CN104087535B - Method for preparing theanine by biological process - Google Patents
Method for preparing theanine by biological process Download PDFInfo
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- CN104087535B CN104087535B CN201410325370.2A CN201410325370A CN104087535B CN 104087535 B CN104087535 B CN 104087535B CN 201410325370 A CN201410325370 A CN 201410325370A CN 104087535 B CN104087535 B CN 104087535B
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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 79
- 229940026510 theanine Drugs 0.000 title claims abstract description 38
- 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 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 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 10
- 108060008539 Transglutaminase Proteins 0.000 claims description 10
- 102000003601 transglutaminase Human genes 0.000 claims description 10
- 239000002253 acid Substances 0.000 claims description 8
- 238000006911 enzymatic reaction Methods 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
- 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 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 239000011734 sodium Substances 0.000 claims description 6
- 241000589516 Pseudomonas Species 0.000 claims description 5
- 229910021529 ammonia Inorganic materials 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000011953 bioanalysis Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 229910052564 epsomite Inorganic materials 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 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
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- 150000001412 amines Chemical class 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 abstract description 3
- 230000010307 cell transformation Effects 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 3
- 230000004151 fermentation Effects 0.000 abstract description 3
- 102000009127 Glutaminase Human genes 0.000 abstract description 2
- 108010073324 Glutaminase Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 20
- 108090000790 Enzymes Proteins 0.000 description 20
- 244000269722 Thea sinensis Species 0.000 description 6
- 235000013616 tea Nutrition 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 3
- 235000013923 monosodium glutamate Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229940073490 sodium glutamate Drugs 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-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
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- QMVPMAAFGQKVCJ-UHFFFAOYSA-N citronellol Chemical compound OCCC(C)CCC=C(C)C QMVPMAAFGQKVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229960001031 glucose Drugs 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004321 preservation 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
- QMVPMAAFGQKVCJ-SNVBAGLBSA-N (R)-(+)-citronellol Natural products OCC[C@H](C)CCC=C(C)C QMVPMAAFGQKVCJ-SNVBAGLBSA-N 0.000 description 1
- 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
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 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
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 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
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia 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
- 210000000577 adipose tissue Anatomy 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-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
- 235000019270 ammonium chloride Nutrition 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
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001430 anti-depressive effect 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
- 230000009286 beneficial effect Effects 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
- JGQFVRIQXUFPAH-UHFFFAOYSA-N beta-citronellol Natural products OCCC(C)CCCC(C)=C JGQFVRIQXUFPAH-UHFFFAOYSA-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
- 230000036772 blood pressure Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000000484 citronellol Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- -1 ethamine hydrochloric acid Salt Chemical class 0.000 description 1
- XWBDWHCCBGMXKG-UHFFFAOYSA-N ethanamine;hydron;chloride Chemical compound Cl.CCN XWBDWHCCBGMXKG-UHFFFAOYSA-N 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 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
- 229930182830 galactose Natural products 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 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
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000582 semen Anatomy 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
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000006276 transfer reaction Methods 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
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 that bioanalysis prepares theanine, particularly relate to a strain and can be used for
Efficiently produce the Pseudomonas nitroreducens strain of theanine.
Background technology
Theanine (L-theanine) is the distinctive a kind of aminoacid of Folium Camelliae sinensis, and its content directly determines tea
The quality of leaf.Additionally, theanine has blood pressure lowering, antitumor, protection nerve, puts the most simultaneously
Pine, antidepressant, resisting fatigue, raising immunity, alleviate the effect such as body fat.L-thiamine is many
Plant physiological function and good stability and safety so that it is have in terms of food, health product
Demand and application widely.
Thus, the method that people have attempted multiple manufacture theanine all the time, currently mainly have
Three kinds of manufacture methods.First, it is the method extracted from Folium Camelliae sinensis.But due to the tea in stem tea
Histidine content only has about 1.5%, and photosynthesis can make a theanine loss part, causes
Yield is the lowest.The method having invented chemical organic synthesis theanine subsequently.But this method may be residual
Stay toxicant, and product includes D, L-type theanine isomer, need to carry out splitting
L-type product can be obtained so that separation and purification operation complexity.The third manufacture method, is to utilize
Microbial enzyme method produces.Report microbe-derived transglutaminase Production by Enzymes at present
The method of theanine, as from disclosed in Pseudomonas and 2005 from citronellol
From in bacillus, mycete or yeast disclosed in pseudomonas GEA and 2007
One kind or two or more transglutaminase, discloses a kind of utilization from hay bud for 2008
The glutamyl transpeptidase of spore bacillus produces the method for theanine, and the method is safe and reliable, but exists
And need prepare enzyme powder and organic solvent may be used, cause industrial process operation complexity etc. to ask
Topic.Have no that any utilization carries out cell transformation from Pseudomonas nitroreducens and produces tea ammonia at present
The report of acid.The transglutaminase enzyme process from Pseudomonas nitroreducens is utilized to prepare tea ammonia
Acid, enzyme source preparation method is simple, owing to this enzyme belongs to endocellular enzyme, can directly utilize cell transformation
And thalline or enzyme immobilizatio carry out the biological preparation of theanine, there is enzyme-to-substrate and easily divide
From exploitation advantages such as, converted product purity are high, and the substrate that can carry out high concentration is converted preparation
Theanine.
