CN104087535A - Method for preparing theanine by biological process - Google Patents
Method for preparing theanine by biological process Download PDFInfo
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- CN104087535A CN104087535A CN201410325370.2A CN201410325370A CN104087535A CN 104087535 A CN104087535 A CN 104087535A CN 201410325370 A CN201410325370 A CN 201410325370A CN 104087535 A CN104087535 A CN 104087535A
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- Prior art keywords
- theanine
- pseudomonas nitroreducens
- strain
- enzyme
- glutamine
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- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 title claims abstract description 85
- 229940026510 theanine Drugs 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000031018 biological processes and functions Effects 0.000 title abstract 2
- 241000204735 Pseudomonas nitroreducens Species 0.000 claims abstract description 21
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 18
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 claims abstract description 9
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- 229930182816 L-glutamine Natural products 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 238000006911 enzymatic reaction Methods 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 6
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 229940073490 sodium glutamate Drugs 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- XWBDWHCCBGMXKG-UHFFFAOYSA-N ethanamine;hydron;chloride Chemical compound Cl.CCN XWBDWHCCBGMXKG-UHFFFAOYSA-N 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 11
- 230000010307 cell transformation Effects 0.000 abstract description 3
- 102000009127 Glutaminase Human genes 0.000 abstract description 2
- 108010073324 Glutaminase Proteins 0.000 abstract description 2
- 238000000855 fermentation Methods 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- GLZPCOQZEFWAFX-UHFFFAOYSA-N Geraniol Chemical compound CC(C)=CCCC(C)=CCO GLZPCOQZEFWAFX-UHFFFAOYSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- 229960002743 glutamine Drugs 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 description 1
- 239000005792 Geraniol Substances 0.000 description 1
- GLZPCOQZEFWAFX-YFHOEESVSA-N Geraniol Natural products CC(C)=CCC\C(C)=C/CO GLZPCOQZEFWAFX-YFHOEESVSA-N 0.000 description 1
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- DATAGRPVKZEWHA-UHFFFAOYSA-N L-gamma-glutamyl-n-ethylamine Natural products CCNC(=O)CCC(N)C(O)=O DATAGRPVKZEWHA-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229960001040 ammonium chloride Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 229940044197 ammonium sulfate Drugs 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 229940113087 geraniol Drugs 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000000324 neuroprotective effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- -1 semi-lactosi Chemical compound 0.000 description 1
- 229940001516 sodium nitrate Drugs 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for preparing theanine by a biological process, which comprises the following steps: preparing cells by using Pseudomonas nitroreducens NTLC4.002 with the collection number of CCTCC NO:M2014254 as the initial strain, and preparing natural theanine by cell transformation. The novel theanine strain applicable to theanine production is separated and screened from the Chinese traditional fermentation product; and the glutaminase generated by the strain can be used for efficiently producing theanine by acting on glutamine and ethylamine under the conditions of pH 9-10 and 35-40 DEG C, wherein the conversion rate can reach more than 80%.
Description
Technical field
The present invention relates to a kind of method of Biological preparation theanine, relate in particular to the Pseudomonas nitroreducens strain that a strain can be used for High-efficient Production theanine.
Background technology
Theanine (L-theanine) is the distinctive seed amino acid of tealeaves, and its content directly determines the quality of tealeaves.In addition, theanine also there is hypotensive, antitumor, neuroprotective simultaneously, loosen, the effect such as antidepressant, antifatigue, raising immunizing power, ameliorate body fat.The different physiological roles of L-thiamine and satisfactory stability and security, make it aspect food, healthcare products, have demand and application widely.
Thus, people have attempted the method for multiple manufacture theanine all the time, mainly contain at present three kinds of manufacture method.The first, be the method for extracting from tealeaves.But because the theanine content in stem tea only has 1.5% left and right, and photosynthesis can make a theanine loss part, causes output very low.Invent subsequently the method for chemical organic synthesis theanine.But this method may residual toxicity material, and resultant comprises D, L-type theanine isomer, need to split and just can obtain L-type product, makes separation and purification complicated operation.The third manufacture method is to utilize microbial enzyme method to produce.Report at present the method for microbe-derived L-Glutamine deaminase Production by Enzymes theanine, as from Pseudomonas and 2005 disclosed from geraniol pseudomonas GEA and 2007 disclosed from the one kind or two or more L-Glutamine deaminase in bacillus, mould or yeast, within 2008, disclose a kind of utilization and produced the method for theanine from the glutamyltranspeptidase of subtilis, the method is safe and reliable, need to prepare enzyme powder and may use organic solvent but exist, causing the problems such as industrial production complicated operation.Have no at present any utilization and carry out the report of cell transformation production theanine from Pseudomonas nitroreducens.Utilize and prepare theanine from the L-Glutamine deaminase enzyme process of Pseudomonas nitroreducens, preparation method is simple in enzyme source, because this enzyme belongs to intracellular enzyme, can directly utilize the immobilization of cell transformation and thalline or enzyme to carry out the biology preparation of theanine, having enzyme-to-substrate easily separates, the high exploitation advantage of converted product purity, and the substrate conversion that can carry out high density is prepared theanine.
