CN104560849A - Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid - Google Patents
Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid Download PDFInfo
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Abstract
The invention provides a constructing method and application of a recombinant glutamyl transpeptidase coexpression plasmid as well as a method for efficiently accumulating theanine. Through coexpression of chaperonin and GST-labelled glutamyl transpeptidase, a genetically-engineered escherichia coli bacterium BL21/C4 with very high enzyme activity of the glutamyl transpeptidase is successfully constructed, and after the coexpression, the optimal enzyme activity reaches 15,500 U/L. By the constructing method, the enzyme expression and the enzyme activity can be significantly improved, transformation of a substrate is promoted, and the production capacity of the theanine is further increased; the process cycle is shortened (the enzyme activity is high); the production cost is reduced (the enzyme consumption cost is lowered in enzymatic reaction); the method is easy to operate, easy to popularize and high in stability and is suitable for large-scale industrial production; a new thought is provided for producing other valuable products by a biological enzyme method.
Description
Technical field
The present invention relates to gene engineering technology field, specifically, is the Recombinant organism about structure one strain with high glutamyltranspeptidase transferase active, by the production method of enzymatic conversion synthesis theanine.
Background technology
L-thiamine (N-ethyl-γ glutamine, theanine) is a kind of natural amino acid, is the characteristic amino acid in tealeaves, is also effective taste compound in tealeaves.L-thiamine is mainly used in: (1) as a kind of emerging foodstuff additive, the additive of the quality improver that can be used as tea drink, the additive improving flavour of food products, functional food, the additive etc. of " mood food "; Widely use in country, areas such as America and Europe, Japan, TaiWan, Chinas.The chocolate of theanine, jelly, pudding, chewing gum, health tea and various refreshment drink is added as Japan has developed.(2) as medicine intermediate field, theanine can be used as in antitumor, hypotensive, calmness, the antifatigue etc. of calming the nerves medicine.In addition L-thiamine is now as the effective constituent in tranquilizer, plays preventive effect to diseases such as Parkinson's disease, senile dementia, afferent nerve dysfunctions.
The production method of L-thiamine mainly contains tealeaves extraction method, chemical synthesis, fermentable enzyme transforming process.(1) tealeaves extraction method is not high due to the content of Theanine in Tea, extracts theanine be worth without actual production from tealeaves.So plant extraction method is actually spent ion exchange resin method from the debris extracted after tea-polyphenol extract theanine.Shortcoming is that production process is complicated, product purity is low, output is little, price is high.(2) chemosynthesis L-thiamine needs the conditions such as high temperature, high pressure and chemical catalyst, and reaction conditions requires higher, and by product is many, and generation is all DL-type raceme, also needs to carry out splitting just to obtain L-type product.Therefore cost can be made to improve and difficult quality control, and easily mix toxic substance, product should not be used for food service industry application, simultaneously complex manufacturing, and easily causes environmental pollution.(3) L-thiamine that fermentable enzyme transforming process is produced is all L-type, and production cost is low, can produce in a large number, because biological enzyme synthesis has the specificity of height, so closer to natural L-thiamine in quality product, its development prospect is boundless.Enzyme addition conversion method is produced L-thiamine and is also had the advantages such as purity is high, by product is few, purification step is few, throughput is strong, and therefore fermentable enzyme transforming process is direct, the most most economical L-thiamine production method that current researchist both at home and abroad generally acknowledges.
The enzyme utilizing fermentable enzyme transforming process to produce L-thiamine mainly contains L-Glutamine deaminase and gamma glutamyl transpeptidase (γ-GGT), this experiment is that research and utilization Escherichia coli fermentation produces gamma glutamyl transpeptidase, the gamma glutamyl transpeptidase that this intestinal bacteria high yield height enzyme is lived, energy efficiently converting glutamine and ethamine are L-thiamine; In the last few years, although there is research that the γ-ggt gene (full length sequence or mutation deletes sequence) in e. coli k-12 source is imported to expression in escherichia coli, by adopting the coexpression of the carrier of different fusion tags or large small subunit, but restructuring glutamyltranspeptidase is not usually to have activated inclusion body or not have activated zymogen forms to be expressed out.Trace it to its cause, mainly the post translational processing process of glutamyltranspeptidase complexity, cause the large small subunit after expressing and transporting correctly not fold and can not effectively interact, therefore can not form natural maturing enzyme.Such fact makes efficient process LAN have bioactive glutamyltranspeptidase becomes a difficult problem.Thus, seek new method improvement engineering strain and seem particularly important with the resultant quantity fundamentally improving theanine.
