CN1493685A - Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain - Google Patents
Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain Download PDFInfo
- Publication number
- CN1493685A CN1493685A CNA031529569A CN03152956A CN1493685A CN 1493685 A CN1493685 A CN 1493685A CN A031529569 A CNA031529569 A CN A031529569A CN 03152956 A CN03152956 A CN 03152956A CN 1493685 A CN1493685 A CN 1493685A
- Authority
- CN
- China
- Prior art keywords
- transglutaminase
- reagent
- glutamine transaminase
- fermentation
- cctcc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A streptomyce shygeroscopicus (CCTCC No.M203062) able to generate glutamine transaminase wigh high output is extracted from the soil of milk cow farm through plate screening, primary fermenting, flask culture, comparing test with standard color tape, and enzyme activity test. Said glutamine transaminase is prepared by deep fermentation.
Description
Technical field
A kind of Transglutaminase EC2.3.2.13 high yield bacterium and screening method thereof and produce Transglutaminase EC2.3.2.13 with this strain fermentation method belong to technical field of bioengineering, and particularly a plant height produces the streptomyces hygroscopicus of Transglutaminase EC2.3.2.13.
Background technology
Along with human living standard's progressively raising, for the requirement of food also from before simply having adequate food and clothing to the nutritional type transition.And protein ought to become the focus that we pay close attention to as one of most important composition in the food.Because numerous food product protein is formed undesirable because of amino acid, or lack suitable structure and conformation, make and these potein deficiency excellent function character, also influenced the nutritive value of food product bodies simultaneously as solvability, foaminess, emulsifying property and emulsifying stability.These protein are carried out chemically modified improve their functional property, people once did extensive studies, but consider the safety issue of chemical reagent and the detrimentally affect that may cause food nutrition thereof, these methods finally fail generally to be accepted by people and use.And enzymatic modification because safety, fast, specificity is strong, food nutrition is not had destruction, and cost is low, more and more obtains people's approval.Up to the present, with enzyme process protein is modified to concentrate in a large number and utilize some enzyme of proteolysis that protein is carried out local hydrolysis, with solvability and the foaminess of improving them, and these are far can not reach people to the requirement of Food Quality.In these areas, using the Transglutaminase EC2.3.2.13 modified protein, is one of the most promising direction of the new protein food product of exploitation
Transglutaminase EC2.3.2.13 (Transglutaminase, EC2.3.2.13, be called for short TG) can the catalytic proteins molecule between or within the acyl group shift reaction, by these catalyzed reactions, can between the range protein molecule or within cause covalent cross-linking, therefore the major function of Transglutaminase EC2.3.2.13 in the foodstuffs industry course of processing comprises: (1) carries out modification to various food proteins, and various chemical reactions do not take place protection Methionin, seal lipoids and lipid-soluble substance; (2) form heat resistanceheat resistant and anti-water film, avoid heat-treating during gelling protein; (3) improve elasticity and water-holding power; (4) change solubility and various functional property, thereby it is higher and contain the food protein of human body institute indispensable amino acid to produce nutritive value.For many years, the commercialization Transglutaminase EC2.3.2.13 mainly extracts from animal tissues, but because its separation and purge process are complicated, and the source is rare, thereby cost an arm and a leg ().In recent years, people begin to turn on the research and utilization microbial fermentation and produce transglutamin-ase 9 transaminase (Microbial Transglutaminase is called for short MTG), and make it to be applied to foodstuffs industry.Through the food after the processing of glutamine of microbe transaminase, its functional property has obtained obvious improvement.
Summary of the invention
The purpose of this invention is to provide a kind of Transglutaminase EC2.3.2.13 high yield bacterium and screening method thereof and produce Transglutaminase EC2.3.2.13 with this strain fermentation method, prepared Transglutaminase EC2.3.2.13 can be used as protein linking agent use in the foodstuffs industry.
Technical scheme of the present invention, system separates a strain streptomyces hygroscopicus (Streptomyces hygroscopicus) WSH03-13 (this research department's preservation) that obtains from Wuxi milk cow factory soil, be deposited in Wuhan China typical culture collection center on July 8th, 2003, be numbered CCTCC NO:M203062.
The screening method of streptomyces hygroscopicus CCTCC NO:M203062 is through flat-plate bacterial colony feature preliminary screening from milk cow factory soil, adopt one grade fermemtation shake-flask culture one by one then, utilize the reference colour band to carry out contrast test, select the darker part bacterial strain of color, carry out enzyme activity determination, and transglutaminase activity size and obtaining relatively.
