decarboxylase to be added is 2-4% by weight of the mother liquor; the input amount of the coenzyme PLP is 0.02-0.04% of the weight of the mother liquor.
5. The method according to claim 1 or 4, wherein in step (2), the tryptophan decarboxylase is prepared by:
1) connecting a gene TDC which is shown as SEQ ID NO.1 and codes the tryptophan decarboxylase of the rice into a plasmid pEGX-4t-J to obtain a recombinant expression vector; then introducing the obtained recombinant expression vector into escherichia coli to obtain recombinant bacteria;
2) inoculating the recombinant bacteria into a culture medium for fermentation culture, and performing fermentation culture until the OD of the fermentation culture solution is 100 times diluted600The value is 0.18-0.20, and a fermentation culture solution is obtained; adding an inducer into the fermentation culture solution for induction culture to obtain an induction culture; and (3) carrying out high-pressure homogenization treatment on the induction culture, and separating supernatant fluid to obtain the tryptophan decarboxylase.
6. The method according to claim 5, wherein in step 2), the composition of the culture medium is: 12g/L of glycerin, 5g/L of yeast extract, 5g/L of peptone and KH2PO4 2g/L，K2HPO4 15g/L，(NH4)2SO4 1.5g/L，MgSO4 0.5g/L，CaCl2 0.015g/L，ZnCl2 0.06g/L
7. The method according to claim 5, wherein in step 2), the conditions of the fermentation culture are as follows: the pH value is controlled at 7.0, the tank pressure is 0.05MPa, the temperature is 33 ℃, and the ventilation ratio is 1: 1.
8. The method according to claim 5, wherein in the step 2), the conditions of the high-pressure homogenization treatment are as follows: homogenizing pressure 15,000PSI, homogenizing flow 400L/Hr, and controlling temperature at 30 deg.C.
9. The method according to claim 1, wherein in step (3), the separated tryptamine further comprises a purification step, specifically: decolorizing tryptamine with activated carbon, re-dissolving with hydrochloric acid to obtain tryptamine salt, filtering, adding sodium hydroxide to adjust pH to 12, precipitating tryptamine, washing with distilled water for 3 times, and drying.
10. The method according to claim 1, wherein in step (4), the separated D-tryptophan further comprises a purification step, specifically: dissolving D-tryptophan, passing through a ceramic membrane, and recrystallizing.
Background
Tryptophan (TRP) is an essential amino acid that the human body cannot synthesize by itself, and is taken only from the diet, and its main function is to participate in protein synthesis and is also a precursor of various bioactive compounds. Tryptophan has two enantiomers, L-tryptophan and D-tryptophan. Since only L-amino acids constitute proteins, D-amino acids cannot synthesize proteins, so that long-term studies have been favored for L-amino acids. In recent years, with the progress of scientific research and the development of various new drugs, the importance of D-amino acids has been recognized. D-tryptophan has special physiological properties, is applied to the medicine, food, feed industry and agriculture, can be used as a non-nutritive sweetener, a feed additive, a plant growth agent and the like, and is an important synthetic precursor of an anticancer agent and an immunosuppressant in the medicine industry. D-tryptophan is relatively less studied than L-tryptophan, has not been produced on a large scale, and is mainly dependent on import, and therefore, the price of D-tryptophan is expensive.
Tryptamine (Tryptamine) is a monoamine alkaloid that may act as a neuromodulator and neurotransmitter. It is an agonist of hTAAR1, or a non-selective serotonin receptor agonist and hydroxytryptamine-norepinephrine-dopamine releasing agent (SNDRA), and has the following structural formula:
at present, tryptamine products in China mainly depend on imports (Chenning and the like, 2017), and the price is quite expensive.
Therefore, the method for converting L-tryptophan with lower market value into D-tryptophan and tryptamine with high value has wide market application prospect.
D-tryptophan is prepared from L-tryptophan, and most of the existing preparation processes firstly synthesize DL-tryptophan and then prepare the D-tryptophan by different resolution methods. But the problems of complicated process, high cost of used raw materials, difficult obtainment and the like generally exist.
Tryptamine is prepared from L-Tryptophan, and at present, Tryptophan Decarboxylase (TDC), which is Pyridoxal-5' -phosphate (PLP) -dependent decarboxylase, is mainly used and can catalyze the decarboxylation of Tryptophan to generate tryptamine (Roc et al, 2014; L et al, 1997). However, the catalytic activities of tryptophan decarboxylases from different sources are different, and the existing tryptophan decarboxylases produced by prokaryotic expression systems also require column chromatography purification, so that it is difficult to realize large-scale production of tryptophan decarboxylases.
Disclosure of Invention
In view of the above prior art, it is an object of the present invention to provide a method for converting L-tryptophan into tryptamine and D-tryptophan. By adopting the method, low-value L-tryptophan can be converted into high-value tryptamine and D-tryptophan in the same reaction system; the conversion efficiency of the L-tryptophan is high, and the purity of the tryptamine and the D-tryptophan obtained by conversion is high.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for converting L-tryptophan to tryptamine and D-tryptophan comprising the steps of:
(1) adding L-tryptophan into a sodium hydroxide solution, stirring and dissolving, adding salicylaldehyde, carrying out racemization reaction in a water bath at 90-95 ℃ until the optical rotation of the solution is +1 to +1.5 degrees, stopping the reaction, cooling to room temperature, and adjusting the pH to 6.0 +/-0.5 to obtain a mother solution containing D-tryptophan and L-tryptophan;
(2) adding tryptophan decarboxylase and coenzyme PLP into the mother liquor, and specifically catalyzing the conversion of L-tryptophan into tryptamine under the conditions of 40-50 ℃ and pH of 6.0 +/-0.5 to obtain reaction liquid containing D-tryptophan and tryptamine;
(3) adjusting the pH value of the reaction liquid to 12.0 to separate out the tryptamine in the reaction liquid to form a precipitate, and separating to obtain the tryptamine;
(4) adjusting pH of the reaction solution after separating out tryptamine to 4.5-5 to separate out D-tryptophan, and separating to obtain D-tryptophan.
Preferably, in the step (1), the concentration of the sodium hydroxide solution is 1-3 mol/L.
Preferably, in the step (1), the adding ratio of the L-tryptophan, the sodium hydroxide solution and the salicylaldehyde is (95-105) g: (500- > 520) ml: (5-6) ml.
Preferably, in the step (2), the input amount of the tryptophan decarboxylase is 2-4% of the weight of the mother liquor; the input amount of the coenzyme PLP is 0.02-0.04% of the weight of the mother liquor.
Preferably, in the step (2), the tryptophan decarboxylase is prepared by the following method:
1) connecting a gene TDC which is shown as SEQ ID NO.1 and codes the tryptophan decarboxylase of the rice into a plasmid pEGX-4t-J to obtain a recombinant expression vector; then introducing the obtained recombinant expression vector into escherichia coli to obtain recombinant bacteria;
2) inoculating the recombinant bacteria into a culture medium for fermentation culture, and performing fermentation culture until the OD of the fermentation culture solution is 100 times diluted600The value is 0.18-0.20, and a fermentation culture solution is obtained; adding an inducer into the fermentation culture solution for induction culture to obtain an induction culture; and (3) carrying out high-pressure homogenization treatment on the induction culture, and separating supernatant fluid to obtain the tryptophan decarboxylase.
