CN115160164A - Preparation method and application of composite alanine methyl ester hydrochloride - Google Patents
Preparation method and application of composite alanine methyl ester hydrochloride Download PDFInfo
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 title claims abstract description 83
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 title claims abstract description 80
- 239000002131 composite material Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 64
- 238000006243 chemical reaction Methods 0.000 claims abstract description 34
- 239000007787 solid Substances 0.000 claims abstract description 33
- 108010016626 Dipeptides Proteins 0.000 claims abstract description 28
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
- 235000004279 alanine Nutrition 0.000 claims abstract description 17
- 238000002425 crystallisation Methods 0.000 claims abstract description 16
- 230000008025 crystallization Effects 0.000 claims abstract description 16
- 238000005886 esterification reaction Methods 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 9
- 239000000706 filtrate Substances 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 239000013078 crystal Substances 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 claims 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 abstract description 56
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000010924 continuous production Methods 0.000 abstract 1
- 230000032050 esterification Effects 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 239000000047 product Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 39
- 229960003767 alanine Drugs 0.000 description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- IYUKFAFDFHZKPI-DFWYDOINSA-N methyl (2s)-2-aminopropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@H](C)N IYUKFAFDFHZKPI-DFWYDOINSA-N 0.000 description 12
- 238000002390 rotary evaporation Methods 0.000 description 12
- -1 alpha-amino acid ester Chemical class 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 108700016155 Acyl transferases Proteins 0.000 description 6
- 102000057234 Acyl transferases Human genes 0.000 description 6
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 235000008206 alpha-amino acids Nutrition 0.000 description 6
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 5
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 229930182816 L-glutamine Natural products 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000012065 filter cake Substances 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- QDQVXVRZVCTVHE-YFKPBYRVSA-N propan-2-yl (2s)-2-aminopropanoate Chemical compound CC(C)OC(=O)[C@H](C)N QDQVXVRZVCTVHE-YFKPBYRVSA-N 0.000 description 3
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000002288 cocrystallisation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LOJYQMFIIJVETK-WDSKDSINSA-N Gln-Gln Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(O)=O LOJYQMFIIJVETK-WDSKDSINSA-N 0.000 description 1
- IJMWOMHMDSDKGK-UHFFFAOYSA-N Isopropyl propionate Chemical group CCC(=O)OC(C)C IJMWOMHMDSDKGK-UHFFFAOYSA-N 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 235000016236 parenteral nutrition Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
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- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
- C07C227/40—Separation; Purification
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- C07C227/42—Crystallisation
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
The invention relates to a preparation method and application of composite alanine methyl ester hydrochloride. Firstly, carrying out esterification on alanine to form an alanine methyl ester hydrochloride solution, then concentrating and introducing glutamine to accelerate crystallization and separation of the alanine methyl ester hydrochloride solution, filtering or centrifuging to obtain a composite alanine methyl ester hydrochloride solid, wherein the yield of the composite alanine methyl ester hydrochloride solid can reach 95% by repeatedly applying filtrate or centrifugate; the compound alanine methyl ester hydrochloride and glutamine are used as base materials to synthesize the glutamine dipeptide by adopting an enzyme method, and the molar conversion rate is as high as 85%. The method has the advantages of cheap reaction raw materials, high conversion rate, simple and easy operation process, realization of continuous production by carrying a matched evaporator and a matched centrifugal machine device, and application potential and economic value in the field of food health products.
Description
Technical Field
The invention relates to the technical field of preparation methods for preparing composite organic compounds, in particular to a preparation method and application of composite alanine methyl ester hydrochloride.