Carry out contrast test with the reference colour band. draw the 100ul fermented liquid with microsyringe, put in the test tube, Φ 8 * 100, add 50ul A reagent, put in 37 ℃ of water-baths and reacted 10 minutes, the B reagent that adds 100ul is more relatively judged alive the having or not and height of enzyme according to the depth and the standard colour band of color.
N-α-CBZ-Gln-Gly of reagent A: 100mg is dissolved in the NaOH solution of 2ml 0.2moL/L, three (methylol) aminomethane (Tris-HCl) the damping fluid 4ml that adds 0.2mol/L pH6.0,0.1mol/L oxammonium hydrochloride 2ml, 0.01mol/L reduced glutathion 2ml, and regulate pH to 6.0.
The HCL of reagent B:3mol/L, 12% trichoroacetic acid(TCA) (TCA), 5%FeCL
3Mixed by 1: 1: 1.
Get the work of 1mL enzyme and be the fermented liquid of 2.0u/mL, be diluted to 0,0.2,0.4 respectively with distilled water, 0.6,0.8,1.0,2.0u/mL enzyme stepladder degree, according to the above-mentioned A reagent react that adds earlier, add the operating process of B reagent react more then, obtain a series of colors colour band from shallow to deep.
A Transglutaminase EC2.3.2.13 enzyme unit alive (1u) definition (u/mL): per minute catalysis forms the enzyme amount of 1 μ molL-L-glutamic acid-γ-single Hydroxylamine HCL in the time of 37 ℃.
Adopting streptomyces hygroscopicus CCTCC NO:M203062 is starting strain, makes Transglutaminase EC2.3.2.13 through seed culture and liquid submerged fermentation.
Plate isolation substratum (g/L):
Arginine 0.83, glycerine 12.50, sodium-chlor 1.0, sal epsom 0.50, agar 15.0, soil diffusion juice 200, distilled water 800 is transferred pH7.0; Add the cycloheximide 0.05 through sterile filtration when being cooled to 60 ℃ after the sterilization, nystatin is cultivated a week down for 0.08,30 ℃.
The preparation of soil diffusion juice: get 300 gram soil, add 1000ml distilled water, autoclaving 30 minutes, post precipitation is got supernatant liquor, filters, and puts in the refrigerator and preserves.
Slant medium (g/L): wort 100 (10 ° of Be), yeast extract paste 5, glucose 5, agar 15, pH7.0 cultivated 5 days for 30 ℃.
Seed culture medium (g/L): glycerine 20, peptone 20, yeast extract paste 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, anhydrous potassium dihydrogenphosphate 2, pH7.0,30 ℃, shake 200 rev/mins of bottle rotating speeds, incubation time is 20～24 hours.
Fermention medium (g/L): glycerine 15～20, glucose sugar 10～15, peptone 20～25, yeast extract paste 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, anhydrous potassium dihydrogenphosphate 2, lime carbonate 5,7.0,30 ℃ of pH shake 200 rev/mins of bottle rotating speeds, and fermentation time is 44～48 hours.
Beneficial effect of the present invention: proposed a kind of high yield bacterium of Transglutaminase EC2.3.2.13, can prepare Transglutaminase EC2.3.2.13 with this bacterial strain through liquid submerged fermentation, enzyme work reaches 5.0u/ml, can be used as protein linking agent use in the foodstuffs industry.
The biological material specimens preservation.
Streptomyces hygroscopicus WSH03-13, preservation date on July 8th, 2003, depositary institution's title and abbreviation: Chinese typical culture collection center C CTCC, deposit number is NO:M203062.
Embodiment
Scrape from the inclined-plane and to get a ring bacterial classification, place the 500ml triangular flask that the 100ml seed culture medium is housed to carry out seed culture.Condition is incubation time 24 hours, 30 ℃ of temperature, shaking speed 200rpm.Insert in the 500ml triangular flask that the 50ml fermention medium is housed with 8% inoculum size again and carry out fermentation culture.Condition is incubation time 48 hours, 30 ℃ of temperature, shaking speed 200rpm.Can obtain the fermented liquid of enzyme 5.0u/ml alive.
Claims (8)
1. Transglutaminase EC2.3.2.13 high yield bacterium, classification called after streptomyces hygroscopicus (Streptomyceshygroscopicus) WSH03-13, its deposit number is CCTCC NO:M203062.
