CN101760499A - Enzymatic production method of theanine - Google Patents
Enzymatic production method of theanine Download PDFInfo
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- CN101760499A CN101760499A CN200810121716A CN200810121716A CN101760499A CN 101760499 A CN101760499 A CN 101760499A CN 200810121716 A CN200810121716 A CN 200810121716A CN 200810121716 A CN200810121716 A CN 200810121716A CN 101760499 A CN101760499 A CN 101760499A
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Abstract
The invention discloses an enzymatic production method of theanine. The method comprises the following steps of: acetylating chemically synthesized DL-theanine as a raw material; screening cunninghamella echinulata strains from soil; mutagenizing the cunninghamella echinulata strains by ultraviolet rays to obtain amino-acylase strains; and utilizing the obtained high-yield amino-acylase strains to three-dimensionally and purposely acetylate so as to decompose DL-amino acid and prepare L- theanine. Compared with other methods, the method has the advantages of low production cost, mild reaction conditions, higher optical purity, simple production process, low cost, and the like, is extremely easy to operate and has favorable industrial application prospects.
Description
Technical field
The invention relates to the Production by Enzymes of theanine, belong to biological technical field.
Background technology
The very long record of drinking tea is arranged on the Chinese history, and the custom of drinking tea in a lot of places in the world passes in the past from China.Chinese famous tea is enjoyed a very good reputation in the world.The discovery the earliest and the utilization of tea are from medicinal.Tea the Englishman is: the liquid of health, the drink of soul; Be described as " state's drink " in China.Modern science studies confirm that in a large number, tealeaves contains and the closely-related biochemical composition of HUMAN HEALTH really, tealeaves not only has to refresh oneself and clears away heart-fire, clearing away summer heat, eliminates indigestion and phlegm, goes greasy fat-reducing, the relieving restlessness that clears away heart-fire, detoxifcation to sober up, promote the production of body fluid to quench thirst, fall that fire makes eye bright, only pharmacological action such as dysentery dehumidifying, also to modern disease, as diseases such as Radiation sickness, cardiovascular and cerebrovascular disease, cancers, certain pharmacological effect is arranged.As seen, more than the tealeaves pharmacological effect, acting on extensively, is that Other Drinks can not be substituted.
The major ingredient that tealeaves has pharmacological action is tea-polyphenol, trimethyl-xanthine, lipopolysaccharides etc.Concrete effect has: 1. help to delay senility.2, help to suppress cardiovascular disorder.3, help prevention and anticancer.4, help prevention and treatment radiation injury.5, help to suppress and resist viral bacterium.6, help beauty and skin care.7, help refreshment.8, help diuresis to recover from fatigue.9, help lipopenicillinase aid digestion.10, helping to protect tooth makes eye bright.Tea set has so many effect, so tea leaf extract is the object of investigator's primary study and concern always.
Theanine (L-Theanine) systematic naming method is: N-ethyl-L glutamine, be nineteen fifty the wine family more two doctors Lang in the beautiful process of revealing the bright refreshing flavour composition of tea of research, the seed amino acid that does not participate in constitutive protein matter that isolation identification goes out from young sprout is the feature amino acid of tealeaves.It accounts for 40%～60% of total free aminoacids in green tea, be the maximum amino acid of content in the tealeaves, and content is abundant in senior green tea, newly picked and processed tea leaves, accounts for about 1%～2% of dried tea content.
Though China occupy prostatitis, the world in the production of tealeaves and consumption, the research of theanine is relatively lagged behind.Japan's research starting in this respect is outstanding than morning and contribution.Why theanine can be used widely in food, and its reason mainly is because the improvement that theanine has good security, stability and theanine production technology.At present, the preparation method of theanine mainly is by chemosynthesis, groups of cells training, microbial fermentation, and the method for merely extracting theanine from tealeaves or its pregnant solution.
Utilize the catechin in the low-grade green tea extraction tealeaves, its waste is used to produce the main method that theanine is current direct extraction separation theanine. and chromatographic technique enrichment theanine has replaced traditional reagent precipitator method fully at present. and ion exchange adsorption enrichment theanine is to utilize theanine can carry out separation and purification with this fundamental characteristics of acidic cation-exchange resin exchange absorption under acidic conditions.
