CN102719367A - Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase - Google Patents
Catalytic synthesis method of L-theanine by using microorganism-produced gamma-glutamyl amino carboxamide synthase Download PDFInfo
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- CN102719367A CN102719367A CN2012102358892A CN201210235889A CN102719367A CN 102719367 A CN102719367 A CN 102719367A CN 2012102358892 A CN2012102358892 A CN 2012102358892A CN 201210235889 A CN201210235889 A CN 201210235889A CN 102719367 A CN102719367 A CN 102719367A
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Abstract
The invention discloses a catalytic synthesis method of L-theanine by using a microorganism-produced gamma-glutamyl amino carboxamide synthase. The catalytic synthesis method comprises the following steps of: screening from tea tree rhizosphere soil to obtain gamma-glutamyl amino carboxamide synthase-sourced strain, nearly Sporidiobolus pararoseus T-C2, with the preservation number of CCTCC NO: M2012232, so as to obtain a purified gamma-glutamyl amino carboxamide synthase; and performing enzymatic synthesis by using the gamma-glutamyl amino carboxamide synthase to obtain the L-theanine, thereby obtaining a method for synthesizing the L-theanine by using the gamma-glutamyl amino carboxamide synthase with high enzyme activity. The synthesis efficiency is much higher than that of the existing enzymatic synthesis technology of the L-theanine by using a microbial enzyme; the safe, simple, convenient, efficient and economical production is realized; and a theoretical basis and a practical basis are provided for subsequent large-scale microbial enzymatic fermentation production of the L-theanine.
Description
Technical field
The present invention relates to a kind of nearly rose look lock shadow yeast (Sporidiobolus pararoseus) T-C2, relate in particular to the method that the γ-paddy carbamyl amine synthetic enzyme that utilizes this microbial fermentation production comes the synthetic L-theanine of catalysis.
Background technology
L-theanine (L-Theanine) is a distinctive nonprotein total free aminoacids in the tea tree; It is one of quality main in the tealeaves and functional component; Be widely used in fields such as food, medicine and fodder additives because of it has multiple physiological action such as the calmness of calming the nerves, immunomodulatory, and be chosen as one of natural product of tool potentiality by U.S.'s heath food industry in 2002.
The natural origin approach of L-theanine is few, and the intravital L-theanine of tea tree is to be substrate with L-glutamic acid and ethamine, under the effect of theanine synthetase, forms, and it and tea tree nitrogen metabolism have close association.Its preparation at present mainly comprises several methods such as direct separation purifying, chemosynthesis and biosynthesizing from tealeaves:
(1) separation and purification: owing to also there is the soluble substance of other higher concentrations in the tealeaves, like theine and tea-polyphenol etc., to the research of present stage, the most key difficult point is how separation and purification obtains high purity L-theanine so.
(2) chemosynthesis: the theanine chemosynthesis provides a kind of simple, convenient, cheap working method.But the chemosynthesis meeting produces more by product, and the product that obtains is the raceme of L-configuration and D-form, and is long with the reaction time, pollute greatly, and economic benefit is not ideal enough.
(3) biosynthesizing: consider that from aspects such as product tooling cost, Product Safety and quality producies biosynthesizing is the development trend of preparation L-theanine.At present, L-theanine biosynthesizing both at home and abroad mainly comprises tea tree cell suspension culture, tea tree callus culture, reaches methods such as the mikrobe enzymatic synthesizes.Now external L-theanine biosynthesizing mainly adopts mikrobe enzymatic synthesis method to produce.
The research of the microbial enzyme of synthetic L-theanine mainly concentrates on four kinds of L-Glutamine deaminase, glutamine synthetase, gamma glutamyl transpeptidase and γ-paddy carbamyl amine synthetic enzyme.Above-mentioned four kinds of microbial enzymes all have the γ-Gu Anxianji forwarding function, promptly all can glutamyl be shifted to ethamine and synthetic L-theanine under optimum conditions, but synthesis mechanism are different.Wherein, Stimulina transpeptidase route of synthesis must be a substrate with Stimulina and ethamine, other three kinds then can L-glutamic acid and ethamine be substrate, the coupling yeast fermentation produces system as ATP, just can synthesize the L-theanine.Because the Stimulina market price is too high; And compare L-Glutamine deaminase and glutamine synthetase; γ-paddy carbamyl amine synthetic enzyme has higher avidity to ethamine, does not produce the by product Stimulina in the reaction, makes it become L-theanine enzymatic synthetic main direction of studying.
