Summary of the invention
The synthetic method that the purpose of this invention is to provide a kind of new theanine, this method have cost of material cheapness, production cost is low, selectivity is high, speed of response is fast, yield is high characteristics, realize the suitability for industrialized production of this product.
The synthetic method of theanine provided by the invention comprises the steps:
1) R
1OCOCH
2CH
2CH (NH
2) COOH (L-L-glutamic acid-γ-ester) CL is at C
1-4Alcoholic solution or the aqueous solution of alcohol in, add alkali after controlled temperature to 0-40 ℃, add (R
2CO)
2O or R
2To there not being raw material, concentrated after-filtration obtains R to COCl in 0-50 ℃ of reaction
1OCOCH
2CH
2CH (NHCOR
2) COOH (solution of N-acidylate-L-glutamic acid-γ-ester); Or
HOCOCH
2CH
2CH (NHCOR
3) COOH (N-acidylate-L-glutamic acid) adds C
1-6Alcohol, complete molten back react under an acidic catalyst condition and is finished, and adds in the saturated inorganic alkali alcosol and an acidic catalyst makes PH neutrality, filters the concentrated R that obtains in back
1OCOCH
2CH
2CH (NHCOR
3) solution of COOH (N-acidylate-L-glutamic acid-γ-ester);
2) R
1OCOCH
2CH
2CH (NHCOR
3) solution of COOH (N-acidylate-L-glutamic acid-γ-ester) and ethamine or ethylamine solution carry out 0-50 ℃ down reaction obtain N-acidylate-theanine solution;
3) N-acidylate-theanine solution and L-L-Aminoacylase are reacted complete to hydrolysis in 20-60 ℃, filter rear filtrate and concentrate, add C
1-4Alcohol or pure aqueous solution crystallization, obtain the theanine crude product after the filtration;
4) the theanine crude product uses 70-95%C
1-4The aqueous solution of alcohol carry out recrystallization, obtain the theanine elaboration;
R
1=C
1-6Alkyl, R
2=C
2H
5R
3=C
1-8Alkyl.
L-L-glutamic acid-γ-ester: alkali=1: 0.1~10 (weight);
L-L-glutamic acid-γ-ester: the ethylamine solution of ethamine or 50-80%=1: 1~100; (weight/volume, grams per milliliter);
N-acidylate-L-glutamic acid: the ethylamine solution of ethamine or 50-80%=1: 1~100; (weight/volume, grams per milliliter);
L-L-glutamic acid-γ-ester: L-L-Aminoacylase=1: 0.1~10 (weight ratio);
N-acidylate-L-glutamic acid: L-L-Aminoacylase=1: 0.1~10 (weight ratio);
L-immobilized aminoacylase vigor 2000~6000u/g.
Described C
1-4Alcohol be methyl alcohol or ethanol.
Described C
1-6Alcohol be methyl alcohol or ethanol.
Described an acidic catalyst is the vitriol oil.
Described mineral alkali is NaOH, KOH, NaHCO
3, Na
2CO
3
The synthetic method of the theanine of selection provided by the invention comprises the steps:
1) L-L-glutamic acid is dissolved in the dehydrated alcohol, is cooled to 0-10 ℃, adds the vitriol oil, and room temperature reaction 8-10 hour, add alkaline solution to neutral, cooled and filtered, filtration cakes torrefaction obtains the L-ethyl glutamate;
2) the L-ethyl glutamate is dissolved in the aqueous ethanolic solution of 30-80%, add alkali, the dissolving postcooling is to 0-10 ℃, added the acetic anhydride room temperature reaction 3 hours, concentrate desalination, obtain L-acetylglutamate ethyl ester solution, add 70% ethylamine solution, stirring reaction is complete to aminating reaction, obtains L-acetyl theanine solution;
3) L-acetyl theanine solution is through concentrating, to neutral, temperature 20-60 ℃ adds L-L-Aminoacylase and carries out enzymic hydrolysis, stirring reaction 10 hours is complete to hydrolysis, remove by filter L-L-Aminoacylase, filtrate concentrates, and adds 95% ethanol crystallization, obtain L-theanine crude product, the aqueous ethanolic solution recrystallization of process 80-95% obtains finished product L-theanine;
Wherein, the weight of L-L-glutamic acid and dehydrated alcohol: volume=1: 5~15 (grams per milliliter);
The weight of the L-L-glutamic acid and the vitriol oil: volume=1: 0.3~2 (grams per milliliter);
L-ethyl glutamate: alkali=1: 0.5~3 (weight);
The weight of L-L-glutamic acid and acetic anhydride: volume=1: 1~2 (grams per milliliter);
L-ethyl glutamate: 70% ethylamine solution=1: 5~15 (weight/volume, grams per milliliter);
L-ethyl glutamate: L-immobilized aminoacylase=1: 0.5~4 (weight);
L-immobilized aminoacylase vigor is 6000u/g;
Described alkali is triethylamine, sodium bicarbonate, NaOH or KOH.