More preferably, in step 1), the plasmid pEGX-4t-J is constructed by the following method:
taking pEGX-4T-1 as a starting plasmid, carrying out enzyme digestion on 3250 th site and 4869 th site of the plasmid pEGX-4T-1 by utilizing pflm I and btg I respectively, and connecting an artificial chain with the length of 100bp after double enzyme digestion; the plasmid pEGX-4t-J has a length of 3449bp, and has a speI cleavage site only at position 3249 and a Bsc 91I cleavage site only at position 3349.
More preferably, in step 2), the composition of the culture medium is: 12g/L of glycerin, 5g/L of yeast extract, 5g/L of peptone and KH2PO4 2g/L，K2HPO4 15g/L，(NH4)2SO4 1.5g/L，MgSO4 0.5g/L，CaCl2 0.015g/L，ZnCl20.06g/L。
More preferably, in step 2), the conditions of the fermentation culture are as follows: controlling the pH value to be 7.0, the tank pressure to be 0.05MPa, the temperature to be 33 ℃, the ventilation ratio to be 1: 1.
more preferably, in step 2), the conditions for inducing culture are: adding inducer IPTG to make the final concentration of IPTG 100mg/L, induction temperature 22 deg.C, and induction culture time 2-6 h.
More preferably, in the step 2), the conditions of the high-pressure homogenization treatment are as follows: homogenizing pressure 15,000PSI, homogenizing flow 400L/Hr, and controlling temperature at 30 deg.C.
Tryptophan decarboxylase prepared by the above method, which is mainly present in the separated supernatant; the content of tryptophan decarboxylase in the supernatant is 0.02 mu mol/L; the enzyme activity is 1.10-1.45U by measuring the generation amount of tryptamine through a liquid phase.
Preferably, in the step (2), the reaction time for specifically catalyzing the conversion of the L-tryptophan into the tryptamine is generally controlled to be 6-10 h; in the industrial production, the content of L-tryptophan in the reaction system is generally monitored to control, and when the content of L-tryptophan in the reaction system is reduced to 0.1 percent, the reaction is continued for 1 hour, and then the reaction can be finished.
Preferably, in the step (3), the separated tryptamine further comprises a purification step, specifically: decolorizing tryptamine with activated carbon, re-dissolving with hydrochloric acid to obtain tryptamine salt, filtering, adding sodium hydroxide to adjust pH to 12, precipitating tryptamine, washing with distilled water for 3 times, and drying.
Preferably, in the step (4), the separated D-tryptophan further comprises a purification step, specifically: dissolving D-tryptophan, passing through a ceramic membrane, and recrystallizing.
The invention has the beneficial effects that:
(1) the invention realizes the conversion of L-tryptophan into D-tryptophan and tryptamine in the same reaction system for the first time, the conversion rate of L-tryptophan is high, and the purity of the converted D-tryptophan and tryptamine is high.
(2) Firstly, racemizing L-tryptophan to obtain mother liquor containing D-tryptophan and L-tryptophan, and controlling the proportion of D-tryptophan/L-tryptophan in the mother liquor by controlling the optical rotation of the solution after racemization; then adding tryptophan decarboxylase to specifically catalyze the L-tryptophan to be converted into tryptamine; when the optical rotation of the solution after racemization is +1 to +1.5 degrees, the efficiency of the subsequent catalytic conversion of L-tryptophan by tryptophan decarboxylase is highest, and the enzymatic conversion rate of the L-tryptophan can be obviously improved.
(3) The tryptophan decarboxylase used for catalyzing L-tryptophan is obtained by adopting an optimized prokaryotic expression system through industrial production, and has low production cost and high enzyme activity.
Drawings
FIG. 1: the expression vector pGEX-4t-J of the invention has a structural schematic diagram.
FIG. 2: the invention discloses a structural schematic diagram of a prokaryotic expression vector of a tryptophan decarboxylase gene.
FIG. 3: codon relative fitness map before optimization.
FIG. 4: and (4) optimizing a codon relative fitness graph.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The terms:
room temperature: the term "room temperature" as used herein means a temperature of 20 ℃ to 30 ℃.
As introduced in the background section, L-tryptophan has a low market value and D-tryptophan and tryptamine have a high market value, and conversion of low-value L-tryptophan to high-value D-tryptophan and tryptamine would have broad market application prospects.
In the prior art, L-tryptophan is subjected to racemization to obtain DL-tryptophan, and then the DL-tryptophan is subjected to resolution, wherein the resolution method mainly comprises enzymatic resolution, chemical resolution, membrane resolution and the like. However, the above resolution methods generally have the problems of complicated process, high cost of used raw materials, difficult obtainment, low conversion rate of L-tryptophan and the like.
The preparation of tryptamine from L-tryptophan is mainly catalyzed by tryptophan decarboxylase. However, it has two main problems: firstly, the existing tryptophan decarboxylase has high production cost and the enzyme activity needs to be further improved; secondly, if L-tryptophan is completely used as a substrate, the conversion rate of L-tryptophan is still low even if the input amount of tryptophan decarboxylase is more and the reaction time is longer. The test shows that: completely using L-tryptophan as a substrate, and reacting for 17 hours under the catalysis of enough tryptophan decarboxylase, wherein the conversion rate of the L-tryptophan is only about 65 percent at most.
Furthermore, the prior art preparation of D-tryptophan from L-tryptophan and the preparation of tryptamine from L-tryptophan are generally two parallel routes, with no intersection between the two. At present, no report is found on the conversion of L-tryptophan into D-tryptophan and tryptamine in the same reaction system.
Based on this, the present invention proposes a method for converting L-tryptophan into tryptamine and D-tryptophan. Firstly, racemizing dissolved L-tryptophan to obtain mother liquor containing D-tryptophan and L-tryptophan; the invention unexpectedly discovers that the conversion rate of L-tryptophan converted into tryptamine through enzyme catalysis is obviously improved in the presence of D-tryptophan. The invention further inspects the influence of the proportion of D-tryptophan and L-tryptophan in the mother liquor on the conversion rate of L-tryptophan, and finds that the optical rotation of racemized solution is +1 degrees to +1.5 degrees, and the conversion rate of L-tryptophan is optimal.
After racemization and tryptophan decarboxylase catalysis treatment, most of L-tryptophan can be converted into D-tryptophan and tryptamine, and the generated tryptamine and D-tryptophan are respectively separated by adopting a mode of adjusting pH. Through calculation, the method of the invention has the advantages that the enzyme catalysis reaction lasts for 6-10h, the conversion rate of the L-tryptophan can reach 93.9-98.9%, and compared with the prior art, the conversion rate of the L-tryptophan is obviously improved.
Conversion (%) of L-tryptophan ═ M1－M2)/M1×100％；
In the formula, M1The input amount of the L-tryptophan is the input amount; m2Is the amount of L-tryptophan remaining after the reaction; m1And M2The weight units of (a) and (b) are consistent.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier. Wherein:
the tryptophan decarboxylase used in the present invention can be purchased from a commercial source; can also be prepared by itself.