This shows; With regard to the market attention rate and demand of present L-theanine; Seek a kind of mikrobe γ-paddy carbamyl amine synthetic enzyme that can produce synthetic L-theanine, and the method for setting up the synthetic L-theanine of γ-paddy carbamyl amine synthetic enzyme catalysis have important significance for theories and more practical value to the future development of its industry.Yet, because of receiving the restriction of research method and technique means,, do not make a breakthrough as yet at present both at home and abroad to gordian technique difficult points such as the screening of enzyme source, cultivation and the immobilization of γ-paddy carbamyl amine synthetic enzyme and the synthetic L-theanine of enzymatic thereof.Therefore, carry out production by biological γ-paddy carbamyl amine synthetic enzyme catalysis and synthesize the hot issue that the research of L-theanine has become current L-theanine preparation.
Summary of the invention
Technical problem to be solved by this invention is: to the deficiency of above-mentioned prior art; A kind of nearly rose look lock shadow yeast (Sporidiobolus pararoseus) T-C2 is provided, the method for the synthetic L-theanine of γ-paddy carbamyl amine synthetic enzyme catalysis of this nearly rose look lock shadow yeast (Sporidiobolus pararoseus) T-C2 fermentative prodn of utilization also is provided simultaneously.
Nearly rose look lock shadow yeast (Sporidiobolus pararoseus) T-C2 of the present invention; Be preserved in Chinese typical culture collection center on June 13rd, 2012, deposit number is CCTCCNO:M2012232, preservation address: China; Wuhan, Wuhan University.
The nearly rose look lock of bacterial strain of the present invention shadow yeast T-C2, bacterium colony is the rose pink colour, and form is the oval convexity, and is opaque, smooth surface, the edge is smooth, and the thalline thickness is prone to provoke, and many ovalizes of thalline or club shape have clamp connexion, can bear the bud of growing sturdily in the young bacterium colony; Find that through biochemistry detection the generation test of sugar-fermenting, nitrate reduction and kind of starch material all is negative, urease test is positive; Through molecular biology identification, obtain 555 bp sequences, the BLAST compare of analysis shows that this bacterial strain is the most approaching with nearly rose look lock shadow yeast (Sporidiobolus pararoseus).
Respectively belong to key with reference to " fungi identification handbook ", Chinese fungi will and yeast, learn evaluation by the JLodder yeast classification and analyze, it is classified as nearly rose look lock shadow yeast (Sporidiobolus pararoseus) by key.
Bacterial strain T-C2 of the present invention is inoculated in the enrichment medium ferments, can obtain γ-paddy carbamyl amine synthetic enzyme fermented liquid, fermentation condition is that inoculum size is 5-10%, and temperature is 28-30 ℃, and rotating speed is 120-180r/min, shaking culture 2-4 days.Consisting of of this enrichment medium: 0.25% methylamine hydrochloride, 0.25 % methyl alcohol, 0.2 % NaCl, 0.1 % KCl, 0.03 % MgSO
47H
2O, 0.005 % KH
2PO
4, 0.005 % K
2HPO
4, 1 * 10
﹣ 7% VB
12And 0.03 % yeast powder, pH7.0-7.4.
The present invention also provides the method that is obtained the γ-paddy carbamyl amine synthetic enzyme of purifying by above-mentioned fermented liquid; Be with above-mentioned fermented liquid in 4 ℃, the centrifugal 15-20min of 12000 r/min, supernatant discarded, embathe twice with the phosphate buffered saline buffer of 10 mmol/L pH 6.0 after; Again in 4 ℃, the centrifugal 15-20min of 12000 r/min; Supernatant discarded is resuspended in the cell that obtains in a small amount of damping fluid then, and damping fluid is not lived cell and got final product; In carrying out cytoclasis 2-3min with the UW of 20 kHZ on ice, obtain crude enzyme liquid again; Crude enzyme liquid is regulated the pH value be 8-9; The ammonium sulfate that under 30 ℃ of conditions, adds the 30-35% saturation ratio is saltoutd; Dissolve to initial volume with the heavy solution weight of albumen precipitation, carry out γ-paddy carbamyl amine synthetic enzyme that the SephadexG-75 column chromatography obtains purifying after the ultrafiltration desalination.