Summary of the invention
The present invention is the invention completed in view of the above problems, its object is to provide simple to operate
And the method that effectively bioanalysis prepares theanine.
The technical solution of the present invention is:
A kind of bacterial strain producing transglutaminase, is characterized in that: its Classification And Nomenclature is nitro reduction
Pseudomonas (Pseudomonas nitroreducens) NTLC4.002, deposit number: CCTCC
NO:M2014254.
A kind of bioanalysis prepares the method for theanine, it is characterized in that: comprise the following steps:
(1) cell is prepared:
The Pseudomonas nitroreducens using deposit number to be CCTCC NO:M2014254
NTLC4.002 is starting strain；
Culture medium, is calculated as with g/L: glucose 10-18, yeast extract 0.7-1.2, paddy ammonia
Acid sodium 9-15, MgSO4·7H2O 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, under conditions of 200rpm, cultivate 16h；
Frozen centrifugation, it is thus achieved that thalline；
(2) cell conversion process prepares natural theanine:
The cell of preparation is placed in containing 0.2～0.4M glutamine and 1.5～2.5M ethamine
PH is the 50～100mM Na of 9～102B4O7In-NaOH sodium borate buffer liquid, 35～
Carry out the enzyme reaction of 2～8 hours at 40 DEG C, prepare theanine.
Beneficial effects of the present invention: inventor etc., in order to solve the problems referred to above, send out from Chinese tradition
In ferment goods, separation screening obtains the bacterial strain that can be used for producing theanine that a strain is novel, and it is produced
Transglutaminase, at pH9～10, under conditions of 35～40 DEG C, by acting on glutamy
Amine and ethamine, can produce theanine efficiently, and conversion ratio is up to more than 80%.
According to the present invention, it is provided that the efficient new method manufacturing theanine, theanine can be made
Produce simple, and beneficially industrialized production.
Biological material specimens preservation
It is that the present inventor waits the novel strain finding first to identify, classification that above-mentioned theanine produces bacterium
Named Pseudomonas nitroreducens NTLC4.002, Latin name: Pseudomonas
nitroreducens；Depositary institution: China typical culture collection center, address: China is military
Chinese university, preservation date: on June 13rd, 2014, deposit number: CCTCC NO:
M2014254。
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is to represent to utilize the Pseudomonas nitroreducens claiming dry weight method and turbidimetry for Determination raw
Long curve.
Fig. 2 is the chart representing the theanine content detected by high performance liquid chromatography.
Detailed description of the invention
Below, describe embodiments of the present invention in detail, but the technical scope of the present invention is unrestricted
In following embodiment, on the premise of not changing its main points, various change can be made and implement.
It addition, the technical scope of the present invention extends to the scope of equalization.
The theanine of the present invention refers to theanine, is the distinctive a kind of ammonia of Folium Camelliae sinensis
Base acid, is effective taste compound in green tea, can serve as quality, flavor improving agent and function
Food additive.
The 1-ethanamine derivatives used in the present invention, can enumerate ethamine, ethylamine hydrochloride etc..
Pseudomonas nitroreducens NTLC4.002 used by the present invention is to be found by the present inventor etc.
New strains, be have produce transglutaminase theanine produce bacterium.It addition, institute of the present invention
Pseudomonas nitroreducens NTLC4.002 identify, be by the mushroom according to certain rule
Learn character, the analysis of biochemical property and the mensuration of 16s RNA sequence and draw.
The transglutaminase of the present invention is the enzyme from Pseudomonas nitroreducens NTLC4.002.
This enzyme in the basic conditions, has transfer vigor, can be catalyzed glutamyl transfer reaction.Should
The enzymatic activity source of reaction is present in thalline, directly can obtain thalline, i.e. enzyme source by centrifugal.
Glutaminase active in the present invention, can transfer to salt by glutamyl by this enzyme
Acid azanol on, the Pidolidone-γ quantitatively generated-mono-hydroximic acid and measure.The unit of this enzyme is fixed
Justice is the enzyme amount required for Pidolidone-γ-mono-hydroximic acid of generation 1 μm ol per minute.