The L-tryptophan used in the invention is white to yellowish crystals or crystalline powder, has an optical rotation of-29.4 DEG to-32.8 DEG, a content of 98.5% to 101.5%, and a pH of 5.5 to 7.0.
Example 1: preparation of rice tryptophan decarboxylase:
(1) taking plasmid pGEX-4T-1 as a starting plasmid, firstly performing single enzyme digestion at a 3250 site by adopting pflm I, cutting 2 ends at the 3 'end, cutting 6 ends at the 5' end, adding A at the 3 'end and adding CTAGT at the 5' end; protecting again; performing single enzyme digestion at 4869 with btg I, cutting 4 pieces at 5' end, adding AAGA, and removing all protection; the artificial strand of 100bp in length, which is the 5 '-CTAGT … … G-3', is added, and the sequence of the artificial strand is omitted, so long as it does not contain both SpeI and Bsc 91I cleavage sites, and the omitted sequence may be arbitrarily selected.
The plasmid pGEX-4t-J is subjected to double enzyme digestion and identified by Spe I and Bsc 91I, and 3349 and 100 of pEGX-4t-J appear after double enzyme digestion, so that the construction of the expression vector pGEX-4t-J is proved to be successful (figure 1).
(2) Performing double enzyme digestion on plasmid pGEX-4t-J by using Spe I and Bsc 91I, and integrating the optimized TDC gene shown by SEQ ID NO.1 to the expression vector pGEX-4t-J subjected to double enzyme digestion by using DNA ligase to obtain a recombinant expression vector pEGX-TDC (figure 2); and then the obtained recombinant expression vector is introduced into escherichia coli B21(DE3) to obtain a recombinant bacterium.
(3) Inoculating the recombinant bacteria into a culture medium for fermentation culture, wherein the conditions of the fermentation culture are as follows: controlling the pH value to be 7.0, the tank pressure to be 0.05MPa, the temperature to be 33 ℃, the ventilation ratio to be 1:1, OD after fermentation culture to 100-fold dilution of fermentation culture solution600The value is 0.18-0.20, and a fermentation culture solution is obtained; (aeration ratio: sterile air volume per unit volume of culture solution in one minute; e.g., 18m in a container3The fermentation tank of the culture solution was purged with 18L of sterile air per minute, and the aeration ratio was 1: 1. )
Cooling the fermentation culture solution to 22 ℃, adding an inducer IPTG into the fermentation culture solution to ensure that the final concentration of the IPTG is 100mg/L, and carrying out induction culture for 2h to obtain an induction culture; homogenizing the induced culture under high pressure, separating and collecting supernatant to obtain rice tryptophan decarboxylase (which is water-soluble and mainly exists in the supernatant);
the culture medium comprises the following components: 12g/L of glycerin, 5g/L of yeast extract, 5g/L of peptone and KH2PO4 2g/L，K2HPO415g/L，(NH4)2SO4 1.5g/L，MgSO4 0.5g/L，CaCl2 0.015g/L，ZnCl2 0.06g/L。
The conditions of the high-pressure homogenization treatment were as follows:
(1) homogenizing pressure: pressure 15,000 PSI;
(2) homogenizing flow rate: 400L/Hr;
(3) sanitation level: the materials of the parts contacting the materials are 316L stainless steel, 17-4PH stainless steel, tungsten carbide, ultra-high molecular polyethylene, PEEK and the like approved by FDA & GMP, and support CIP;
(4) temperature control: 30 ℃;
(5) hydraulic power transmission;
(6) the feeding mode is as follows: the system is directly connected with the pipeline.
The rice tryptophan decarboxylase is an application form of supernatant separated after high-pressure homogenization, and the content of the rice tryptophan decarboxylase in the supernatant is determined to be 0.02 mu mol/L by using a plant Tryptophan Decarboxylase (TDC) detection kit (purchased from Shanghai Zhen Ke Biotech Co., Ltd.); the enzyme activity is 1.10-1.45U by measuring the generation amount of tryptamine through a liquid phase.
The method for preparing tryptophan decarboxylase has the following advantages:
(1) the invention clones coding gene of tryptophan decarboxylase from rice leaves with deficient nutrition. In order to make the coding gene more suitable for a prokaryotic expression system, the invention carries out codon optimization on the original coding gene (shown in SEQ ID NO. 2), and the optimized coding gene of tryptophan decarboxylase is shown in SEQ ID NO. 1; the codon relative fitness of the optimized tryptophan decarboxylase encoding gene is obviously improved (see figure 3 and figure 4); the optimized tryptophan decarboxylase encoding gene is adopted for prokaryotic expression, and the expression level of the target methionine decarboxylase is obviously improved.
(2) The invention takes pGEX-4T-1 as a starting plasmid and pGEX-4T-1 as an inducible plasmid, and can express the target gene only after the induction by adding an inducer, thereby controlling the expression time of the target gene. The invention further modifies pGEX-4T-1, and plasmid pEGX-4T-1 is connected with a segment of 100bp base after double enzyme digestion, thus the modification treatment has the advantages of reducing the size of the plasmid, and the original plasmid is 4969bp and only 3449bp after modification. Furthermore, two enzyme cutting sites of SpeI and Bsc 91I are respectively arranged at 3249 and 3349, so that the insertion of the target gene is convenient.
(3) The present invention optimizes the fermentation culture medium composition, and the optimized culture medium is suitable for the growth and propagation of recombinant bacteria.
(4) In the prior art, the tryptophan decarboxylase expressed by pronucleus generally needs to be purified by adopting column chromatography, the purification process is complex, and the industrial production is difficult to realize. The method separates and takes the supernatant after the induced culture is subjected to high-pressure homogenization treatment, and the supernatant can be directly used as tryptophan decarboxylase without the process of column chromatography purification.
Example 2: method for converting L-tryptophan into tryptamine and D-tryptophan
(1) Adding L-tryptophan into 2mol/L sodium hydroxide solution, stirring to dissolve, and adding salicylaldehyde, wherein the adding amount ratio of the L-tryptophan to the sodium hydroxide solution to the salicylaldehyde is 100 g: 510 ml: 5.5 ml; carrying out racemization reaction in water bath at 90-95 ℃ until the optical rotation of the solution is +1 to +1.5 degrees, stopping the reaction, cooling to room temperature, and adjusting the pH value to 6.0 +/-0.5 to obtain mother liquor containing D-tryptophan and L-tryptophan;
(2) adding tryptophan decarboxylase and coenzyme PLP into the mother liquor, wherein the adding amount of the tryptophan decarboxylase is 3 percent of the weight of the mother liquor, and the tryptophan decarboxylase is added in the form of the supernatant separated in the example 1; the input amount of the coenzyme PLP is 0.03 percent of the weight of the mother liquor; specifically catalyzing L-tryptophan to be converted into tryptamine under the conditions of 45 ℃ and pH6.0 +/-0.5, wherein the reaction time is 8 hours, and obtaining reaction liquid containing D-tryptophan and tryptamine;
(3) adjusting the pH value of the reaction liquid to 12.0 to separate out the tryptamine in the reaction liquid to form a precipitate, and separating to obtain the tryptamine; decolorizing tryptamine with activated carbon, adding hydrochloric acid for redissolving to generate tryptamine salt, temporarily storing, filtering, adding sodium hydroxide to adjust pH to 12, precipitating tryptamine again, washing with distilled water for 3 times, and drying to obtain tryptamine product.