The synthetic method of the theanine of selection provided by the invention comprises the steps:
1) the L-acetylglutamate mixes with methyl alcohol or propyl carbinol, stirs and is cooled to 0-10 ℃, adds the vitriol oil, and room temperature reaction 8-10 hour, be neutralized to neutrality with saturated mineral alkali alcoholic solution, cooled and filtered, mother liquor concentrates, and obtains L-acetylglutamate methyl ester solution;
2) L-acetylglutamate methyl esters or the positive butyl acetate solution of L-acetylglutamate add 70% ethylamine solution, and stirring reaction is complete to aminating reaction, obtain L-acetyl theanine solution;
3) L-acetyl theanine solution is through concentrating, to neutral, temperature 20-60 ℃ adds L-L-Aminoacylase and carries out enzymic hydrolysis, stirring reaction 10 hours is complete to hydrolysis, remove by filter L-L-Aminoacylase, filtrate concentrates, and adds 95% ethanol crystallization, obtain L-theanine crude product, the aqueous ethanolic solution recrystallization of process 80-95% obtains finished product L-theanine;
The weight of L-acetylglutamate and methyl alcohol or propyl carbinol: volume=1: 5~15 (grams per milliliter);
The weight of the L-acetylglutamate and the vitriol oil: volume=1: 0.3~2 (grams per milliliter);
L-acetylglutamate: alkali=1: 0.5~3 (weight);
L-acetylglutamate methyl esters or the positive butyl ester of L-acetylglutamate: 70% ethylamine solution=1: 5~15 (weight/volume, grams per milliliter;
L-acetylglutamate: L-immobilized aminoacylase=1: 0.5~4 (weight);
L-immobilized aminoacylase vigor 6000u/g;
Described alkali is triethylamine, sodium bicarbonate, NaOH or KOH.
The synthetic method of the theanine of selection provided by the invention comprises the steps:
1) L-L-glutamic acid is dissolved in the dehydrated alcohol, is cooled to 0-10 ℃, adds the vitriol oil, and room temperature reaction 8-10 hour, add triethylamine solution to neutral, cooled and filtered, filtration cakes torrefaction obtains the L-ethyl glutamate;
2) the L-ethyl glutamate is dissolved in the aqueous ethanolic solution of 30-70%, add sodium bicarbonate, the dissolving postcooling is to 0-10 ℃, added the acetic anhydride room temperature reaction 3 hours, concentrate desalination, obtain L-acetylglutamate ethyl ester solution, add 70% ethylamine solution, stirring reaction is complete to aminating reaction, obtains L-acetyl theanine solution.