The chemical synthesis of theanine mainly is L-glutamic acid-ethamine synthesis method at present: L-glutamic acid is at room temperature dewatered is converted into the cyclic lactames structure, controls certain condition then, ethamine is combined with the L-glutamic acid that changes into ring texture form theanine.Adopt the chemosynthesis means to prepare theanine and have that price is low, cost is low, be suitable for industrialized characteristics.But the chemosynthesis manufacturing all is DL-type raceme, need split and just can obtain L-type product.
In recent years,, utilize microbial fermentation to produce enzyme, and then the production that utilizes the extract of enzyme to carry out object more and more is widely used along with development of biology and application.Compare with chemical synthesis, the microbe fermentation method of producing theanine has diverse characteristic.Because the height specificity that enzymatic reaction had can be converted into needed product fast with reaction substrate, and the transformation efficiency height, by product is few, and is also little to the pollution of environment, is a kind of method that is worthy to be popularized and studies.
Summary of the invention
One of purpose of the present invention has provided a kind of enzymatic production method of theanine.
Two of purpose of the present invention is the mould that has obtained a kind of high yield L-Aminoacylase from screening, i.e. cunninghamella echinulata bacterium.The L-Aminoacylase that this bacterium produced can be widely used among the deacetylation.
Technical solution of the present invention is as follows:
1. the screening and culturing of cunninghamella echinulata bacteria strain
Behind the sample natural air drying that picks up from soil, pulverize with mortar, carry out lock out operation according to gradient dilution method and spread plate method.Coated flat board places incubator to cultivate, treat that bacterium colony grows after, observe with uv analyzer, Analysis and Identification goes out to sting the little Ke Yinhan bacterium of spore.
This bacterium colony of picking is connected to fresh solid plate, with the sterilized water wash-out of the bacterial strain on the flat board, is connected to jolting cultivation in the nutrient solution.Get the above-mentioned seed liquor that is diluted to 10ml and be divided into the 2ml small test tube of packing into, under dark condition, carry out uviolizing and handle, be inverted for 28 ℃-30 ℃ and cultivate 1-3 days to there being single bacterium colony to occur.The single preferably bacterium colony of picking growing way carries out shake flask fermentation repeatedly and sieves again.Sieve again by fermentation, we are transferred to the test tube slant substratum with the high bacterial strain of yield of enzyme that obtains, and cultivate 28h-36h and cover with mycelia to the test tube slant in 28 ℃ of constant incubators.And then be made into the cultivation of spore suspension shaking table, collect the mycelium pellet that obtains.
2.DL-the acetylize of theanine
Add the DL-theanine in basic solution, stirring and dissolving drips acetic anhydride, and the PH that adds alkali control solution simultaneously is a weakly alkaline, uses the ice bath controlled temperature during reaction, with triketohydrindene hydrate color reaction control reaction end.Regulating PH then is strongly-acid, and decompression, desalination add acetone again and separate out N-acetyl-DL-theanine.
3. enzyme process splits the separation and purification of DL-theanine and resolved product
Preparation N-acetyl-DL-theanine solution, it is neutral regulating pH value, adds in the wet thallus placement constant temperature oscillator to transform.Filter, regulate PH, decolouring.It is neutral with distillation washing post then that the cation exchange resin column of JK008 is processed into alkalescence, and the feed liquid upper prop is collected the effluent liquid of L-theanine, and regulating PH be acidity, and condensing crystal obtains the L-theanine.Unconverted N-acetyl-DL-theanine elutes with distilled water, proceeds after treatment to split.
The present invention is a raw material with chemical industry synthetic raw material DL-theanine; through after the acetylize; the high yield L-Aminoacylase bacterial strain stereospecificity ground deacetylation that the cunninghamella echinulata bacterium that utilization screens from soil obtains through ultraviolet mutagenesis prepares the L-theanine thereby split the DL-propylhomoserin.Compare with other method, this method has advantages such as production cost is low, reaction conditions is gentle, optical purity is higher, production technique is simple, with low cost, very easily in operation, has favorable industrial application prospect.