Background
The glutamine dipeptide as the amino acid parenteral nutrition agent can be synthesized by a chemical method and a microbial enzyme method, and the invention patents CN101659691A and CN1786019A disclose the chemical synthesis method of the glutamine dipeptide, but the synthesis route is long, the environmental pollution is large, and the process cost is high. Therefore, the raw materials adopted by enterprises in this year mostly comprise alpha-amino acid ester acyltransferase, L-alanine and ester derivatives thereof and glutamine. The L-alanine ester derivatives comprise alanine methyl ester, alanine isopropyl ester, alanine methyl ester hydrochloride and the like, the invention patent CN 106834394B directly adopts alanine and glutamine as base materials to synthesize the glutamine dipeptide, the molar conversion rate is 64.5 percent, the invention patent CN105274174A adopts alanine methyl ester, alanine isopropyl ester and glutamine as base materials to synthesize the glutamine dipeptide, and the L-alanine ester derivatives are under the condition that the alanine isopropyl ester is excessive by about one time (m is Propanoic acid isopropyl ester ：m Glutamine = 2), 93.5% conversion of glutamine-reduced glutamine-glutamine dipeptide (i.e. molar conversion of 62.9%); under the condition of 40% excess of alanine methyl ester, the molar conversion rate of glutamine dipeptide is 62.9%; the invention patent CN104561202A prepares the glutamine dipeptide from the self-made alanine methyl ester and glutamine, the mol conversion rate is 59.14%, it can be seen that the variety of the L-alanine ester derivative and the base material proportion have great influence on the conversion rate of the glutamine dipeptide, and the alanine ester derivative which can be converted into the glutamine dipeptide more efficiently needs to be prepared.
Disclosure of Invention
The invention discloses a preparation method and application of composite alanine methyl ester hydrochloride, wherein partial glutamine is compounded in L-alanine methyl ester hydrochloride to form cocrystal, so that the enzyme catalysis efficiency in the subsequent preparation process of glutamine dipeptide can be greatly improved.
The invention firstly provides a preparation method of composite alanine methyl ester hydrochloride, which comprises the following steps:
s1, dripping dehydrated hydrochloric acid into alanine, stirring and refluxing with alcohol at 80 ℃ for esterification reaction for 10-20h to obtain alanine methyl ester hydrochloride solution (initial solid pure of the solution)Content is m 0 )；
S2, evaporating and concentrating at 60-80 ℃ for 1-3 h to obtain a concentrated solution;
s3, adding glutamine seed crystals into the concentrated solution of the S2, stirring and crystallizing for 1-60 min, and then filtering or centrifuging to obtain composite alanine methyl ester hydrochloride solid and filtrate or centrifugate;
repeating the steps of S2, S3 and S4 on the filtrate or centrifugate of S4 and S3; obtaining the compound alanine methyl ester hydrochloride which consists of the following components in parts by weight:
80-95 parts by weight of alanine methyl ester hydrochloride;
4.5 to 10 weight portions of alanine;
0.5 to 10 parts by weight of glutamine.
In the preparation method described above, it is further preferable that the complex alanine methyl ester hydrochloride of step S4 is composed of the following components in parts by weight:
85 to 92.5 weight portions of alanine methyl ester hydrochloride;
7-9.5 parts by weight of alanine;
0.5 to 5 parts by weight of glutamine.
In the above-mentioned production process, it is further preferred that the concentration end point of the step S2 is the initial net solid content (m) of the alanine methyl ester hydrochloride solution 0 ) 2.5 to 3.5 times of the total weight of the powder.
In the preparation method described above, it is further preferable that, when the end point of the concentration in the step S2 is 2.5 to 3 times of the initial net solid content of the alanine methyl ester hydrochloride solution, the concentrated liquid in the step S3 is crystallized and then is separated by filtration or centrifugation; and if the concentration end point of the step S2 is 3-3.5 times of the initial net solid content of the alanine methyl ester hydrochloride solution, carrying out crystallization on the concentrated liquid in the step S3 and then carrying out centrifugal separation.
In the above-mentioned production method, it is further preferable that the glutamine seed crystal in the step S3 is added in an amount of 0.5 to 11.1% based on the initial net solid content of the alanine methyl ester hydrochloride solution.