Summary of the invention
To be solved by this invention be to provide a kind of chaperone or chaperone combination respectively with the system of glutamyltranspeptidase coexpression, for building the bacterial strain of high expression glutamyltranspeptidase, so that enzymatic conversion synthesis theanine.
Have the Recombinant organism that glutamyltranspeptidase enzyme is lived, this genetic engineering bacterium is with e. coli bl21 (DE3) for Host Strains, and in Host Strains, cotransformation has chaperone expression plasmid pG-Tf2 and glutamyltranspeptidase expression plasmid.
The invention also discloses described Recombinant organism and produce the application in theanine.
Utilizing glutamyltranspeptidase recombinant plasmid to produce a method for theanine, is obtain corotation beggar, this corotation of inducing culture beggar with chaperone expression plasmid pG-Tf2 and glutamyltranspeptidase expression plasmid cotransformation intestinal bacteria; With the corotation beggar through inducing culture for enzyme source, glutamine and ethamine are conversion of substrate, enzyme' s catalysis theanine.
In aforesaid method, inducing culturing condition is preferably as follows: by the access of cotransformation daughter bacteria liquid containing in penbritin and chlorampenicol resistant liquid TB substratum, wherein, be added with the inductor of 10ng/ml tsiklomitsin as chaperone in TB substratum; Shaking culture to OD600 value is after 1.2, adds the IPTG that final concentration is 0.5mM, induces 16h at 20 DEG C.
The technical scheme utilizing glutamyltranspeptidase recombinant plasmid to produce theanine provided by the invention obtains on the basis of following great many of experiments.：
(1) by 5 kinds of chaperone expression plasmids respectively with glutamyltranspeptidase expression plasmid cotransformation e. coli bl21 (DE3);
(2) glutamyltranspeptidase and the chaperone protein induced expression in five kinds of coexpression system;
In (3) five kinds of coexpression system, lived by the expression amount and enzyme detecting glutamyltranspeptidase, filter out
The cotransformation sublist of pG-Tf2 chaperone expression plasmid reveals best glutamyltranspeptidase enzyme lives;
(4) optimization of pG-Tf2 chaperone expression plasmid and the best coexpression condition of glutamyltranspeptidase;
(5) genetic engineering bacterium that the high glutamyltranspeptidase enzyme of abduction delivering is lived is cultivated, with glutamine and ethamine for substrate, high-efficiency enzyme promoted synthesis theanine.
In step (1), 5 kinds of selected chaperone expression plasmids see attached list 1, all with chlorampenicol resistant; Glutamyl transpeptidase expression plasmid is that the name that this laboratory retains is called Pgex-4t-1/gst-ggt (reference: Xu Junfeng in addition, single Jian Hua, Zhao Ningwei, Yin Zhimin. the structure of intestinal bacteria γ 2ggt prokaryotic expression carrier pGEX-4T-1/ γ-ggt and protein expression thereof. Agriculture of Anhui science, 2007,35 (30): 9467 – 9469), with amicillin resistance; Double convenient screening corotation beggar.
Table 1.5 kind of intestinal bacteria chaperone plasmid brief introduction
In table 1,5 kinds of plasmids all can be bought from Takara company.In step (2), the method for its abduction delivering is: the corotation beggar that step (2) obtains is inoculated in the TB liquid nutrient medium containing penbritin and chlorampenicol resistant, 37 DEG C, 220rpm/min shaking culture 12h; The 5 kinds of cotransformation bacterium activated are contained in the TB liquid nutrient medium of 100ml penbritin and chlorampenicol resistant in the access of 1:50 ratio and (for the corotation beggar of the chaperone expression plasmid containing araB promotor, in its substratum, adds the pectinose of 0.5-4mg/ml; For the corotation beggar of the chaperone expression plasmid containing Pzt1 promotor, in its substratum, add 1-10ng/ml tsiklomitsin; For the corotation beggar of the chaperone expression plasmid simultaneously containing araB and Pzt1, in its substratum, both added the pectinose of 0.5-4mg/ml, added again 1-10ng/ml tsiklomitsin), 35 DEG C of shaking culture are to suitable OD600 value; Add appropriate IPTG, 160rpm/min, at 20 DEG C, cultivate 15h, collected by centrifugation thalline.