The present invention also provides the method for the synthetic L-theanine of γ-paddy carbamyl amine synthetic enzyme that utilizes the purifying that aforesaid method obtains simultaneously; This method is γ-paddy carbamyl amine synthetic enzyme that above-mentioned purifying obtains to be added the 0.01-0.02mL γ-ratio of paddy carbamyl amine synthetic enzyme in every liter of L-theanine synthetic system join in the test tube that L-theanine synthetic system is housed and react; Be under the condition of 8-9 behind the reaction 10-12h at 30-35 ℃, pH value; Test tube is immersed termination reaction in the boiling water; After being cooled to room temperature, the centrifuging and taking supernatant obtains the L-theanine.Wherein, consisting of of above-mentioned L-theanine synthetic system: 30 mmol/L L-glutamic acid, 150 mmol/L ethylamine hydrochlorides, 15 mmol/L ATP, 30 mmol/L MgCl
2, the 100 mmol/L imidazole buffers of pH7.75, and 0.1mg/mL CTAB.
The present invention starts with from mikrobe γ-paddy carbamyl amine synthetic enzyme enzyme source that synthetic L-theanine is produced in screening; The microorganism strains that utilization filters out is produced γ-paddy carbamyl amine synthetic enzyme; It is added production L-theanine in the synthetic L-theanine system of the enzymatic of optimizing; And judge the enzyme activity height according to the output of the synthetic L-theanine of its enzymatic, obtain best production γ-paddy carbamyl amine synthetic enzyme method.The nearly rose look lock shadow yeast culture condition that the present invention obtains is simple, need not strict control growing environment, and the existing comparatively sophisticated research of yeast coupling ATP fermentation reaction basis, has the excellent development prospect.And utilize that the mikrobe own activity produces have an active enzyme of external theanine synthetase; Under manually operated special conditions; As long as give suitable substrate, energy and suitable ambient conditions; Carry out fermentative prodn, just can obtain L-theanine safe, pollution-free, that have no side effect.Achievement of the present invention can be applicable in the follow-up L-theanine production; On green, free of contamination basis, improve output; Realized safe, easy, efficient, economization production, inspired ferment production the theory and practice foundation is provided for carrying out the extensive microbial enzyme of L-theanine from now on.
Embodiment:
Substratum among the following embodiment is formed as follows, and substratum is formed if no special instructions, is all conventional preparation.
Screening culture medium: 0.25% methylamine hydrochloride, 0.25% methyl alcohol, 0.2 % NaCl, 0.1 % KCl, 0.03 % MgSO
47H
2O, 0.005% KH
2PO
4, 0.005% K
2HPO
4, and 1 * 10
﹣ 7% VB
12, pH7.0-7.4;
The purifying substratum: same screening culture medium, other adds 20g/L agar;
Enrichment medium: same screening culture medium, other adds 0.03% yeast powder;
Potassium phosphate buffer: pH 6.0 contains 10% glycerine, 1mmol/L MgCl
2, and 1mmol/L 2 mercapto ethanol;
The preparation of embodiment 1 bacterial strain of the present invention
(1) strain domestication
From the good tea tree rhizosphere 15-25cm soil of Hunan Inst. of Tea's high bridge experiment tea plantation upgrowth situation, take a sample; The 10g fresh soil sample packed into contain in the triangular flask of 90mL sterilized water; Break up with granulated glass sphere, place the constant temperature shaking table, 25 ℃, the 150 r/min 1h that vibrates; Process soil supension, leave standstill.By 2% volume ratio the soil supension supernatant is added in the screening culture medium; 30 ℃, 150 r/min shaking culture are after 5-14 days, and the substratum (the OD610 value is greater than 0.1) of muddiness is forwarded in the fresh screening culture medium, are cultured to muddiness again; This process 3-5 time repeatedly promptly obtains taming bacterium liquid.
(2) strains separation purifying
To tame bacterium liquid with sterilized water stepwise dilution to 10 successively
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6Six extent of dilution, separate application and are done parallel appearance on flat board, 30 ℃ constant temperature culture 3-7 days.Choose the suitable multiple of colony growth density (10
-4Or 10
-5), select the separation of on the purifying substratum, ruling of single bacterium colony.Cultivate after 3-7 days for 30 ℃, bacterium colony is repeated once by preliminary purification, isolates bacterial strain of the present invention after 3-7 days, and the inclined-plane line is preserved.