(4) And adding hydrochloric acid into the reaction liquid after separating out the tryptamine, adjusting the pH value to 4.5-5.0 to separate out the D-tryptophan, separating to obtain the D-tryptophan, separating out the D-tryptophan, passing through a ceramic membrane after dissolving, and recrystallizing to obtain the D-tryptophan product.
Example 3: method for converting L-tryptophan into tryptamine and D-tryptophan
(1) Adding L-tryptophan into 1mol/L sodium hydroxide solution, stirring to dissolve, and adding salicylaldehyde, wherein the adding amount ratio of L-tryptophan to sodium hydroxide solution to salicylaldehyde is 95 g: 520 ml: 5ml of the solution; carrying out racemization reaction in water bath at 90-95 ℃ until the optical rotation of the solution is +1 to +1.5 degrees, stopping the reaction, cooling to room temperature, and adjusting the pH value to 6.0 +/-0.5 to obtain mother liquor containing D-tryptophan and L-tryptophan;
(2) adding tryptophan decarboxylase and coenzyme PLP into the mother liquor, wherein the adding amount of the tryptophan decarboxylase is 4 percent of the weight of the mother liquor; the input amount of the coenzyme PLP is 0.04 percent of the weight of the mother liquor; specifically catalyzing L-tryptophan to be converted into tryptamine under the conditions of 50 ℃ and pH6.0 +/-0.5, wherein the reaction time is 10 hours, and obtaining a reaction solution containing D-tryptophan and tryptamine;
(3) adjusting the pH value of the reaction liquid to 12.0 to separate out the tryptamine in the reaction liquid to form a precipitate, and separating to obtain the tryptamine; decolorizing tryptamine with activated carbon, adding hydrochloric acid for redissolving to generate tryptamine salt, temporarily storing, filtering, adding sodium hydroxide to adjust pH to 12, precipitating tryptamine again, washing with distilled water for 3 times, and drying to obtain tryptamine product.
(4) And adding hydrochloric acid into the reaction liquid after separating out the tryptamine, adjusting the pH value to 4.5-5.0 to separate out the D-tryptophan, separating to obtain the D-tryptophan, separating out the D-tryptophan, passing through a ceramic membrane after dissolving, and recrystallizing to obtain the D-tryptophan product.
Example 4: method for converting L-tryptophan into tryptamine and D-tryptophan
(1) Adding L-tryptophan into 3mol/L sodium hydroxide solution, stirring to dissolve, and adding salicylaldehyde, wherein the adding amount ratio of the L-tryptophan to the sodium hydroxide solution to the salicylaldehyde is 105 g: 500 ml: 6 ml; carrying out racemization reaction in water bath at 90-95 ℃ until the optical rotation of the solution is +1 to +1.5 degrees, stopping the reaction, cooling to room temperature, and adjusting the pH value to 6.0 +/-0.5 to obtain mother liquor containing D-tryptophan and L-tryptophan;
(2) adding tryptophan decarboxylase and coenzyme PLP into the mother liquor, wherein the adding amount of the tryptophan decarboxylase is 2 percent of the weight of the mother liquor; the input amount of the coenzyme PLP is 0.02 percent of the weight of the mother liquor; specifically catalyzing L-tryptophan to be converted into tryptamine under the conditions of 40 ℃ and pH6.0 +/-0.5, wherein the reaction time is 6 hours, and obtaining a reaction solution containing D-tryptophan and tryptamine;
(3) adjusting the pH value of the reaction liquid to 12.0 to separate out the tryptamine in the reaction liquid to form a precipitate, and separating to obtain the tryptamine; decolorizing tryptamine with activated carbon, adding hydrochloric acid for redissolving to generate tryptamine salt, temporarily storing, filtering, adding sodium hydroxide to adjust pH to 12, precipitating tryptamine again, washing with distilled water for 3 times, and drying to obtain tryptamine product.
(4) And adding hydrochloric acid into the reaction liquid after separating out the tryptamine, adjusting the pH value to 4.5-5.0 to separate out the D-tryptophan, separating to obtain the D-tryptophan, separating out the D-tryptophan, passing through a ceramic membrane after dissolving, and recrystallizing to obtain the D-tryptophan product.
The quality tests were carried out on the tryptamine products and D-tryptophan products prepared in examples 2 to 4, wherein the quality standards of tryptamine are shown in Table 1 and the quality standard of D-tryptophan is shown in Table 2.
Table 1: tryptamine quality standard
Detecting items
Standard of merit
Traits
Yellowish or off-white crystalline powder
Content (wt.)
≥98.0％
Loss on drying
≤0.50％
Ash content
≤0.50％
Heavy metals
≤20ppm
Arsenic (As)
Less than or equal to 1.5ppm (external inspection)
Table 2: quality standard of D-tryptophan
Item
Index (I)
Content (wt.)
98.5％～101.5％
Specific rotation degree
+30.0°～+33.0°
pH
5.0～7.0
Light transmittance
Internal control
Loss on drying
≤0.3％
Residue on ignition
≤0.1％
Chloride compound
≤0.02％
Sulfates of sulfuric acid
≤0.02％
Iron salts
≤20ppm
Heavy metals
≤10ppm
The tryptamine product was tested as follows:
1. sense organ
A proper amount of sample is taken and placed on white paper, and the white paper is spread by a glass rod and visually inspected and smelled by nose.
2. The content is as follows: not less than 98.0 percent
2.1 instruments
2.1.1 high performance liquid chromatograph Waters e-2695
2.1.2 ultraviolet Detector UV2489
2.1.3 ultrasonic cleaning machine
2.1.4 suction filtration device: 1L of
2.2 reagents
2.2.1 mobile phase A: accurately weighing 4.1g of potassium dihydrogen phosphate, dissolving in about 500mL of pure water, adding 2.8mL of triethylamine, diluting to 1L with pure water, mixing, filtering with 0.22um filter membrane, and ultrasonic degassing in an ultrasonic machine for 30 min.
2.2.2 mobile phase B: chromatographically pure methanol.
2.2.3 sample solution: accurately weighing 0.01g (accurate to 0.0002g) of the sample into a 100mL volumetric flask, adding a mobile phase (A: B ═ 60:40) to dissolve and dilute to the mark, and shaking up.
2.3 chromatographic conditions
A chromatographic column: YMC-Triart C18
Mobile phase: a, B and 60:40
Flow rate: 1.0mL/min
Column temperature: room temperature or 30 deg.C
Detection wavelength: 225nm
And (4) calculating a result: external standard method
3. Loss on drying: less than or equal to 0.5 percent
3.1 instruments
3.1.1 analytical balance: sensory quantity 0.1mg
3.1.2 constant temperature drying oven: the temperature is accurate to +/-0.1 DEG C
3.1.3 weighing bottle: 70 x 35mm
3.2 operation
Drying the flat weighing bottle in a 105 ℃ oven to constant weight, taking out, transferring into a drier, cooling to room temperature, precisely weighing m1. About 1g of the sample was put into the weighing flask, and m was precisely weighed2Then drying in a 105 ℃ oven for 3 hours, taking out, transferring into a drier, cooling to room temperature, precisely weighing m3。
3.3 calculation of results
In the formula:
x-loss of drying of sample,% ]%
m1-weighing the mass of the bottle, g
m2-weighing the mass of the bottle and the sample, g
m3-weighing the bottle and the sample after drying, g.