Claims (5)
1. the Production by Enzymes of theanine is characterized in that may further comprise the steps:
(1) screening and culturing of cunninghamella echinulata bacteria strain
Behind the sample natural air drying that picks up from soil, pulverize with mortar, carry out lock out operation according to gradient dilution method and spread plate method.Coated flat board places incubator to cultivate, treat that bacterium colony grows after, observe with uv analyzer, Analysis and Identification goes out to sting the little Ke Yinhan bacterium of spore.This bacterium colony of picking is connected to fresh solid plate, with the sterilized water wash-out of the bacterial strain on the flat board, is connected to jolting cultivation in the nutrient solution.Get the above-mentioned seed liquor that is diluted to 10ml and be divided into the 2ml small test tube of packing into, under dark condition, carry out uviolizing and handle, be inverted for 28 ℃-30 ℃ and cultivate 1-3 days to there being single bacterium colony to occur.The single preferably bacterium colony of picking growing way carries out shake flask fermentation repeatedly and sieves again.
Sieve again by fermentation, we are transferred to the test tube slant substratum with the high bacterial strain of yield of enzyme that obtains, and cultivate 28h-36h and cover with mycelia to the test tube slant in 28 ℃ of constant incubators.And then be made into the cultivation of spore suspension shaking table, collect the mycelium pellet that obtains.
(2) acetylize of DL-theanine
Add the DL-theanine in basic solution, stirring and dissolving drips acetic anhydride, and the PH that adds alkali control solution simultaneously is a weakly alkaline, uses the ice bath controlled temperature during reaction, with triketohydrindene hydrate color reaction control reaction end.Regulating PH then is strongly-acid, and decompression, desalination add acetone again and separate out N-acetyl-DL-theanine.
(3) enzyme process splits the separation and purification of DL-theanine and resolved product
Preparation N-acetyl-DL-theanine solution, it is neutral regulating pH value, adds in the wet thallus placement constant temperature oscillator to transform.Filter, regulate PH, decolouring.It is neutral with distillation washing post then that the cation exchange resin column of JK008 is processed into alkalescence, and the feed liquid upper prop is collected the effluent liquid of L-theanine, and regulating PH be acidity, and condensing crystal obtains the L-theanine.Unconverted N-acetyl-DL-theanine elutes with distilled water, proceeds after treatment to split.
2. go out to sting the little Ke Yinhan bacterium of spore according to said Analysis and Identification in the claim (1), little Ke Yinhan bacterium is characterised in that its thorn spore: bacterium colony just is white in color, back canescence.When a large amount of conidiums formed, it is Powdered that the surface is.Optimum growth temperature is the 28-30 degree.The branch of mycelia is in great numbers, about 12 microns of diameter.The two bifurcated branches of mitogenetic sporophore sympodium or plan, the top forms a vesicle, subglobular.Stigma is being given birth on the vesicle surface, forms conidium on the stigma, above the about 4 microns spinule of dense growth, colourless.
3. be connected in the nutrient solution jolting and cultivate according to said in the claim (1), it is characterized in that being connected in the nutrient solution 28 ℃ of-30 ℃ of joltings (120r/min) by 5% inoculum size cultivates 24h.
4. carry out uviolizing handle under dark condition according to said in the claim (1), it is characterized in that the ultraviolet lamp with 15W, fixed distance is about 30 centimetres, and irradiation time is 1 minute.
5. according to transforming in the said adding wet thallus placement constant temperature oscillator in the claim (3), it is characterized in that invert point is 50 ℃, oscillation frequency is 130r/min, and transformation time is 30h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810121716A CN101760499A (en) | 2008-10-23 | 2008-10-23 | Enzymatic production method of theanine |
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| CN200810121716A CN101760499A (en) | 2008-10-23 | 2008-10-23 | Enzymatic production method of theanine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020124351A1 (en) * | 2018-12-18 | 2020-06-25 | 黄华成 | Application of glutamine derivative in preparation of animal feed additive |
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- 2008-10-23 CN CN200810121716A patent/CN101760499A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020124351A1 (en) * | 2018-12-18 | 2020-06-25 | 黄华成 | Application of glutamine derivative in preparation of animal feed additive |
| US11964929B2 (en) | 2018-12-18 | 2024-04-23 | Wisorig Technologies Pte. Limited | Application of glutamine derivative in preparation of animal feed additive |
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Application publication date: 20100630 |