3) L-acetyl theanine solution is through concentrating, to neutral, temperature adds L-L-Aminoacylase for 42 ℃ and carries out enzymic hydrolysis, stirring reaction 10 hours is complete to hydrolysis, remove by filter L-L-Aminoacylase, filtrate concentrates, and adds 95% ethanol crystallization, obtain L-theanine crude product, the aqueous ethanolic solution recrystallization of process 80-95% obtains finished product L-theanine;
Wherein, the weight of L-L-glutamic acid and dehydrated alcohol: volume=1: 5~15 (grams per milliliter);
The weight of the L-L-glutamic acid and the vitriol oil: volume=1: 0.3~2 (grams per milliliter);
L-ethyl glutamate: alkali=1: 0.5~3 (weight);
The weight of L-L-glutamic acid and acetic anhydride: volume=1: 1~2 (grams per milliliter);
L-ethyl glutamate: 70% ethylamine solution=1: 5~15 (weight/volume, grams per milliliter);
L-ethyl glutamate: L-immobilized aminoacylase=1: 0.5~4 (weight);
L-immobilized aminoacylase vigor is 6000u/g.
The synthetic method of the theanine of selection provided by the invention comprises the steps:
1) the L-acetylglutamate mixes with methyl alcohol or propyl carbinol, stirs and is cooled to 0-10 ℃, adds the vitriol oil, and room temperature reaction 8-10 hour, be neutralized to neutrality with saturated KOH or NaOH ethanolic soln, cooled and filtered, mother liquor concentrates, and obtains L-acetylglutamate methyl ester solution;
2) L-acetylglutamate methyl esters or the positive butyl acetate solution of L-acetylglutamate add 70% ethylamine solution, and stirring reaction is complete to aminating reaction, obtain L-acetyl theanine solution;
3) L-acetyl theanine solution is through concentrating; to neutral; temperature adds L-L-Aminoacylase for 42 ℃ and carries out enzymic hydrolysis; stirring reaction 10 hours is complete to hydrolysis; remove by filter L-L-Aminoacylase, filtrate concentrates, and adds 95% ethanol crystallization; obtain L-theanine crude product, the aqueous ethanolic solution recrystallization of process 80-95% obtains finished product L-theanine.
Wherein, the weight of L-acetylglutamate and methyl alcohol or propyl carbinol: volume=1: 5~15 (grams per milliliter);
The weight of the L-acetylglutamate and the vitriol oil: volume=1: 0.3~2 (grams per milliliter);
L-acetylglutamate: alkali=1: 0.5~3 (weight).
L-acetylglutamate methyl esters or the positive butyl ester of L-acetylglutamate: 70% ethylamine solution=1: 5~15; (weight/volume, grams per milliliter)
L-acetylglutamate: L-immobilized aminoacylase=1: 0.5~4 (weight);
L-immobilized aminoacylase vigor 6000u/g.
L-N-acyl glutamic acid-γ-ester obtains L-N-acyl group theanine after 70% ethylamine solution ammonification, also contain a small amount of D-N-acyl group theanine.Adopt the hydrolysis of L-L-Aminoacylase, have only the hydrolysis of L-N-acyl group theanine to remove acyl group, separate out in aqueous ethanolic solution, D-N-acyl group theanine still is dissolved in the alcohol solution, thereby obtains high-quality L-theanine.The present invention uses cheap acyl group to do protecting group in conjunction with the advantage of the L-L-Aminoacylase hydrolysis acyl group of industrialization, after reacting with ethylamine solution, with L-L-Aminoacylase hydrolysis acyl group, just obtains L-theanine again.
The invention provides a kind of synthetic method of new theanine, this method has cost of material cheapness, production cost is low, selectivity is high, speed of response is fast, yield is high characteristics.Raw materials used and reagent is fit to suitability for industrialized production, and operating process is simple, can guarantee quality product.
Embodiment
L-L-glutamic acid-γ-ester can be that raw material prepares as people such as Pan Shirong at " study on the synthesis of L-LEU-glutamic acid methyl ester-L-glutamic acid multipolymer " (biomedical engineering magazine according to currently known methods with L-L-glutamic acid, 1997,2, people such as the method for mentioning 101-104) or what dogface are in " the esterification modification foreign study progress of biosynthesizing polyglutamic acid " (fine chemistry industry, 2002,11,636) the method preparation of mentioning in.L-L-glutamic acid-γ-ethyl ester can directly be bought from Shanghai posthumous title discipline chemical industry company limited etc.