3.4 precision
The relative average deviation of the test results of the same sample cannot exceed 0.3 percent.
4, ash content: less than or equal to 0.5 percent
4.1 reagent: sulfuric acid
4.2 operation
The crucible is constant in weight at the temperature of 600 +/-50 ℃, taken out and placed in a dryer for cooling and accurate weighing m1。
Weighing 1g-2g of sample into a crucible with constant weight, and precisely weighing m21mL of sulfuric acid was added to wet the sample and heated at as low a temperature as possible until the sample was completely charred. Cooling, adding 1mL sulfuric acid to wet the residue, and slowly addingThe heat is not released until white smoke is released. Then, the mixture is heated to 600 +/-50 ℃ until the residue is completely incinerated (1-2 h). Taking out, cooling in a drier, and accurately weighing m3The percentage of residue to sample was calculated.
Unless otherwise specified, if the content of residue exceeds the specified limits, the wetting with sulfuric acid, heating and burning at 600 ℃. + -. 50 ℃ for 30 minutes are repeated until the residual weight of the residue of two successive samples does not exceed 0.5mg, or until the percentage of residue meets the specified limits.
4.3 calculation of results
In the formula:
m1mass of the crucible, g
m2Mass of crucible and sample, g
m3Mass of crucible and residue, g.
4.4 notes
The crucible is ignited to constant weight, namely the crucible is ignited at 600 +/-50 ℃, then is cooled down and precisely weighed, and the weight difference of two times is below 0.3 mg.
When the weight is constant, the number of the crucibles placed in each dryer is preferably not more than 4, the weighing sequence is consistent each time, the cooling time in the dryer is also consistent, the crucibles are quickly and precisely weighed after being taken out from the dryer, and the weight difference between the two times is below 0.3mg, so the crucibles can be regarded as the constant weight.
The purity of the sulfuric acid used for the examination should be taken into account and, if necessary, a blank test may be carried out.
5 heavy metals: less than or equal to 20ppm
5.1 reagents
5.1.1 lead nitrate stock solution: 159.8mg of lead nitrate was dissolved in water to which 1mL of nitric acid had been added, and then diluted with water to 1000 mL.
Standard lead solution: the solution is used on the same day, 10mL of lead nitrate stock solution is sucked, and water is added to the solution to reach 100 mL. The standard lead solution contained 10. mu.g of lead per ml.
Acetate buffer (ph 3.5): 25.0g of ammonium acetate was weighed out accurately, dissolved in 25mL of water, and 38.0mL of 6mol/L hydrochloric acid solution was added. If necessary, the pH of the acetate solution is adjusted to 3.5 with 6mol/L ammonia or 6mol/L hydrochloric acid, diluted to 100ml with water, and mixed.
Thioacetamide test solution: 0.2mL of a thioacetamide solution (4g of thioacetamide dissolved in 100mL of water) was mixed with 1mL of a glycerol base solution (water was added to 200g of glycerol to a total weight of 235g, 140mL of 1mol/L sodium hydroxide and 50mL of water) and the solution was placed in a boiling water bath for 20s and used immediately.
5.2 operation
Sample tube: weighing 1.0g of sample, dissolving in 25mL of water, using a pH meter or a small range pH indicator paper as an external indicator, adjusting the pH value of the solution to 3.0-4.0 by using 1mol/L acetic acid or 6mol/L ammonia water, diluting to 40mL by using water, and mixing.
Standard tubes: sucking 2mL of a standard lead solution into a 50mL colorimetric tube, using a pH meter or a small-range pH indicator paper as an external indicator, adjusting the pH value of the solution to between 3.0 and 4.0 by using 1mol/L acetic acid or 6mol/L ammonia water, diluting the solution to 40mL by using water, and mixing.
Monitoring the tube: 1.0g of the sample was weighed, dissolved in 25mL of water, and 2mL of the standard lead solution was added. Using pH meter or small range pH indicator paper as external indicator, adjusting pH value of the solution to 3.0-4.0 with 1mol/L acetic acid or 6mol/L ammonia water, diluting to 40mL with water, and mixing.
Adding 2mL of acetate buffer solution with the pH value of 3.5 and 1.2mL of thioacetamide solution into the three tubes respectively, adding water to a constant volume of 50mL, uniformly mixing, and standing for 2 minutes. The solution color is seen from top to bottom with white as the bottom, and the sample tube must not be darker than the standard tube sample color. The tube sample color should be monitored to be equal to or darker than the color of the standard tube, otherwise USP method 2 should be used.
6, arsenic: less than or equal to 1.5ppm
And (5) outsourcing detection.
The method for testing the D-tryptophan product is as follows:
1 sense organ
A proper amount of sample is taken and placed on white paper, and the white paper is spread by a glass rod and visually inspected and smelled by nose.
2, content: 98.5 to 101.5 percent
2.1 reagent preparation:
2.1.10.1 mol/L perchloric acid standard solution preparation: 8.5mL of perchloric acid was weighed out, and the solution was poured into 500mL of glacial acetic acid with stirring, and 24mL of acetic anhydride was added thereto, and the volume was adjusted to 1000mL with glacial acetic acid.
2.1.2 calibration: 160mg (called 0.0002g) of potassium hydrogen phthalate dried to constant weight is accurately weighed and placed in a triangular flask, 20mL of glacial acetic acid is added to dissolve the potassium hydrogen phthalate, 1 drop of methyl violet indicator is added, the prepared perchloric acid solution is used for titration until blue is used as an end point, and the average value is obtained by calibration twice.
C1＝m3/0.2042*V2
In the formula:
C1-calibrated HClO4Concentration, mol/L
m3The mass of potassium hydrogen phthalate, g
V2The amount of perchloric acid standard solution, mL
0.2042——KHC8H4O4Millimolar mass of
2.1.30.5% alpha-naphthol benzyl alcohol indicator: 0.5g of alpha-naphthol benzyl alcohol was weighed and dissolved in glacial acetic acid to a constant volume of 100 mL.
2.1.4 methyl violet indicators: 0.5g of methyl violet is weighed and dissolved with glacial acetic acid to a volume of 100 mL.
2.2, operation:
weighing 150mg of a sample dried to constant weight, accurately weighing 0.0002g, adding 3mL of formic acid, dissolving in 50mL of glacial acetic acid, adding 2-3 drops of an alpha-naphthol benzyl alcohol indicator, titrating with 0.1mol/L perchloric acid standard solution until the alpha-naphthol benzyl alcohol indicator is green, and simultaneously carrying out a blank test.
2.3 calculation method:
in the formula:
x-content, is%
C-concentration of perchloric acid Standard solution, mol/L
Titration of the blank consumes mL of perchloric acid standard solution
Titration of the sample consumes mL of perchloric acid standard solution
m-sample weight, g
2.4 notes: if the temperature difference between the titration sample and the perchloric acid calibration exceeds 10 ℃, recalibration is needed, and if the temperature difference does not exceed 10 ℃, the perchloric acid solution concentration can be corrected according to the following formula:
in the formula:
0.0011-coefficient of expansion of glacial acetic acid
C1Calibration of the concentration of perchloric acid
3 specific rotation degree: +30.0 to +33.0 degree
3.1 instruments
3.1.1 polarimeter: the accuracy is 0.01 deg.