In the above-mentioned preparation method, it is further preferable that the number of times of repeating S2, S3, S4 of the filtrate or the centrifugate in the step S4 is not more than 10.
In the above-mentioned preparation method, it is further preferable that the temperature of the stirring crystallization in the step S3 is 20 to 40 ℃.
Further preferably, in the application of the composite alanine methyl ester hydrochloride prepared by the method, the composite alanine methyl ester hydrochloride is used as a substrate and glutamine is used for synthesizing the glutamine dipeptide, and the molar conversion rate is not less than 80%. More preferably, the molar conversion is as high as 85%.
More preferably, in the application, the adding ratio of the composite alanine methyl ester hydrochloride and the glutamine for synthesizing the glutamine dipeptide by using the composite alanine methyl ester hydrochloride as a base material is (0.8-1.5): (1.5-0.8).
More preferably, in the application, the adding ratio of the composite alanine methyl ester hydrochloride and the glutamine for synthesizing the glutamine dipeptide by taking the composite alanine methyl ester hydrochloride as a substrate is 1.5: (0.9-1.1).
The invention has the beneficial effects that:
1. the method has the advantages that the alanine methyl ester hydrochloride solution is directly evaporated, concentrated and purified after being prepared by the esterification reaction, no complicated purification step is introduced, and no water or other organic reagents are introduced, so that the method is simple and feasible;
2. after the alanine methyl ester hydrochloride solution is concentrated, glutamine is added to serve as seed crystals, a co-crystallization result can be formed, the enzyme catalysis efficiency is improved in the process of preparing the glutamine dipeptide, and the molar conversion rate is further improved. The molar conversion rate is not lower than 80 percent; in the optimal embodiment of the application, the molar conversion rate is as high as 85%, and compared with the conversion rate of synthesizing the glutamine dipeptide from the commercialized alanine methyl ester hydrochloride powder, the conversion rate is only 70%, so that the conversion rate is greatly improved, and the industrial application prospect is better.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are only for purposes of illustration and explanation and are not intended to be limiting.
The invention discloses a gene for coding glutamine dipeptide biosynthetic enzyme and application thereof by referring to Chinese invention patent CN 106754985B in the solution of alpha-amino acid ester acyltransferase used in the following embodiments in the application, and provides a method for preparing glutamine dipeptide biosynthetic enzyme by recombinant escherichia coli containing the gene.
Example 1
Preparation of alanine methyl ester hydrochloride solution
2000g of L-alanine and 2850g of concentrated hydrochloric acid are mixed, stirred and dissolved at 60 ℃, 10L of n-heptane is weighed and added into a reaction kettle, stirred, refluxed and dehydrated at 80 ℃ and kept for 2 hours. Concentration was then continued and n-heptane was distilled off. Adding 8000g of methanol into the reaction kettle, heating to 80 ℃, and carrying out stirring reflux reaction for 10 hours to obtain an L-alanine methyl ester hydrochloride solution. The conversion of L-alanine methyl ester hydrochloride by HPLC was 95.9%.
Example 2
Preparation of compound alanine methyl ester hydrochloride
1000g of L-alanine methyl ester hydrochloride solution (solid content is 20%) obtained by the esterification reaction is transferred to a rotary evaporation bottle to be rotationally evaporated at 60 ℃ to 500g, then concentrated solution is transferred to a crystallizing dish, 2.5g of glutamine is added to the crystallizing dish, the mixture is stirred at 25 ℃ for 30min, a large amount of crystals are separated out, and then vacuum filtration is carried out to obtain 283.3g of filter cake and 210.5g of filtrate. Drying the filter cake to obtain the compound alanine methyl ester hydrochloride.
The composite alanine methyl ester hydrochloride obtained in the example 2 consists of the following components in parts by weight: alanine methyl ester hydrochloride 91.9wt%; 7.6wt% of alanine; 0.5wt% of glutamine.