Embodiment 1 produces the separation of glutamy enzyme bacterium
Take a little soil to be separated and join in isolation medium (in terms of g/L): glucose 15,
Yeast extract 12, Semen Maydis pulp 10, MgSO4·7H2O 0.5、K2HPO41, pH7.0.At 37 DEG C,
24h, after dilution spread, it is thus achieved that pure culture, 37 DEG C of trainings is cultivated under conditions of 200rpm
Support 30h, obtain the bacterial strain that 23 strain enzymes work are higher, finally select a strain enzyme work and can reach 0.2U/mL
Bacterial strain as experimental strain.
Embodiment 2 obtains thalline
Use the Pseudomonas nitroreducens SK16.004 obtained by embodiment 1, in fermentation culture
In base (in terms of g/L): glucose 12, yeast extract 0.9, sodium glutamate 12, MgSO4·7H2O
0.5、K2HPO4 0.6、KH2PO40.6, EDTA-Fe 0.1, pH 6.8-7.0,35-37 DEG C,
16h is cultivated under conditions of 200rpm；Frozen centrifugation, it is thus achieved that thalline, is enzyme source.
Embodiment 3 enzyme reaction
Use the enzyme source of embodiment 2, containing glutamine 0.3M, ethamine (or ethamine hydrochloric acid
Salt) the 50mM sodium borate buffer liquid (Na of 1.5M2B4O7-NaOH, pH10) in, at 37 DEG C
Under carry out the enzyme reaction of 2～8 hours, synthesized theanine.
The mensuration of embodiment 4 theanine yield
By the enzyme reaction solution carried out in embodiment 3 through TCA precipitation, dilute, by Agilent liquid
Chromatography, is carried out quantitatively theanine, glutamine, glutamic acid respectively.Analysis condition is such as
Under: INSTRUMENT MODEL: Ag1260, chromatographic column: C18, column temperature: 40 DEG C.
The optimization of embodiment 5 Pseudomonas nitroreducens NTLC4.002 condition of culture
The present inventor etc. have studied the culture medium nitro beyond the culture medium used in embodiment 1
The cultivation of reduction pseudomonas NTLC4.002.Have studied glucose, fructose, galactose,
After the carbon sources such as maltose, sucrose and lactose, it was found that fermented liquid supernatant when using glucose
Liquid enzyme is lived the highest.Have studied ammonium chloride, ammonium sulfate, sodium nitrate, glutamine, glutamic acid
Behind sodium, yeast extract, beef extract and peptone nitrogen source, it was found that use yeast to take out
Extract can reach optimum efficiency.Have studied glucose, yeast extract, sodium glutamate simultaneously
Optium concentration is respectively 10～18 than rear discovery, glucose, yeast extract, sodium glutamate
Under conditions of g/L, 0.7～1.2g/L, 9～15g/L, available enzyme higher fermentation liquid alive.
Embodiment 6 uses the transglutaminase from Pseudomonas nitroreducens NTLC4.002
The optimization of enzyme reaction condition.According to the enzyme reaction condition in embodiment 3, have studied make for
Transglutaminase the suitableeest enzyme reaction condition from Pseudomonas nitroreducens NTLC4.002, finds
Use 0.3M glutamine and 1.5M ethamine, at 37 DEG C, under the conditions of pH10, react 2～8
The theanine of hour available maximum yield.
Claims (2)
1. produce a bacterial strain for transglutaminase, it is characterized in that: its Classification And Nomenclature is nitro
Reduction pseudomonas (Pseudomonas nitroreducens) NTLC4.002, deposit number:
CCTCC NO:M2014254.
2. the method that bioanalysis prepares theanine, is characterized in that: comprise the following steps:
(1) cell is prepared:
The Pseudomonas nitroreducens using deposit number to be CCTCC NO:M2014254
NTLC4.002 is starting strain；
Culture medium, is calculated as with g/L: glucose 10-18, yeast extract 0.7-1.2, paddy ammonia
Acid sodium 9-15, MgSO4·7H2O 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, under conditions of 200rpm, cultivate 16h；
Frozen centrifugation, it is thus achieved that thalline；
(2) cell conversion process prepares natural theanine:
The cell of preparation is placed in containing 0.2～0.4M glutamine and 1.5～2.5M ethamine or second
The pH of amine hydrochlorate is the 50～100mM Na of 9～102B4O7-NaOH sodium borate buffer liquid
In, at 35～40 DEG C, carry out the enzyme reaction of 2～8 hours, prepare theanine.
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| 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 |
| CN113774075B (en) | 2021-09-30 | 2023-08-08 | 天津科技大学 | Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermentation of escherichia coli genetically engineered bacterium |
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| 茶氨酸制备方法研究进展;龚雨顺等;《湖南农业大学学报（自然科学版）》;20080430;第34卷(第2期);232-235 * |
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