3.1.2 analytical balance: the sensory dose is 0.1 mg.
3.1.3 volumetric flask: 50mL
3.1.4 weighing bottle: 70 x 35mm
3.2 reagents
Pure water
3.3 operation
A sample (dried product dried at 105 ℃ for 3 hours) is precisely weighed, dissolved and diluted by adding pure water to prepare a solution (1g → 100mL) containing about 10mg per 1mL, the optical rotation is measured by using a digital automatic polarimeter (pure water is used as a blank correction zero point at first), a 2dm optical rotation tube is firstly washed 3 times by using a test solution, then the test solution is slowly injected (no air bubble exists in the tube), the optical rotation is measured in the polarimeter, the average value of three optical rotation readings is taken, and the temperature of the test solution is measured.
3.4 results calculation
In the formula:
α -optical rotation reading, with right hand as "+" and left hand as "-";
l-optical rotation tube length (dm);
c-concentration of test solution (g/100mL solution);
kt-temperature coefficient (-0.090);
t-temperature of the solution at the time of measurement.
3.5 notes
3.5.1 before each measurement, the solvent is used for blank correction, and after the measurement, the correction is carried out for 1 time again to determine whether the zero point is changed or not during the measurement; if the zero point is found to be changed in the 2 nd calibration, the optical rotation should be measured again.
3.5.2 when preparing the solution and measuring, the temperature should be adjusted to 20 ℃. + -. 0.5 ℃ (or the temperature specified in each variety).
3.5.3 the solution of the liquid or solid substance to be tested should be sufficiently soluble and the test solution should be clear.
3.5.4, the specific rotation of the substance is related to factors such as the measurement light source, the measurement wavelength, the solvent, the concentration and the temperature. Therefore, the measurement conditions should be noted when the specific rotation of a substance is expressed.
3.5.5 sample and blank tubes should be placed in the polarimeter at the same position.
3.5.6 the glass sheet on the measuring tube should be kept bright and clean, and the rubber ring on the measuring tube should be replaced in time when aging occurs.
3.5.7 no bubble should be arranged at the position of the optical rotation tube glass slide, and the bubble is driven to the position of the calabash tube.
4pH：5.0～7.0
4.1 instruments
4.1.1pH meter: to the accuracy of 0.01
4.1.2 graduated cylinder: 50mL, class A
4.2 reagents and solutions: pure water
4.3 analytical procedure
4.3.1 reagent preparation
The pH value of the potassium hydrogen phthalate is 4.00, and the pH value of the mixed phosphate is 6.86.
4.3.2pH Meter calibration
The electrode was inserted into the first buffer and the "calibrate" key was pressed and the first point calibration was completed after the reading stabilized. After the electrode is cleaned, a second buffer is inserted and the "calibrate" key is pressed, and after the reading is stabilized, the "read" key is pressed to complete the second point calibration. And (5) measuring the sample after the calibration is finished.
4.3.3 operations
Taking 0.5g sample, adding 50mL of fresh cold water to dissolve, and measuring with calibrated pH meter.
5 light transmittance
5.1 instruments
5.1.1752 model ultraviolet-visible spectrophotometer
5.1.2 volumetric flask: 50mL
5.2 reagents
2mol/L hydrochloric acid solution: 180mL of hydrochloric acid was measured and diluted with water to 1000 mL.
5.3 operation
0.5g of the sample is weighed, dissolved in 20mL of hydrochloric acid solution and recorded on a spectrophotometer model 752 using a 1cm cuvette at a wavelength of 430nm with reference to the hydrochloric acid solution.
6 weight loss on drying: less than or equal to 0.3 percent
6.1 instruments
6.1.1 analytical balance: sensory quantity 0.1mg
6.1.2 constant temperature drying oven: the temperature is accurate to +/-0.1 DEG C
6.1.3 weighing bottle: 70 x 35mm
6.2 operation
Drying the flat weighing bottle in a 105 ℃ oven to constant weight, taking out, transferring into a drier, cooling to room temperature, precisely weighing m1. About 1g of the sample was put into the weighing flask, and m was precisely weighed2Then drying in a 105 ℃ oven for 3 hours, taking out and transferring into a drier to be cooled to a roomWarm, precision weighing m3。
6.3 calculation of results
In the formula:
x-loss of drying of sample,% ]%
m1-weighing the mass of the bottle, g
m2-weighing the mass of the bottle and the sample, g
m3-weighing the bottle and the sample after drying, g.
6.4 precision
The relative average deviation of the test results of the same sample cannot exceed 0.3 percent.
7 residue on ignition: less than or equal to 0.1 percent
7.1 reagent: sulfuric acid
7.2 operation
The crucible is constant in weight at the temperature of 600 +/-50 ℃, taken out and placed in a dryer for cooling and accurate weighing m1。
Weighing 1g-2g of sample into a crucible with constant weight, and precisely weighing m21mL of sulfuric acid was added to wet the sample and heated at as low a temperature as possible until the sample was completely charred. After cooling, 1mL of sulfuric acid was added to wet the residue and the mixture was heated slowly until no white smoke emerged. Then, the mixture is heated to 600 +/-50 ℃ until the residue is completely incinerated (1-2 h). Taking out, cooling in a drier, and accurately weighing m3The percentage of residue to sample was calculated.
Unless otherwise specified, if the content of residue exceeds the specified limits, the wetting with sulfuric acid, heating and burning at 600 ℃. + -. 50 ℃ for 30 minutes are repeated until the residual weight of the residue of two successive samples does not exceed 0.5mg, or until the percentage of residue meets the specified limits.
7.3 calculation of results
In the formula:
m1mass of the crucible, g
m2Mass of crucible and sample, g
m3Mass of crucible and residue, g.
7.4 notes
The crucible is ignited to constant weight, namely the crucible is ignited at 600 +/-50 ℃, then is cooled down and precisely weighed, and the weight difference of two times is below 0.3 mg.
When the weight is constant, the number of the crucibles placed in each dryer is preferably not more than 4, the weighing sequence is consistent each time, the cooling time in the dryer is also consistent, the crucibles are quickly and precisely weighed after being taken out from the dryer, and the weight difference between the two times is below 0.3mg, so the crucibles can be regarded as the constant weight.
The purity of the sulfuric acid used for the examination should be taken into account and, if necessary, a blank test may be carried out.
8, chloride: less than or equal to 0.02 percent
8.1 reagents
8.1.1 Dilute nitric acid: 105mL of nitric acid was measured and diluted with water to 1000 mL.
8.1.20.1 mol/L silver nitrate: 17.5g of silver nitrate, dissolved in water and diluted to 1000 mL.
8.1.30.02 mol/L hydrochloric acid solution, which needs calibration.