The preparation of embodiment 2 γ-paddy carbamyl amine synthetic enzyme
Get the above-mentioned inclined-plane bacterial strain that obtains, picking one ring places the 40mL enrichment medium, and 30 ℃, 150r/min shaking culture 3 days detect the OD value and get final product greater than 0.1.Again with 4 ℃ of enrichment culture liquid, the centrifugal 18min of 12000r/min, supernatant discarded, embathe twice with the phosphate buffered saline buffer of 10 mmol/L pH 6.0 after; Again in 4 ℃, the centrifugal 18min of 12000r/min; Abandon supernatant, then the cell that obtains is resuspended in (not living cell gets final product) in a small amount of damping fluid, carry out ultrasonic cell disintegration (20 kHZ again on ice; 2-3 min), process crude enzyme liquid.
It is 8 that crude enzyme liquid is regulated the pH value, and the ammonium sulfate that under 30 ℃ of conditions, adds 30% saturation ratio is saltoutd, and dissolves to initial volume with the heavy solution weight of albumen precipitation, carries out the SephadexG-75 column chromatography after the ultrafiltration desalination and gets final product.
The above-mentioned product that obtains is adopted the SDS-polyacrylamide gel electrophoresis; Adopt 5% to concentrate glue, 12% separation gel; Contrast with lower molecular weight standard protein (11-72ku); The zymoprotein molecular weight of measuring product is between the 17-26ku, can confirm that the product that purifying obtains is γ-paddy carbamyl amine synthetic enzyme.
The L-theanine is synthesized in embodiment 3 γ-paddy carbamyl amine synthetic enzyme catalysis
γ-paddy carbamyl amine the synthetic enzyme of purifying is added the 0.02mL γ-ratio of paddy carbamyl amine synthetic enzyme in every liter of L-theanine synthetic system to join in the test tube that L-theanine synthetic system is housed and reacts; At 30 ℃, pH value is behind the reaction 12h test tube to be immersed termination reaction in the boiling water under 8 the condition; After being cooled to room temperature, centrifugal 5 min of 12000r/min get supernatant; Obtain the L-theanine ,-20 ℃ of preservations.
Wherein, L-theanine synthetic system is: 30 mmol/L L-glutamic acid, 150 mmol/L ethylamine hydrochlorides, 15 mmol/L ATP, 30 mmol/L MgCl
2, 100 mmol/L imidazole buffers (pH7.75), and 0.1mg/mL CTAB.
Embodiment 4 γ-paddy carbamyl amine synthetic enzyme enzyme activity detects
Use the HPLC performance liquid chromatography that product is detected, weigh relative enzyme activity height through the L-theanine output that detection obtains.HPLC performance liquid chromatography testing conditions: day island proper Tianjin high performance liquid chromatograph, paired ion chromatography.Chromatographic column: KromasilTMC18 (5 μ m, 200 mm * 4.6 mm); Mobile phase A: 0.1% phosphate aqueous solution (adding 10 mol/L SDS); Mobile phase B: acetonitrile; Flow velocity is 1ml/min; Detector: photodiode array detector, detecting wavelength is 200 nm; 32 ℃ of column temperatures; Sample size is l0 μ L.
Detecting the synthetic output of L-theanine through HPLC is 16.90mmol/L, synthesizes L-theanine compared with techniques, result such as following table 1 with existing mikrobe enzymatic:
Table 1 L-theanine output contrast table
Can know that by last table the output of the synthetic L-theanine of the inventive method is far above the synthetic L-theanine technology of existing mikrobe enzymatic.
Claims (10)
1. bacterial strain that produces γ-paddy carbamyl amine synthetic enzyme; This bacterial strain is nearly rose look lock shadow yeast (Sporidiobolus pararoseus) T-C2; Be preserved in Chinese typical culture collection center on June 13rd, 2012, deposit number is CCTCC NO:M2012232.
2. the application of the described bacterial strain of claim 1 in preparation γ-paddy carbamyl amine synthetic enzyme.
3. a fermentation method prepares the method for γ-paddy carbamyl amine synthetic enzyme fermented liquid; It is characterized in that: the fermentation bacterial strain uses therefor is the described bacterial strain of claim 1; The used substratum that ferments is an enrichment medium, and fermentation condition is: inoculum size is 5-10%, and temperature is 28-30 ℃; Rotating speed is 120-180r/min, shaking culture 2-4 days.
4. fermentation method as claimed in claim 3 prepares the method for γ-paddy carbamyl amine synthetic enzyme fermented liquid, it is characterized in that: the consisting of of said enrichment medium: 0.25% methylamine hydrochloride, 0.25 % methyl alcohol, 0.2 % NaCl, 0.1 % KCl, 0.03 % MgSO
47H
2O, 0.005 % KH
2PO
4, 0.005 % K
2HPO
4, 1 * 10
﹣ 7% VB
12And 0.03 % yeast powder, pH7.0-7.4.