Summary of the invention
The present invention is the invention completing in view of the above problems, and its object is to provide the method for simple to operate and effective Biological preparation theanine.
Technical solution of the present invention is:
A bacterial strain that produces L-Glutamine deaminase, is characterized in that: its Classification And Nomenclature is Pseudomonas nitroreducens (Pseudomonas nitroreducens) NTLC4.002, deposit number: CCTCC NO:M2014254.
A method for Biological preparation theanine, is characterized in that: comprise the following steps:
(1) prepare cell:
Using the Pseudomonas nitroreducens NTLC4.002 that deposit number is CCTCC NO:M2014254 is starting strain;
Substratum, counts with g/L: glucose 10-18, yeast extract 0.7-1.2, Sodium Glutamate 9-15, MgSO47H2O 0.45-1, K2HPO4 0.45-1, KH2PO4 0.45-1, EDTA-Fe 0.06-0.11, pH 6.7-7.2; 35-38 DEG C, cultivates 16 h under the condition of 200 rpm; Frozen centrifugation, obtains thalline;
(2) cell conversion process is prepared natural theanine:
By the cell of preparation 50～100mM Na2B4O7-NaOH sodium borate buffer liquid that to be placed in containing the pH of 0. 2～0. 4M glutamine and 1.5～2.5M ethamine be 9～10, at 35～40 DEG C, carry out the enzyme reaction of 2～8 hours, make theanine.
Beneficial effect of the present invention: contriver etc. are in order to address the above problem, from Chinese traditional zymotic goods, separation screening obtains novel can be used for of a strain and produces the bacterial strain of theanine, the L-Glutamine deaminase that it produces, under pH9～10, the condition of 35～40 DEG C, by acting on glutamine and ethamine, can produce efficiently theanine, transformation efficiency can reach more than 80%.
According to the present invention, the novel method of efficient manufacture theanine can be provided, can make the production of theanine simple, and be beneficial to suitability for industrialized production.
Biological material specimens preservation
It is that the inventor waits the novel strain of finding first qualification that above-mentioned theanine is produced bacterium, and Classification And Nomenclature is Pseudomonas nitroreducens NTLC4.002, Latin name:
pseudomonas nitroreducens; Depositary institution: Chinese Typical Representative culture collection center, address: Wuhan, China university, preservation date: on June 13rd, 2014, deposit number: CCTCC NO:M2014254.
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 represents to utilize the Pseudomonas nitroreducens growth curve that claims dry weight method and turbidimetry for Determination.
Fig. 2 is the chart that represents the theanine content detecting by high performance liquid chromatography.
Embodiment
Describe embodiments of the present invention in detail, but technical scope of the present invention is not limited to following embodiment, is not changing under the prerequisite of its main points, can make various changes and implement below.In addition, technical scope of the present invention prolongs and impartial scope.
Theanine of the present invention refers to theanine, is the distinctive seed amino acid of tealeaves, is effective taste compound in green tea, can be used as quality, flavor-improving agent and function food additive.
The 1-ethanamine derivatives using in the present invention, can enumerate ethamine, ethylamine hydrochloride etc.
The present invention Pseudomonas nitroreducens NTLC4.002 used waits by the inventor the new bacterial strain of finding, is to have the theanine production bacterium that produces L-Glutamine deaminase.In addition, the Pseudomonas nitroreducens NTLC4.002 qualification that the present invention is used, is to draw by learning character, the analysis of biochemical property and the mensuration of 16s RNA sequence according to the mushroom of certain rule.
L-Glutamine deaminase of the present invention is the enzyme from Pseudomonas nitroreducens NTLC4.002.This enzyme, under alkaline condition, has transfer vigor, can catalysis glutamyl shift reaction.The enzymic activity source of this reaction is present in thalline, can be directly by centrifugal acquisition thalline, i.e. enzyme source.
Glutaminase active in the present invention, can transfer to glutamyl on oxammonium hydrochloride by this enzyme, quantitative generated Pidolidone-γ-mono-hydroximic acid and measuring.The unit definition of this enzyme is the needed enzyme amount of Pidolidone-γ-mono-hydroximic acid that per minute generates 1 μ mol.
Embodiment 1 produces the separation of glutamy enzyme bacterium
Get a little soil to be separated and join (in g/L) in isolation medium: glucose 15, yeast extract paste 12, corn steep liquor 10, MgSO47H2O 0. 5, K2HPO41, pH7.0.At 37 DEG C, under the condition of 200rpm, cultivate 24h, after dilution spread, obtain pure growth, cultivate 30h for 37 DEG C, obtain 23 strain enzymes higher bacterial strain alive, the bacterial strain that finally a selected strain enzyme work can reach 0.2U/mL is as experimental strain.