N-acidylate-L-glutamic acid can be that raw material prepares as Shao Weiqing at " preparation of N-p-benzoyl-L-L-glutamic acid " (Chinese Journal of Pharmaceuticals 2004 according to currently known methods with L-L-glutamic acid; 1; people such as method of 10) mentioning or Li Qiuxian are at " N-acylation reaction of L-glutamic acid " (Changchun Polytechnic Univ.'s journal; 2003; 4,19) the method preparation of mentioning in.
N-acidylate-L-glutamic acid-γ-ester and N-acidylate-theanine do not obtain solid materials in reaction process; but exist with the solution form, so be ethamine or ethylamine solution or L-L-Aminoacylase consumption all just L-L-glutamic acid-γ-ester or N-acidylate-L-glutamic acid carry out relevant proportioning and calculate with starting raw material.
Example 1
Take by weighing in 1000 milliliters of three-necked bottles, add L-L-glutamic acid 60 grams, 600 milliliters of dehydrated alcohols are cooled to 10 ℃ under stirring, and drip 30 milliliters of vitriol oils, after drip finishing, room temperature reaction 8 hours drips triethylamine to neutral, cooled and filtered, filtration cakes torrefaction obtains L-L-glutamic acid-γ-ethyl ester 50 grams, yield 70%.
In 1000 milliliters of three-necked bottles, add L-L-glutamic acid-γ-ethyl ester 40g, add 600 milliliters of aqueous ethanolic solutions, add 60 gram sodium bicarbonates, dissolving postcooling to 10 ℃ drips 40 milliliters of acetic anhydrides, drip complete after, room temperature reaction 3 hours, concentrate desalination, obtain just solution of L-N-, add 300 milliliters of 70% ethylamine solutions, stirring reaction is complete to aminating reaction, obtains L-N-acetyl theanine solution.
L-N-acetyl theanine solution is through concentrating; check that solution is to neutral; L-the immobilized aminoacylase that adds equivalent (weight) (is buied from Tianjin TianAn Medicine Industry Co., Ltd; L-immobilized aminoacylase vigor 6000u/g) carries out enzymic hydrolysis; 42 ℃ of stirring reactions 10 hours are complete to hydrolysis; remove by filter L-basic acylase; filtrate concentrates; 95% ethanol crystallization; obtain L-theanine crude product, the aqueous ethanolic solution recrystallization through 80% obtains finished product L-theanine, output 14g; productive rate 35% (in L-L-glutamic acid-γ-ethyl ester) meets japanese food additive standard.
Example 2
Example 1 takes by weighing in 1000 milliliters of three-necked bottles, add L-L-glutamic acid 60 grams, 500 milliliters of propyl carbinols are cooled to below 10 ℃ under stirring, and drip 30 milliliters of vitriol oils, after drip finishing, room temperature reaction 12 hours drips triethylamine to neutral, cooled and filtered, filtration cakes torrefaction obtains L-L-glutamic acid-γ-positive butyl ester 66.6 grams, yield 80%.
In 1000 milliliters of three-necked bottles, add L-L-glutamic acid-γ-positive butyl ester 40g, add 400 milliliters of aqueous ethanolic solutions, add 50 gram sodium bicarbonates, below the dissolving postcooling to 10 ℃, 0 milliliter of acetic anhydride of Dropwise 5, drip finish after, room temperature reaction 2 hours, concentrate desalination, obtain L-N-acetylglutamate-γ-positive butyl acetate solution, add 400 milliliters of 70% ethylamine solutions, stirring reaction is complete to aminating reaction, obtains L-N-acetyl theanine solution.
L-N-acetyl theanine solution is through concentrating; check that solution is to neutral; L-the immobilized aminoacylase that adds equivalent (weight) (is buied from Tianjin TianAn Medicine Industry Co., Ltd; L-immobilized aminoacylase vigor 6000u/g) carries out enzymic hydrolysis; 42 ℃ of stirring reactions 10 hours are complete to hydrolysis; remove by filter L-L-Aminoacylase; filtrate concentrates; 95% ethanol crystallization; obtain L-theanine crude product, the aqueous ethanolic solution recrystallization through 80% obtains finished product L-theanine, output 12.35g; productive rate 36% (in L-L-glutamic acid-γ-positive butyl ester) meets japanese food additive standard.