Example 3
1000g of L-alanine methyl ester hydrochloride solution (solid content is 20%) obtained by the esterification reaction is transferred to a rotary evaporation bottle to be rotationally evaporated at 80 ℃ to 700g, then the concentrated solution is transferred to a crystallization dish, 11.1g of glutamine is added to the crystallization dish, the mixture is stirred at 25 ℃ for 5min, a large amount of crystals are separated out, and then the mixture is centrifuged to obtain 412.6g of solid and 276.2g of centrifugate. Drying the centrifugal solid to obtain the compound alanine methyl ester hydrochloride.
The composite alanine methyl ester hydrochloride obtained in the embodiment 3 consists of the following components in parts by weight: alanine methyl ester hydrochloride 85.7wt%; 9.3wt% of alanine; 5wt% of glutamine.
Example 4
1000g of L-alanine methyl ester hydrochloride solution (solid content is 20%) obtained by the esterification reaction is transferred to a rotary evaporation bottle to be rotationally evaporated at 80 ℃ to 600g, then the concentrated solution is transferred to a crystallization dish, 5g of glutamine is added to the crystallization dish, the mixture is stirred at 25 ℃ for 5min, a large amount of crystals are separated out, and then the crystallization dish is centrifuged to obtain 353.7g of solid and 241.3g of centrifugate. Drying the centrifugal solid to obtain the compound alanine methyl ester hydrochloride.
The composite alanine methyl ester hydrochloride obtained in the example 4 consists of the following components in parts by weight: alanine methyl ester hydrochloride 90.4wt%; 7.9wt% of alanine; 1.7wt% of glutamine.
Example 5
1000g of L-alanine methyl ester hydrochloride solution (solid content is 20%) obtained by the esterification reaction is transferred to a rotary evaporation bottle to be rotationally evaporated at 80 ℃ to 550g, then the concentrated solution is transferred to a crystallization dish, 10g of glutamine is added to the crystallization dish, the mixture is stirred at 25 ℃ for 5min, a large amount of crystals are separated out, and then the crystallization dish is centrifuged to obtain 327.3g of solid and 220.8g of centrifugate. Drying the centrifugal solid to obtain the compound alanine methyl ester hydrochloride.
The composite alanine methyl ester hydrochloride obtained in the example 5 consists of the following components in parts by weight: alanine methyl ester hydrochloride 87.9wt%; 7.5wt% of alanine; glutamine 4.6wt%.
In addition, the effect of various parameters for the preparation of complex alanine methyl ester hydrochloride not listed in examples 2 to 5 is shown in Table 1:
TABLE 1 influence of various parameters of the preparation of Complex alanine methyl ester hydrochloride
Example 6
Experiment for multi-kettle sleeve
Transferring 1000g (solid content is 20%) of L-alanine methyl ester hydrochloride solution obtained by esterification reaction into a rotary evaporation bottle, carrying out rotary evaporation at 80 ℃ to 600g, transferring the concentrated solution into a crystallization vessel, adding 1g of glutamine, stirring at 25 ℃ for 5min, separating out a large amount of crystals, and centrifuging to obtain 350.3g of centrifugal solid in a first kettle and 249.2g of centrifugal liquid; drying the centrifugal solid to obtain composite alanine methyl ester hydrochloride, adding 249.2g of centrifugal liquid, adding 1000g of L-alanine methyl ester hydrochloride solution obtained by esterification reaction again, transferring the solution into a rotary evaporation bottle, carrying out rotary evaporation at 80 ℃ to 750g, transferring the concentrated solution into a crystallization vessel, adding 1.2g of glutamine, stirring at 25 ℃ for 5min, precipitating a large amount of crystals, and centrifuging to obtain 438.5g of centrifugal solid and 249.2g of centrifugal liquid in a second kettle; the operation is further repeated for two times to complete the four-kettle application experiment. As shown in Table 2;
TABLE 2 purity and yield of filter cake from each kettle
The yield of alanine methyl ester hydrochloride can be increased by using each kettle, but the content of alanine methyl ester hydrochloride can be slightly reduced, so the total number of times of application cannot exceed 10 times, and the effect of the test performed by using four kettles in the embodiment is better.