8.2 operation
Taking 1.78g of sample, placing the sample in a 50mL Nashi colorimetric tube, adding water to dissolve the sample to 25mL (neutralizing the solution by nitric acid if necessary), adding 10mL of dilute nitric acid, adding water to make the volume of the sample be about 40mL, and shaking up to obtain a sample tube;
precisely sucking 0.02mol/L hydrochloric acid solution 0.5mL, placing in another Nashi colorimetric tube, adding dilute nitric acid 10mL, adding water to make about 40mL, and shaking up to obtain a standard tube;
adding 1.0mL of 0.1mol/L silver nitrate into the sample tube and the standard tube respectively, adding water to make the volume of the sample tube and the standard tube to be 50mL, shaking uniformly, standing in a dark place for 5 minutes until the mixture is on a black background, and vertically observing from top to bottom, wherein the turbidity displayed by the sample tube is not thicker than that of the standard tube.
8.3 notes
A too low temperature, preferably 30 ℃ to 40 ℃, affects the turbidity generation. Direct light causes decomposition of silver chloride, so it is necessary to keep it in dark.
9 sulfate salt: less than or equal to 0.02 percent
9.1 reagents
9.1.10.01 mol/L sulfuric acid solution, which needs calibration.
9.1.23 mol/L hydrochloric acid solution: 270mL of hydrochloric acid was measured and diluted with water to 1000 mL.
9.1.312% barium chloride: 120g of barium chloride is accurately weighed and dissolved in water, and water is added to the solution until the volume is 1000 mL.
9.2 operation
Taking 0.48g of sample, placing the sample in a 50mL Nashi colorimetric tube, adding water to dissolve the sample to 40mL, adding 2mL of hydrochloric acid solution, and shaking up to obtain a sample tube;
precisely sucking 0.1mL of 0.01mol/L sulfuric acid solution, placing the solution in another 50mL of nano colorimetric tube, adding water to dissolve the solution to 40mL, adding 2mL of hydrochloric acid solution, and shaking up to obtain a standard tube;
respectively adding 5mL of 12% barium chloride into the sample tube and the standard tube, adding water to 50mL, shaking uniformly, placing for 10 minutes, placing under a black background, vertically observing from top to bottom, and comparing the turbidity of the sample tube with that of the standard tube, so that the sample tube cannot be thicker.
9.3 notes
The 12% barium chloride solution is prepared before use, and is fully shaken immediately after the barium chloride is added to prevent the turbid concentration from being influenced by local over-concentration.
The turbidity is affected by too low a temperature, preferably 25 ℃ to 35 ℃.
10 iron salt: less than or equal to 20ppm
10.1 reagents
10.1.1 standard iron stock solution: 863.4mg of ferric ammonium sulfate [ FeNH4(SO4) 2.12H 2O ] is accurately weighed, dissolved in a small amount of water, 10mL of 2N (1mol/L) sulfuric acid solution is added, and the volume is adjusted to 100mL by adding water.
10.1.2 standard iron solution: sucking 10mL to 1000mL volumetric flask of the stock solution, adding 10mL of 2N (1mol/L) sulfuric acid solution, adding water to a constant volume of 1000mL, and uniformly mixing for later use. (10. mu.g of iron per mL of this standard iron solution)
10.1.3 Dilute hydrochloric acid: 234mL of hydrochloric acid was taken and diluted to 1000mL of water.
10.1.430% ammonium thiocyanate solution: 30g of ammonium thiocyanate is dissolved in water to be 100 mL.
10.1.5 ammonium persulfate
10.2 operation
1.0g of sample is taken, 2mL of hydrochloric acid is added into residues after burning and ashing, the residues are placed on a water bath and evaporated to dryness, 4mL of dilute hydrochloric acid is added, 30mL of water is added after slight heat dissolution, and the mixture is transferred into a 50mL nano colorimetric tube which is a sample tube;
precisely sucking 2.0mL of standard iron solution, placing the standard iron solution in a 50mL Nashi colorimetric tube, adding 4mL of dilute hydrochloric acid, and adding water to obtain 35mL of the standard iron solution, wherein the standard iron solution is a standard tube;
0.05g of ammonium persulfate and 3mL of 30% ammonium thiocyanate solution are respectively added into the two tubes, a proper amount of water is added to dilute the mixture into 50mL, the mixture is shaken uniformly, a white background is placed to vertically observe the mixture from top to bottom, and the color of the sample tube is not darker than that of a standard tube.
10.3 notes
Both light and temperature affect the stability of the iron thiocyanate, so immediate colorimetry is required.
11 heavy metals: less than or equal to 10ppm
11.1 reagents
11.1.1 lead nitrate stock solution: 159.8mg of lead nitrate was dissolved in water to which 1mL of nitric acid had been added, and then diluted with water to 1000 mL.
11.1.2 standard lead solution: the solution is used on the same day, 10mL of lead nitrate stock solution is sucked, and water is added to the solution to reach 100 mL. The standard lead solution contained 10. mu.g of lead per ml.
11.1.3 acetate buffer (ph 3.5): 25.0g of ammonium acetate was weighed out accurately, dissolved in 25mL of water, and 38.0mL of 6mol/L hydrochloric acid solution was added. If necessary, the pH of the acetate solution is adjusted to 3.5 with 6mol/L ammonia or 6mol/L hydrochloric acid, diluted to 100ml with water, and mixed.
11.1.4 Thioacetamide test solution: 0.2mL of a thioacetamide solution (4g of thioacetamide dissolved in 100mL of water) was mixed with 1mL of a glycerol base solution (water was added to 200g of glycerol to a total weight of 235g, 140mL of 1mol/L sodium hydroxide and 50mL of water) and the solution was placed in a boiling water bath for 20s and used immediately.
11.1.5 phenolphthalein indicator liquid: 1g of phenolphthalein is taken and dissolved in 100mL of ethanol.
11.1.6 Ammonia test solution: 400mL of concentrated ammonia solution is taken and diluted to 1000mL by adding water.
11.2 operation
Sample tube: taking residue left under the residue on ignition, adding 0.5mL of nitric acid, evaporating to dryness until nitrogen oxide is removed, cooling, adding 2mL of hydrochloric acid, placing on a water bath, evaporating to dryness, adding 15mL of water, dropwise adding an ammonia test solution until phenolphthalein indicator solution is neutral, adding 2mL of acetate buffer solution, slightly heating for dissolution, transferring to a Nashi colorimetric tube, and adding water for dilution to 25 mL.
Standard tubes: taking a reagent for preparing a sample tube solution, putting the reagent into a porcelain dish, adding 2mL of acetate buffer solution after evaporation to dryness, adding 15mL of water, transferring into a nano colorimetric tube, adding 1mL of standard lead solution, and adding water to dilute to 25 mL.
2.0mL of thioacetamide solution was added to each of the two tubes, mixed well, and allowed to stand for 2 minutes. The solution color is seen from top to bottom with white as the bottom, and the sample tube must not be darker than the standard tube sample color. The monitoring tube sample color should be equal to or darker than the color of the standard tube.
Through detection, the tryptamine products and the D-tryptophan products prepared in the embodiments 2 to 4 of the invention both meet the standard.
Comparative example 1:
the optical rotation of the solution after racemization in the step (1) of example 2 was adjusted to-2 to-3, and the other conditions were the same as in example 2.
Comparative example 2:
the optical rotation of the solution after racemization in the step (1) of example 2 was adjusted to +4 to +5, and the other conditions were the same as in example 2.