Example 3
In 1000 milliliters of three-necked bottles, add L-L-glutamic acid 60 grams, 600 milliliters of dehydrated alcohols are cooled to 10 ℃ under stirring, drip 30 milliliters of vitriol oils, after drip finishing, room temperature reaction 8 hours drips triethylamine to neutral, cooled and filtered, filtration cakes torrefaction obtain L-L-glutamic acid-γ-ethyl ester 50 grams, yield 70%.
In 1000 milliliters of three-necked bottles, add L-L-glutamic acid-γ-ethyl ester 40g, add 200 milliliters of entry, add 40 gram sodium bicarbonates, dissolving postcooling to 10 ℃ drips 15 milliliters of Benzoyl chlorides, drip complete after, room temperature reaction 2 hours, concentrate desalination, obtain L-N-benzoyl L-glutamic acid-γ-ethyl ester solution, add 400 milliliters of 70% ethylamine solutions, stirring reaction is complete to aminating reaction, obtains L-N-benzoyl theanine solution.
L-N-benzoyl theanine solution is through concentrating; check that solution is to neutral; L-the immobilized aminoacylase that adds equivalent (weight) (is buied from Tianjin TianAn Medicine Industry Co., Ltd; L-immobilized aminoacylase vigor 6000u/g) carries out enzymic hydrolysis; 42 ℃ of stirring reactions 16 hours are complete to hydrolysis; remove by filter L-L-Aminoacylase; filtrate concentrates; 95% ethanol crystallization; obtain L-theanine crude product, the aqueous ethanolic solution recrystallization through 80% obtains finished product L-theanine, output 15g; productive rate 37.5% (in L-L-glutamic acid-γ-ethyl ester) meets japanese food additive standard.
Example 4
L-N-acetylglutamate 40g, methyl alcohol 400ml stir to be cooled to and drip the 20ml vitriol oil below 10 ℃, drip and finished the back room temperature reaction 8 hours, be neutralized to neutrality with saturated NaOH alcoholic solution (concentration), cooled and filtered, mother liquor concentrates, and obtains L-N-acetylglutamate-γ-methyl ester solution, adds 300 milliliters of 70% ethylamine solutions, stirring reaction is complete to aminating reaction, obtains L-N-acetyl theanine solution.
L-N-acetyl theanine solution is near neutral through concentrating; 42 ℃ add equivalent immobilized aminoacylases, stirring reaction 10 hours to hydrolysis fully, remove by filter L-Aminoacylase, filtrate concentrates, 95% ethanol is separated out crystallization obtains L-theanine crude product; aqueous ethanolic solution recrystallization through 80% obtains finished product L-theanine 12 grams; yield 32.6% (in the L-N-acetylglutamate) meets japanese food additive standard.
Example 5
L-N-acetylglutamate 40g, 200 milliliters of stirrings of propyl carbinol are cooled to and drip the 20ml vitriol oil below 10 ℃, drip and finished the back room temperature reaction 12 hours, be neutralized to neutrality with saturated KOH alcoholic solution (concentration), cooled and filtered, mother liquor concentrates, and obtains L-N-acetylglutamate-γ-positive butyl acetate solution, adds 300 milliliters of 70% ethylamine solutions, stirring reaction is complete to aminating reaction, obtains L-N-acetyl theanine solution.
L-N-acetyl theanine solution is near neutral through concentrating; 42 ℃ add equivalent immobilized aminoacylases, stirring reaction 10 hours to hydrolysis fully, remove by filter L-Aminoacylase, filtrate concentrates, 95% ethanol is separated out crystallization obtains L-theanine crude product; aqueous ethanolic solution recrystallization through 80% obtains finished product L-theanine 13.3 grams; yield 36.1% (in the L-N-acetylglutamate) meets japanese food additive standard.