In step (3), glutamyltranspeptidase expression amount measuring method is WesternBlot, and its enzyme activity determination carries out according to the GGT kit method of Shanghai biotechnology company limited.The corotation beggar of the best glutamyltranspeptidase enzyme of the tool pG-Tf2 chaperone alive expression plasmid of screening is the result determined by repeatedly repeating comparative experiments.
In step (4), change the temperature of tetracyclin inducer and IPTG concentration and induction, the pG-Tf2 chaperone expression plasmid determined by the glutamyltranspeptidase enzyme work of comparing under different condition and the best coexpression condition of glutamyltranspeptidase.
In step (5), the genetic engineering bacterium BL21/C4 of the enzyme' s catalysis theanine adopted is through the high glutamyltranspeptidase enzyme that has that step (4) optimizes and lives, and its enzyme liquid is directly as the source of glutamyltranspeptidase.
Beneficial effect: using gene engineering technique of the present invention by high expression Production by Enzymes theanine, uses the inventive method the expression amount of obvious high enzyme and enzyme to live, promotes the conversion of substrate, and then improve the turnout of theanine.Meanwhile, shorten process cycle (enzymic activity is good), reduce production cost (reducing enzyme consumption cost in enzymatic reaction), easy to operate, easily amplify, good stability, be suitable for large-scale industrial production, for other value product of biological Production by Enzymes provides new thinking.
Accompanying drawing explanation
Fig. 1 is the WesternBlot results contrast of the glutamyltranspeptidase of expressing in five kinds of coexpression system.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
1, the structure of glutamyltranspeptidase and five kinds of intestinal bacteria partner system coexpression system:
By the escherichia coli cloning bacterium frozen storing liquid containing four kinds of chaperone expression plasmids (see table 1) respectively by volume the ratio of 1:100 to transfer containing 4ml and four kinds of chaperone expression plasmids (see table 1) in the test tube with LB liquid nutrient medium corresponding to resistance; 200rpm, after 37 DEG C of shaking culture 12h, plasmid is little carries; Mixed in 1:1 ratio with glutamyltranspeptidase expression plasmid pGEX-4T-1/ γ-ggt respectively by after little carrying 5 kind of chaperone expression plasmid, mixing the nucleic acid contained in rear 5 kinds of mixed solutions is 50ng; These 5 kinds of plasmid mixed solutions are transformed into respectively in 100 μ l e. coli bl21 (DE3) competent cells; The LB liquid medium of 890 μ l, 37 DEG C of preheatings is added, renewal cultivation 2-3 hour in shaking table in the competent cell after conversion; Draw corotation e. coli bl21 (DE3) the cell 200 μ l after 5 kinds of renewal cultivations, be coated on 5 LB solid medium flat boards containing penbritin and paraxin respectively, be inverted in 37 DEG C of incubators and cultivate 15-17 hour; Picking 6-10 clone on each flat board afterwards, and the 4ml that transfers respectively contains in the test tube of penbritin and chlorampenicol resistant LB liquid medium, 220rpm, after 37 DEG C of shaking culture 12h, plasmid is little carries; Plasmid after little for each clone carrying is carried out agarose gel electrophoresis, and checking is two turns successful corotation beggar, and then completes the structure of glutamyltranspeptidase and five kinds of intestinal bacteria partner system coexpression system, proves to successfully construct.
2, glutamyltranspeptidase and the expression of chaperone in five kinds of coexpression system:
The frozen bacterium of corotation beggar successfully constructed 5 kinds contains in the test tube of 4ml penbritin and chlorampenicol resistant liquid nutrient medium in the ratio access of 1:50,220rpm, 37 DEG C of shaking culture 12h; The 5 kinds of corotation bacterium activated are contained in the Erlenmeyer flask of 100ml penbritin and paraxin liquid TB substratum in the ratio access of 1:50 and (for the corotation beggar of the chaperone expression plasmid containing araB promotor, in its substratum, adds the pectinose of 0.5-4mg/ml; For the corotation beggar of the chaperone expression plasmid containing Pzt1 promotor, in its substratum, add 1-10ng/ml tsiklomitsin; For the corotation beggar of the chaperone expression plasmid simultaneously containing araB and Pzt1, in its substratum, both added the pectinose of 0.5-4mg/ml, added 1-10ng/ml tsiklomitsin simultaneously), 35 DEG C of shaking culture are to suitable OD
600value; Add appropriate IPTG, 160rpm/min, at 20 DEG C, cultivate 15h.