Comparative example 3:
the catalytic reaction temperature in step (2) of example 2 was adjusted to 35 ℃ and the pH was adjusted to 7.5, and the other conditions were the same as in example 2.
Comparative example 4:
the catalytic reaction temperature in step (2) of example 2 was adjusted to 55 ℃ and the pH was adjusted to 5.5, and the other conditions were the same as in example 2.
The conversion rates of L-tryptophan in examples 2 to 4 and comparative examples 1 to 4 were examined, and the conversion rate (%) of L-tryptophan was equal to (M)1－M2)/M1×100％；
In the formula, M1The input amount of the L-tryptophan is the input amount; m2Is the amount of L-tryptophan remaining after the reaction; m1And M2The weight units of (a) and (b) are consistent.
The results are as follows:
table 3: conversion of L-tryptophan
Group of
Conversion of L-tryptophan
Example 2
97.5％
Example 3
98.9％
Example 4
94.2％
Comparative example 1
72.5％
Comparative example 2
81.5％
Comparative example 3
82.9％
Comparative example 4
64.5％
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Kazai Biotech Co., Ltd, Xintai City, Shantou City, Kazai Biotech Co., Ltd
<120> a method for converting L-tryptophan into tryptamine and D-tryptophan
<130> 2021
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1533
<212> DNA
<213> Artificial sequence
<400> 1
ctagttgttg atatgggttc tctggacacc aacccgaccg ctttctctgc tttcccggct 60
ggtgaaggtg aaaccttcca gccgctgaac gctgacgacg ttcgttctta cctgcacaaa 120
gctgttgact tcatctctga ctactacaaa tctgttgaat ctatgccggt tctgccgaac 180
gttaaaccgg gttacctgca ggacgaactg cgtgcttctc cgccgaccta ctctgctccg 240
ttcgacgtta ccatgaaaga actgcgttct tctgttgttc cgggtatgac ccactgggct 300
tctccgaact tcttcgcttt cttcccgtct accaactctg ctgctgctat cgctggtgac 360
ctgatcgctt ctgctatgaa caccgttggt ttcacctggc aggcttctcc ggctgctacc 420
gaaatggaag ttctggctct ggactggctg gctcagatgc tgaacctgcc gacctctttc 480
atgaaccgta ccggtgaagg tcgtggtacc ggtggtggtg ttatcctggg taccacctct 540
gaagctatgc tggttaccct ggttgctgct cgtgacgctg ctctgcgtcg ttctggttct 600
gacggtgttg ctggtctgca ccgtctggct gtttacgctg ctgaccagac ccactctacc 660
ttcttcaaag cttgccgtct ggctggtttc gacccggcta acatccgttc tatcccgacc 720
ggtgctgaaa ccgactacgg tctggacccg gctcgtctgc tggaagctat gcaggctgac 780
gctgacgctg gtctggttcc gacctacgtt tgcgctaccg ttggtaccac ctcttctaac 840
gctgttgacc cggttggtgc tgttgctgac gttgctgctc gtttcgctgc tggttgcacc 900
tctacccgtc gtaccccggc tgctcgtgct tctgctcgtt cttctggtac cacctctacc 960
gcttggtctg cttggacccc gtctgcttaa gctccgacca acggttaata accggcttct 1020
accgctccgg cttctacctg cgctaccccg accgcttctc cggctccgtc tcgtccgacc 1080
cgttctacct ctcgtaccac cccggctacc ccggctcgtt ctccgacctc tcgtacctgc 1140
cgttctgctt ctgctgctgc ttctggtggt tcttcttctg gttggtcttg cgctccgacc 1200
gcttctccgt cttgccgttc tacctctggt gctacctctc cgtggccgcg ttgctctcgt 1260
acctcttctg ctgctaccac cggttctcgt tcttcttgcc gtggtacctc tctgtcttct 1320
gcttctggtt ctggtccggg tcgtcgtcgt cgtcgtgacg gtggtggtcg tcgtcgtggt 1380
gaaccgcgtg ctgacggtgc tgctgaacag gaccgtcagg gtgttcgtgg tgctcacggt 1440
ggtcgtcgtc aggttcgtgc tgctctgcgt ggtggtctgg ttgctgctgg tcgtgcttct 1500
cgtgctgaac gtgttggtgc tcaccaggaa gac 1533
<210> 2
<211> 1533
<212> DNA
<213> Rice
<400> 2
ctagttgttg atatgggcag cttggacacc aaccccacgg ccttctccgc cttccccgcc 60
ggcgagggtg aaaccttcca gccgctcaac gccgatgatg tccggtccta cctccacaag 120
gcggtggact tcatctcgga ctactacaag tccgtggagt ccatgccggt gctgcccaat 180
gtcaagccgg ggtacctgca ggacgagctc agggcctcgc cgccgacgta ctcggcgccg 240
ttcgacgtca ccatgaagga gctccggagc tccgtcgtcc ccgggatgac gcactgggcg 300
agccccaact tcttcgcgtt tttcccctcc acgaatagtg cggccgccat tgccggcgac 360
ctcatcgcgt cggcgatgaa cacggtcggg ttcacgtggc aggcgtcgcc ggcggccacc 420
gagatggagg tgctcgcgct ggactggctc gcgcagatgc tcaacctgcc gacgagcttc 480
atgaaccgca ccggcgaggg gcgtggcacc ggcggtgggg ttattctggg gacgaccagc 540
gaggcgatgc tcgtcacgct cgttgccgcg cgcgacgccg cgctgcggcg gagcggcagc 600
gacggcgtgg cgggactcca ccggctcgcc gtgtacgccg ccgaccagac gcactccacg 660
ttcttcaagg cgtgccgcct cgccgggttt gatccggcga acatccggtc gatccccacc 720
ggggccgaga ccgactacgg cctcgacccg gcgaggctgc tggaggcgat gcaggccgac 780
gccgacgccg ggctggtgcc cacctacgtg tgcgccacgg tgggcaccac gtcgtccaac 840
gccgtcgacc cggtgggcgc cgtggccgac gtcgcggcga ggttcgccgc tgggtgcacg 900
tcgacgcggc gtacgccggc agcgcgtgca tctgcccgga gttcaggcac cacctcgacg 960
gcgtggagcg cgtggactcc atcagcatga gcccccacaa atggctgatg acctgcctcg 1020
actgcacctg cctctacgtg cgcgacaccc accgcctcac cggctccctc gagaccaacc 1080
cggagtacct caagaaccac gccagcgact ccggcgaggt caccgacctc aaggacatgc 1140
aggtcggcgt cggccgccgc ttccgggggc tcaagctctg gatggtcatg cgcacctacg 1200
gcgtcgccaa gctgcaggag cacatccgga gcgacgtcgc catggccaag gtgttcgagg 1260
acctcgtccg cggcgacgac aggttcgagg tcgtcgtgcc gaggaacttc gctctcgtct 1320
gcttcaggat cagggccggg ccggcgccgc cgccgcgacg gaggaggacg ccgacgaggc 1380
gaaccgcgag ctgatggagc ggctgaacaa gaccggcaag gcgtacgtgg cgcacacggt 1440
ggtcggcggc aggttcgtgc tgcgcttcgc ggtgggctcg tcgctgcagg aagagcatca 1500
cgtgcggagc gcgtgggagc tcatcaagaa gac 1533