The composite alanine methyl ester hydrochloride obtained in the example 6 consists of the following components in parts by weight: alanine methyl ester hydrochloride 92.1 parts by weight; 7.3 parts by weight of alanine; 0.6 part by weight of glutamine.
Example 7
Synthesis of glutamine dipeptide from composite alanine methyl ester hydrochloride
Adding 500g of L-glutamine and 700g of composite alanine methyl ester hydrochloride into 4L of water, adjusting the pH value to 9.0 by using NaOH aqueous solution, then adding 10mL of alpha-amino acid ester acyltransferase solution into a reaction system, dropwise adding HCl after reacting for 30min to adjust the pH value to 6.0, heating to 70 ℃ to inactivate the enzyme, testing the content of glutamine dipeptide in the reaction solution by HPLC, and calculating the molar conversion rate to be 85.1%.
Comparative example 1
Commercial alanine methyl ester hydrochloride synthesis of glutamine dipeptide
Adding 500g of L-glutamine and 700g of commercial alanine methyl ester hydrochloride into 4L of water, adjusting the pH value to 9.0 by using NaOH aqueous solution, then adding 10mL of alpha-amino acid ester acyltransferase solution into a reaction system, reacting for 30min, dropwise adding HCl to adjust the pH value to 6.0, heating to 70 ℃ to inactivate the enzyme, and measuring the content of glutamine dipeptide in the reaction solution by HPLC (high performance liquid chromatography), wherein the molar conversion rate is calculated to be 70%.
Comparative example 2
Glutamine co-crystallization component not introduced in advance in composite alanine methyl ester hydrochloride and glutamine dipeptide synthesized by same
Transferring 1000g of L-alanine methyl ester hydrochloride solution (solid content is 20%) obtained by the esterification reaction into a rotary evaporation bottle, carrying out rotary evaporation at 80 ℃ to 700g, transferring the concentrated solution into a crystallization vessel, stirring at 20 ℃ for 90min, precipitating a large amount of crystals, and centrifuging to obtain 380.7g of centrifugal solid and 318.6g of centrifugal liquid; drying the centrifugal solid to obtain glutamine-free alanine/alanine methyl ester hydrochloride composite crystals.
Adding 200g of L-glutamine and 280.5g of the alanine/alanine methyl ester hydrochloride composite crystal into 1.6L of water, adjusting the pH to 9.0 by using NaOH aqueous solution, then adding 4mL of alpha-amino acid ester acyltransferase solution into a reaction system, reacting for 30min, dropwise adding HCl to adjust the pH to 6.0, heating to 70 ℃ to inactivate the enzyme, and measuring the content of glutamine dipeptide in the reaction solution by HPLC (high performance liquid chromatography), wherein the molar conversion rate is calculated to be 71.8%.
Comparative example 3
Preparation of single L-alanine methyl ester hydrochloride and synthesis of glutamine dipeptide
Transferring 1000g (solid content is 20%) of L-alanine methyl ester hydrochloride solution obtained by esterification reaction into a rotary evaporation bottle, adding 1000g of cyclohexane, carrying out rotary evaporation at 80 ℃ to 700g, transferring the concentrated solution into a crystallization vessel, stirring at 20 ℃ for 90min, precipitating a large amount of crystals, and then centrifuging to obtain 370.9g of centrifugal solid and 328.6g of centrifugal solution; drying the centrifugal solid to obtain single alanine methyl ester hydrochloride crystal.