The glutamyltranspeptidase enzyme of 3, expressing in five kinds of coexpression system is lived and the WesternBlot of expression of enzymes amount detects (1) enzyme activity determination method: the cell centrifugation of coexpression under various condition is received bacterium, collected after centrifugation bacterial sediment, and (original bacteria liquid amasss: it is resuspended that ratio PBS) adds PBS damping fluid in 1:5; By concentrated bacterium liquid ultrasonication (200hz, 23min) after resuspended; The bacterium liquid got after 1 μ l fragmentation adds the ultrapure water of 99 μ l, and the broken bacterium liquid after being diluted by this 100 μ l joins 2ml enzyme and lives in reaction kit A liquid, in 37 DEG C of water-bath 10min; Joined by 200 μ l reaction terminating liquids in the reaction solution bacterium liquid mixed solution after temperature bath, room temperature places 5min; Draw the ultrapure water that 20 these mixed solutions of μ l add 180 μ l, the mixed solution after this being diluted adds in 96 orifice plates, detects the light absorption value at 410nm place in microplate reader.The glutamyltranspeptidase transferring enzyme enzyme of a unit is lived and is defined as under optimum condition, and catalysis in one minute discharges the enzyme amount needed for 1 micromole p-NA.Our measurement result display, for the corotation beggar containing pG-Tf2 chaperone expression plasmid, under the chaperone combination groES-groEL-trigger-factor help that pG-Tf2 expression plasmid is expressed, after coexpression, glutamyltranspeptidase enzyme is lived and is up to 14438+27U/l, has all exceeded other chaperone plasmid.
(2) the WesternBlot measuring method of expression of enzymes amount: after fragmentation terminates, by cytoclasis liquid at 12000rpm, centrifugal 15min under 4 DEG C of conditions; After centrifugal end, collect centrifuged supernatant and prepare Supernatant samples, preparation deposit sample; And measure the total protein concentration in every increment product by Bradford method, get equal protein and carry out SDS-PAGE; After electrophoresis terminates by protein delivery to (time 100min, constant current 350Ma) on nitrocellulose filter; TBS (containing 5% skim-milk) is used to close 1h, TBST washing, each 5min, totally 3 times subsequently.Add primary antibodie, overnight incubation under 4 DEG C of conditions, after antibody incubation spends the night, then wash 3 times with TBST, each 5min, hatch half an hour, the infrared imaging system observed result of Odyssey with the fluorescence two of Ddy800 is anti-, after scanning, calculate signal band density value.From Fig. 1 result, in the full bacterium sample of 5 kinds of corotation beggars and single expression, the gray-value variation trend of target protein band is identical with foregoing enzyme variation tendency alive, this is visibly different to the expressional function of glutamyltranspeptidase after illustrating that various chaperone is combined in cell inner expression.WB result illustrates equally, chaperone combination groES-groEL-trigger-factor is the chaperone combination that best promotion glutamyltranspeptidase is expressed, pG-Tf2 is the chaperone expression plasmid that best promotion glutamyltranspeptidase is expressed, cotransformation bacterium liquid called after BL21/C4.
4, the exploration of the optimum coexpression condition of corotation beggar BL21/C4
The glycerol stock of corotation beggar BL21/C4 is contained in the test tube of 4ml penbritin and chlorampenicol resistant liquid nutrient medium in the ratio access of 1:50,220rpm, 37 DEG C of shaking culture 12h, the ratio access of the BL21/C4 bacterium liquid activated in 1:50 is contained in the Erlenmeyer flask of 100ml penbritin and chlorampenicol resistant liquid TB substratum (because chaperone group expression plasmid pGTf-2 contains Pzt1 promotor, in order to the initial period starting at fermention medium to cultivate just induces the expression of groES-groEL-trigger-factor chaperone combination, 10ng/ml is added respectively in its TB substratum, 30ng/ml, 70ng/ml tsiklomitsin is as the inductor of chaperone), 200rpm, 35 DEG C of shaking culture to suitable OD600 value is after 1.2, add appropriate IPTG (in order to raw at the appropriate abduction delivering of chaperone, the expression of induction target protein glutamyltranspeptidase, 0.3mM, 0.5mM, the IPTG of 0.7mM is added in each experimental group), under the condition of 160rpm/min, (in order to the inducing temperature finding the suitableeest glutamyltranspeptidase to express under proper temperature, 16 DEG C, 20 DEG C, 28 DEG C of three temperature condition are measured separately) induce 8h or 16h respectively.By table 2 result, we can draw: 20 DEG C, the IPTG induced concentration of the tsiklomitsin induced concentration of 10ng/ml and 0.5mM, glutamyltranspeptidase enzyme after induction 16h live and be optimum.Under these top conditions, the highest glutamyltranspeptidase enzyme is lived and is reached 15488+183U/l, and living with the glutamyltranspeptidase enzyme of single expression condition of the best and compare, is its about 2 times; Compared with living with the glutamyltranspeptidase enzyme obtained under the best glutamyltranspeptidase fermentation condition of e. coli k-12 wild type strain, it is 387 times of wild-type.