5. γ-paddy carbamyl amine synthetic enzyme the fermented liquid that obtains like claim 3 or 4 said methods.
6. the γ that makes by the described fermented liquid of claim 5-paddy carbamyl amine synthetic enzyme crude enzyme liquid.
7. γ as claimed in claim 6-paddy carbamyl amine synthetic enzyme crude enzyme liquid; It is characterized in that: this crude enzyme liquid be with fermented liquid in 4 ℃, the centrifugal 15-20min of 12000 r/min, supernatant discarded, embathe twice with the phosphate buffered saline buffer of 10 mmol/L pH 6.0 after; Again in 4 ℃, the centrifugal 15-20min of 12000 r/min; Supernatant discarded is resuspended in the cell that obtains in a small amount of damping fluid then, and damping fluid is not lived cell and got final product; In carrying out cytoclasis 2-3min with the UW of 20 kHZ on ice, get final product again.
8. utilize claim 6 or 7 described γ-paddy carbamyl amine synthetic enzyme crude enzyme liquid purifying to obtain the method for γ-paddy carbamyl amine synthetic enzyme; It is characterized in that: this method is said crude enzyme liquid to be regulated the pH value be 8-9; The ammonium sulfate that under 30 ℃ of conditions, adds the 30-35% saturation ratio is saltoutd; Dissolve to initial volume with the heavy solution weight of albumen precipitation, after the ultrafiltration desalination, adopt the SephadexG-75 column chromatography to obtain again.
9. utilize the method for the synthetic L-theanine of γ-paddy carbamyl amine synthetic enzyme of the described purifying of claim 8; It is characterized in that: this method is γ-paddy carbamyl amine synthetic enzyme that above-mentioned purifying obtains to be added the 0.01-0.02mL γ-ratio of paddy carbamyl amine synthetic enzyme in every liter of L-theanine synthetic system join in the test tube that L-theanine synthetic system is housed and react; Be under the condition of 8-9 behind the reaction 10-12h at 30-35 ℃, pH value; Test tube is immersed termination reaction in the boiling water, be cooled to room temperature after, the centrifugal 4-6min of 12000 r/min; Get supernatant, obtain the L-theanine.
10. the method for synthetic L-theanine as claimed in claim 9 is characterized in that: said L-theanine synthetic system is: 30 mmol/L L-glutamic acid, 150 mmol/L ethylamine hydrochlorides, 15 mmol/L ATP, 30 mmol/L MgCl
2, the 100 mmol/L imidazole buffers of pH7.75, and 0.1mg/mL CTAB.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111073830A (en) * | 2019-11-29 | 2020-04-28 | 河南巨龙生物工程股份有限公司 | Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine |
| CN114213276A (en) * | 2021-11-18 | 2022-03-22 | 翁源广业清怡食品科技有限公司 | Method for extracting and purifying theanine from enzyme catalytic reaction |
| US11453898B1 (en) | 2021-09-30 | 2022-09-27 | Henan Julong Biological Engineering Co., Ltd | Genetically engineered bacterium of Escherichia coli and method for fermentation production of L-theanine thereof |
| CN115777810A (en) * | 2022-12-07 | 2023-03-14 | 四川农业大学 | Method for producing Tibetan tea by using aspergillus extracellular enzyme preparation |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111073830A (en) * | 2019-11-29 | 2020-04-28 | 河南巨龙生物工程股份有限公司 | Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine |
| CN111073830B (en) * | 2019-11-29 | 2021-07-23 | 河南巨龙生物工程股份有限公司 | Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine |
| US11453898B1 (en) | 2021-09-30 | 2022-09-27 | Henan Julong Biological Engineering Co., Ltd | Genetically engineered bacterium of Escherichia coli and method for fermentation production of L-theanine thereof |
| CN114213276A (en) * | 2021-11-18 | 2022-03-22 | 翁源广业清怡食品科技有限公司 | Method for extracting and purifying theanine from enzyme catalytic reaction |
| CN114213276B (en) * | 2021-11-18 | 2023-09-05 | 翁源广业清怡食品科技有限公司 | Method for extracting and purifying theanine from enzyme catalytic reaction |
| CN115777810A (en) * | 2022-12-07 | 2023-03-14 | 四川农业大学 | Method for producing Tibetan tea by using aspergillus extracellular enzyme preparation |
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