2. the screening method of a streptomyces hygroscopicus CCTCC NO:M203062, it is characterized in that from milk cow factory soil through flat-plate bacterial colony feature preliminary screening, adopt one grade fermemtation shake-flask culture one by one then, utilize the reference colour band to carry out contrast test, select the darker part bacterial strain of color, carry out enzyme activity determination, and transglutaminase activity size and obtaining relatively.
3. method according to claim 2, it is characterized in that carrying out contrast test with the reference colour band, draw the 100ul fermented liquid with microsyringe, put in the test tube, Φ 8 * 100, add 50ul A reagent, put in 37 ℃ of water-baths and react 10 minutes, the B reagent that adds 100ul is more relatively judged alive the having or not and height of enzyme according to the depth and the standard colour band of color;
N-α-CBZ-Gln-Gly of reagent A: 100mg is dissolved in the NaOH solution of 2ml 0.2moL/L, three (methylol) aminomethane (Tris-HCl) the damping fluid 4ml that adds 0.2mol/L pH6.0,0.1mol/L oxammonium hydrochloride 2ml, 0.01mol/L reduced glutathion 2ml, and regulate pH to 6.0;
The HCL of reagent B:3mol/L, 12% trichoroacetic acid(TCA) (TCA), 5%FeCL
3Mixed by 1: 1: 1.
4. the preparation method of a Transglutaminase EC2.3.2.13, it is characterized in that adopting streptomyces hygroscopicus CCTCC NO:M203062 is starting strain, makes Transglutaminase EC2.3.2.13 through seed culture and liquid submerged fermentation.
5. preparation method according to claim 4 is characterized in that seed culture, seed culture medium (g/l): glycerine 20, peptone 20, yeast extract paste 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, anhydrous potassium dihydrogenphosphate 2, pH7.0; Culture condition: incubation time is 20-24 hour, 30 ℃ of temperature.
6. preparation method according to claim 4 is characterized in that liquid submerged fermentation, fermention medium (g/l): glycerine 15-20, glucose 10-15, peptone 20-25, yeast extract paste 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, anhydrous potassium dihydrogenphosphate 2, lime carbonate 5, pH 7.0; Fermentation condition: fermentation time 44-48 hour, 30 ℃ of temperature.
7. method according to claim 4 is characterized in that prepared Transglutaminase EC2.3.2.13 enzyme work reaches 5u/ml.
8. the streptomyces hygroscopicus CCTCC NO:M203062 application of prepared Transglutaminase EC2.3.2.13 of fermenting is characterized in that can be used for the agent of foodstuffs industry protein cross.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031529569A CN1208452C (en) | 2003-09-05 | 2003-09-05 | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031529569A CN1208452C (en) | 2003-09-05 | 2003-09-05 | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1493685A true CN1493685A (en) | 2004-05-05 |
| CN1208452C CN1208452C (en) | 2005-06-29 |
Family
ID=34240735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031529569A Expired - Fee Related CN1208452C (en) | 2003-09-05 | 2003-09-05 | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1208452C (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100447236C (en) * | 2006-10-28 | 2008-12-31 | 江南大学 | Method of adding trypsase to increase output of prepared glutamine transaminase in fermentation |
| CN101984052A (en) * | 2010-12-09 | 2011-03-09 | 江南大学 | Separation and purification method of transglutaminase |
| CN102586164A (en) * | 2012-02-16 | 2012-07-18 | 江南大学 | Streptomyces lividan for efficiently excreting transglutaminase and application thereof |
| CN101698836B (en) * | 2009-11-23 | 2013-04-17 | 江南大学 | Method for supplementing material in the fermentation process of transglutaminase |
| CN103409385A (en) * | 2013-08-07 | 2013-11-27 | 江南大学 | Method for producing proenzyme of transglutaminase through fermentation |
| CN103497904A (en) * | 2013-09-18 | 2014-01-08 | 江南大学 | Genetically engineered bacterium and method for producing glutamine transaminase zymogen by genetically engineered bacterium |
| CN103981130A (en) * | 2014-05-09 | 2014-08-13 | 虞龙 | Glutamine transaminase high-yielding strain and application thereof |
| CN110358708A (en) * | 2019-07-15 | 2019-10-22 | 泰兴市东圣生物科技有限公司 | A kind of high-yield glutamine transaminase bacterial strain and its application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660515B (en) * | 2012-05-10 | 2013-12-11 | 江南大学 | Glutamine transaminase with improved enzymatic activity and thermal stability |
-
2003