Adding 200g of L-glutamine and 280.1g of the alanine/alanine methyl ester hydrochloride composite crystal into 1.6L of water, adjusting the pH to 9.0 by using NaOH aqueous solution, then adding 4mL of alpha-amino acid ester acyltransferase solution into a reaction system, reacting for 30min, dropwise adding HCl to adjust the pH to 6.0, heating to 70 ℃ to inactivate the enzyme, and measuring the content of glutamine dipeptide in the reaction solution by HPLC (high performance liquid chromatography), wherein the molar conversion rate is calculated to be 68.5%.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the details shown in the description and the examples, which are set forth, but are fully applicable to various fields of endeavor as are suited to the particular use contemplated, and further modifications will readily occur to those skilled in the art, since the invention is not limited to the details shown and described without departing from the general concept as defined by the appended claims and their equivalents.
Claims (10)
1. The preparation method of the compound alanine methyl ester hydrochloride is characterized by comprising the following steps:
s1, dripping dehydrated hydrochloric acid into alanine, and stirring and refluxing the dehydrated hydrochloric acid and alcohol at 80 ℃ to perform esterification reaction for 10-20 hours to obtain an alanine methyl ester hydrochloride solution;
s2, evaporating and concentrating at 60-80 ℃ for 1-3 h to obtain a concentrated solution;
s3, adding glutamine seed crystals into the concentrated solution of the S2, stirring and crystallizing for 1-60 min, and then filtering or centrifuging to obtain composite alanine methyl ester hydrochloride solid and filtrate or centrifugate;
s4, repeating the steps of S2, S3 and S4 on the filtrate or centrifugate of the S3; obtaining the compound alanine methyl ester hydrochloride which consists of the following components in parts by weight:
80-95 parts by weight of alanine methyl ester hydrochloride;
4.5 to 10 weight portions of alanine;
0.5 to 10 parts by weight of glutamine.
2. The preparation method of claim 1, wherein the S4 composite alanine methyl ester hydrochloride comprises the following components in parts by weight:
85 to 92.5 weight portions of alanine methyl ester hydrochloride;
7-9.5 parts by weight of alanine;
0.5 to 5 parts by weight of glutamine.
3. The method according to claim 1, wherein the concentration end point of step S2 is 2.5 to 3.5 times the initial net solid content of the alanine methyl ester hydrochloride solution.
4. The method according to claim 1, wherein when the concentration end point in step S2 is 2.5 to 3 times of the initial net solid content of the alanine methyl ester hydrochloride solution, the concentrated liquid in step S3 is crystallized and then separated by filtration or centrifugation; and if the concentration end point of the step S2 is 3-3.5 times of the initial net solid content of the alanine methyl ester hydrochloride solution, carrying out crystallization on the concentrated liquid in the step S3 and then carrying out centrifugal separation.
5. The method according to claim 1, wherein the glutamine seed crystal is added in an amount of 0.5 to 11.1% based on the initial net solid content of the alanine methyl ester hydrochloride solution in the step S3.
6. The method according to claim 1, wherein the filtrate or the centrifugate in the step S4 is repeated S2, S3 and S4 no more than 10 times.
7. The method according to claim 1, wherein the temperature for stirring and crystallizing in the step S3 is 20 to 40 ℃.
8. Use of a complex alanine methyl ester hydrochloride salt prepared according to the method of claim 1, wherein said complex alanine methyl ester hydrochloride salt is used as a substrate with glutamine for the synthesis of glutamine dipeptide at a molar conversion of not less than 80%.
9. The use of claim 8, wherein the ratio of the composite alanine methyl ester hydrochloride used as a base material for synthesizing glutamine dipeptide to the glutamine is (0.8-1.5): (1.5-0.8).
10. The use of claim 9, wherein the ratio of the composite alanine methyl ester hydrochloride used as a primer for the synthesis of glutamine dipeptide to the glutamine added is 1.5: (0.9-1.1).
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