Embodiment 2 obtains thalline
The Pseudomonas nitroreducens SK16.004 that uses embodiment 1 to obtain, in fermention medium (in g/L): glucose 12, yeast extract 0.9, Sodium Glutamate 12, MgSO47H2O 0.5, K2HPO4 0.6, KH2PO4 0.6, EDTA-Fe 0.1, pH 6.8-7.0,35-37 DEG C, cultivates 16 h under the condition of 200 rpm; Frozen centrifugation, obtains thalline, is enzyme source.
Embodiment 3 enzyme reactions
Use the enzyme source of embodiment 2, containing in the 50mM sodium borate buffer liquid (Na2B4O7-NaOH, pH10) of glutamine 0.3M, ethamine (or ethylamine hydrochloride) 1.5M, at 37 DEG C, carry out the enzyme reaction of 2～8 hours, synthesized theanine.
The mensuration of embodiment 4 theanine output
The enzyme reaction solution carrying out in embodiment 3, through TCA precipitation, dilution, by Agilent liquid chromatograph, is carried out quantitatively theanine, glutamine, L-glutamic acid respectively.Analysis condition is as follows: instrument model: Ag1260, chromatographic column: C18, column temperature: 40 DEG C.
The optimization of embodiment 5 Pseudomonas nitroreducens NTLC4.002 culture condition
The inventor etc. have studied the cultivation of the substratum Pseudomonas nitroreducens NTLC4.002 beyond the substratum in use embodiment 1.Having studied after the carbon sources such as glucose, fructose, semi-lactosi, maltose, sucrose and lactose, found that, while using glucose, fermented liquid supernatant liquid enzyme is lived the highest.Having studied after ammonium chloride, ammonium sulfate, SODIUMNITRATE, glutamine, Sodium Glutamate, yeast extract, beef extract and peptone nitrogenous source, found that, use yeast extract can reach best effect.Studied glucose, yeast extract, Sodium Glutamate optimum concn than rear discovery, glucose, yeast extract, Sodium Glutamate are respectively under the condition of 10～18 g/L, 0.7～1.2 g/L, 9～15g/L simultaneously, can obtain enzyme higher fermented liquid alive.
Embodiment 6 uses the optimization from the L-Glutamine deaminase enzyme reaction condition of Pseudomonas nitroreducens NTLC4.002.According to the enzyme reaction condition in embodiment 3, study the suitableeest enzyme reaction condition of L-Glutamine deaminase from Pseudomonas nitroreducens NTLC4.002 that uses, find to use 0.3M glutamine and 1.5M ethamine, at 37 DEG C, under pH10 condition, react the theanine that can obtain maximum yield for 2～8 hours.
Claims (2)
1. a bacterial strain that produces L-Glutamine deaminase, is characterized in that: its Classification And Nomenclature is Pseudomonas nitroreducens (Pseudomonas nitroreducens) NTLC4.002, deposit number: CCTCC NO:M2014254.
2. a method for Biological preparation theanine, is characterized in that: comprise the following steps:
(1) prepare cell:
Using the Pseudomonas nitroreducens NTLC4.002 that deposit number is CCTCC NO:M2014254 is starting strain;
Substratum, counts with g/L: glucose 10-18, yeast extract 0.7-1.2, Sodium Glutamate 9-15, MgSO47H2O 0.45-1, K2HPO4 0.45-1, KH2PO4 0.45-1, EDTA-Fe 0.06-0.11, pH 6.7-7.2; 35-38 DEG C, cultivates 16 h under the condition of 200 rpm; Frozen centrifugation, obtains thalline;
(2) cell conversion process is prepared natural theanine:
By the cell of preparation 50～100mM Na2B4O7-NaOH sodium borate buffer liquid that to be placed in containing the pH of 0. 2～0. 4M glutamine and 1.5～2.5M ethamine or ethylamine hydrochloride be 9～10, at 35～40 DEG C, carry out the enzyme reaction of 2～8 hours, make theanine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561160A (en) * | 2014-12-22 | 2015-04-29 | 南通励成生物工程有限公司 | Method for preparing theanine by using biological method |
| CN104830941A (en) * | 2015-04-29 | 2015-08-12 | 宁夏诚志万胜生物工程有限公司 | Microbial conversion method utilizing mixed bacteria to efficiently synthesize L-theanine |
| CN107164418A (en) * | 2017-06-14 | 2017-09-15 | 浙江中医药大学 | A kind of method that Permeabilized cells prepare 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1688705A (en) * | 2002-08-06 | 2005-10-26 | 太阳化学株式会社 | Process for producing theanine |
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| CN1688705A (en) * | 2002-08-06 | 2005-10-26 | 太阳化学株式会社 | Process for producing theanine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561160A (en) * | 2014-12-22 | 2015-04-29 | 南通励成生物工程有限公司 | Method for preparing theanine by using biological method |
| CN104830941A (en) * | 2015-04-29 | 2015-08-12 | 宁夏诚志万胜生物工程有限公司 | Microbial conversion method utilizing mixed bacteria to efficiently synthesize L-theanine |
| CN107164418A (en) * | 2017-06-14 | 2017-09-15 | 浙江中医药大学 | A kind of method that Permeabilized cells prepare 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 |
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| Publication number | Publication date |
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| CN104087535B (en) | 2017-01-11 |
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