The transferase active of glutamyltranspeptidase after table 2 corotation beggar BL21/C4 coexpression
Note: underlined numbers represents: glutamyltranspeptidase transferring enzyme the highest under each induction time and tetracycline concentration is lived.Numeral overstriking represents: the highest glutamyltranspeptidase transferring enzyme enzyme is lived.
5, coexpression bacterial strain BL21/C4 is utilized efficiently to synthesize theanine
The glutamine of 0.171mol is dissolved in 500ml pure water; By acid-soluble for the ethylamine salt of 2.86mol in above-mentioned reaction solution, regulate the pH to 10.0 of reaction solution; After measuring coexpression, the glutamyltranspeptidase enzyme of BL21/C4 fermented liquid is lived, and the fermented liquid got containing 3000U joins in above-mentioned reaction solution; PH to 10.0 is regulated with pure ethamine; By in the shaking table of this reaction solution to 37 DEG C, under 150rpm/min condition, carry out enzymatic reaction; Reacting in the process of carrying out, every the pH situation of detection reaction liquid half an hour, and regulate pH to 10.0 with pure ethamine; At the 0h of reaction, 2h, 4h, 6h, 9h, 11h get 200 μ l reaction solution samples respectively; To the reaction solution sample of different time be taken at as boiling sample 5min in boiling water; By centrifugal 5min under the condition of sample 12500rpm/min of boiling; Get sample point sample on filter paper in 0.5 μ l each stage, carry out paper chromatography detection; Paper chromatography terminates to carry out triketohydrindene hydrate color reaction to result afterwards.Finally, using the BL21/C4 intact cell after coexpression as glutamyltranspeptidase enzyme source, under maintenance ethamine is greater than 15 to 1 conditions all the time with theanine mol ratio, the glutamine of 0.17M is converted into the theanine of 0.127M, and reaction conversion ratio reaches 75%.
Claims (4)
1. the Recombinant organism that there is glutamyltranspeptidase enzyme and live, it is characterized in that: this genetic engineering bacterium is with e. coli bl21 (DE3) for Host Strains, and in Host Strains, cotransformation has chaperone expression plasmid pG-Tf2 and glutamyltranspeptidase expression plasmid.
2. Recombinant organism BL21/C4 described in claim 1 is producing the application in theanine.
3. utilize glutamyltranspeptidase recombinant plasmid to produce a method for theanine, it is characterized in that: be obtain corotation beggar, this corotation of inducing culture beggar with chaperone expression plasmid pG-Tf2 and glutamyltranspeptidase expression plasmid cotransformation intestinal bacteria; With the corotation beggar through inducing culture for enzyme source, glutamine and ethamine are conversion of substrate, enzyme' s catalysis theanine.
4. method according to claim 3, it is characterized in that: described inducing culturing condition is as follows: by the access of cotransformation daughter bacteria liquid containing in penbritin and chlorampenicol resistant liquid TB substratum, wherein, the inductor of 10ng/ml tsiklomitsin as chaperone is added with in TB substratum; Shaking culture to OD600 value is after 1.2, adds the IPTG that final concentration is 0.5mM, induces 16h at 20 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105219747A (en) * | 2015-11-11 | 2016-01-06 | 南京师范大学 | A kind of preparation method of gamma glutamyl transpeptidase |
| CN110564789A (en) * | 2019-09-12 | 2019-12-13 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
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2014
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219747A (en) * | 2015-11-11 | 2016-01-06 | 南京师范大学 | A kind of preparation method of gamma glutamyl transpeptidase |
| CN110564789A (en) * | 2019-09-12 | 2019-12-13 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
| CN110564789B (en) * | 2019-09-12 | 2021-05-04 | 河南巨龙生物工程股份有限公司 | Method for producing L-theanine by using escherichia coli fermentation |
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