- 2003-09-05 CN CNB031529569A patent/CN1208452C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100447236C (en) * | 2006-10-28 | 2008-12-31 | 江南大学 | Method of adding trypsase to increase output of prepared glutamine transaminase in fermentation |
| CN101698836B (en) * | 2009-11-23 | 2013-04-17 | 江南大学 | Method for supplementing material in the fermentation process of transglutaminase |
| CN101984052A (en) * | 2010-12-09 | 2011-03-09 | 江南大学 | Separation and purification method of transglutaminase |
| CN101984052B (en) * | 2010-12-09 | 2012-09-26 | 江南大学 | Separation and purification method of transglutaminase |
| CN102586164A (en) * | 2012-02-16 | 2012-07-18 | 江南大学 | Streptomyces lividan for efficiently excreting transglutaminase and application thereof |
| CN102586164B (en) * | 2012-02-16 | 2014-06-18 | 江南大学 | Streptomyces lividan for efficiently excreting transglutaminase and application thereof |
| CN103409385A (en) * | 2013-08-07 | 2013-11-27 | 江南大学 | Method for producing proenzyme of transglutaminase through fermentation |
| CN103409385B (en) * | 2013-08-07 | 2015-06-03 | 江南大学 | Method for producing proenzyme of transglutaminase through fermentation |
| CN103497904A (en) * | 2013-09-18 | 2014-01-08 | 江南大学 | Genetically engineered bacterium and method for producing glutamine transaminase zymogen by genetically engineered bacterium |
| CN103981130A (en) * | 2014-05-09 | 2014-08-13 | 虞龙 | Glutamine transaminase high-yielding strain and application thereof |
| CN110358708A (en) * | 2019-07-15 | 2019-10-22 | 泰兴市东圣生物科技有限公司 | A kind of high-yield glutamine transaminase bacterial strain and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1208452C (en) | 2005-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101792727B (en) | Bacillus coagulans and application thereof in L-sodium lactate preparation | |
| CN110229852B (en) | Method for producing gamma-polyglutamic acid by fermenting soybean protein zymolyte | |
| CN100334200C (en) | Glutamic acid capable of having high-yield glutamine | |
| CN1208452C (en) | Glutamine transaminase high productive bacteria and its screening method and fermentation method producing glutamine transaminase using said bacterial strain | |
| CN1246450C (en) | Process for producing chitosan enzyme producing fungus and chitosan oligomer | |
| CN103937691B (en) | One plant production β fructosidases aspergillus oryzae strain and its cultural method and application | |
| CN101886095B (en) | Method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and special culture medium thereof | |
| CN102127515B (en) | Screening and application of L-proline high-producing Brevundimonas sp. (JNPP-1) | |
| CN110129225A (en) | γ~polyglutamic acid producing strains and breeding prepare γ~polyglutamic acid method | |
| CN106867937A (en) | With the bacterial strain of pea protein wastewater liquid fermenting and producing Bacillus subtilis natto microbial inoculum, method and application | |
| CN103981130A (en) | Glutamine transaminase high-yielding strain and application thereof | |
| CN116496911B (en) | Ricasfungin intermediate FR901379 high-yield strain and application thereof | |
| CN105219661A (en) | The special strain therefore of synthesis of oligonucleotides semi-lactosi and the method with its synthesis of oligonucleotides semi-lactosi | |
| CN105175275B (en) | A kind of isolation and purification method of L ornithine | |
| CN103667107B (en) | A kind of manure enterococcin strain producing Pfansteihl | |
| CN110184244A (en) | A kind of Sparassis crispa produces the preparation method of laccase | |
| CN113564215B (en) | Preparation method of biosurfactant with carbon dioxide and/or lignocellulose as substrate | |
| CN109456898A (en) | A kind of the fermentation preparation and its application of chaetomium globosum dextranase | |
| CN105002228B (en) | A method of preparing L-Orn by raw material of arginine | |
| CN1948500A (en) | Preparation method of functional sweetener D-tatai sugar | |
| CN116515795B (en) | Application of Aspergillus tubingensis in preparing phytase and/or degrading phytic acid | |
| CN114369558B (en) | Serratia marcescens and application thereof in naringinase production | |
| CN116463324B (en) | Method for improving enzyme activity of protein glutaminase production | |
| CN116286557B (en) | Salt-tolerant bacillus beijerinckii for producing cellulase and culture method thereof | |
| CN114982893B (en) | Preparation method of konjak fungus oligosaccharide-dissolving instant beverage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |