CN1249222C - Process for raising yield of glutamine transaminase prepared by fermination process by adding amine hydrocarbon - Google Patents
Process for raising yield of glutamine transaminase prepared by fermination process by adding amine hydrocarbon Download PDFInfo
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- CN1249222C CN1249222C CN 200410041693 CN200410041693A CN1249222C CN 1249222 C CN1249222 C CN 1249222C CN 200410041693 CN200410041693 CN 200410041693 CN 200410041693 A CN200410041693 A CN 200410041693A CN 1249222 C CN1249222 C CN 1249222C
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- fermentation
- glutamine transaminase
- transglutaminase
- azanol
- yield
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- Enzymes And Modification Thereof (AREA)
Abstract
The present invention discloses a method for raising the yield of glutamine transaminase prepared by a fermination method by adding hydroxyl amine, which relates to a method for preparing glutamine transaminase by the fermentation of streptomyces hygroscopicus. In the present invention, streptomyces hygroscopicus is used as fermentation strains, inoculated in a fermentation medium after slope culture and seed culture, and made into glutamine transaminase through liquid fermentation. The high expression of glutamine transaminase is realized by adding hydroxyl amine in the fermentation process. The present invention has the advantages of low price of hydroxyl amine, and little dosage and can obviously raise the yield of glutamine transaminase. Under the identical other culture conditions, compared with the controls without the addition of hydroxyl amine, the method can raise yield by about 30% and reach an advanced stage of preparing glutamine transaminase by a fermentation method.
Description
Technical field
A kind of azanol that adds improves the method that fermentation method prepares glutamine transaminase output, relates to streptomyces hygroscopicus fermentative preparation Transglutaminase EC2.3.2.13.
Background technology
Transglutaminase EC2.3.2.13 (Microbial transglutaminase, be called for short MTG), the hydrolysis of glutamine amide group in connection between intramolecular crosslinked, intermolecular crosslinked, protein of catalytic proteins and the amino acid and the protein molecule, thereby further improve protein function character, improve proteinic nutritive value, produce the novel protein food that satisfies people's demand.Transglutaminase EC2.3.2.13 widespread use now is in many aspects: in meat product processing, the meat mincing of some low values are recombinated in the crosslinked action of Transglutaminase EC2.3.2.13, thereby improve its outward appearance, structure, local flavor, to improve its nutritive value and marketable value; In food processing process, Transglutaminase EC2.3.2.13 can protect the Methionin in the food protein to avoid taking place chemical reaction, thus the protection limiting amino acid; In milk-product processing, Transglutaminase EC2.3.2.13 improves proteinic trophicity; In medicine industry, Transglutaminase EC2.3.2.13 embedding lipid or lipid-soluble substance form heat-resisting water-proof film; In the bean food of non-meat, Transglutaminase EC2.3.2.13 improves the elasticity and the water-holding power of food; In textile industry, Transglutaminase EC2.3.2.13 also is used to improve the quality of silk fabric.
The method of producing MTG now mainly is a Production by Microorganism Fermentation, and present business-like Transglutaminase EC2.3.2.13 product is mainly produced by companies such as Japanese aginomotos.Domesticly still rest on conceptual phase with the microorganisms producing Transglutaminase EC2.3.2.13, bacterial classification mainly is a streptomyces mobaraensis, and production level is very low.
Summary of the invention
The objective of the invention is a kind of azanol that adds and improve the method that fermentation method prepares glutamine transaminase output.
Technical scheme of the present invention
Bacterial classification
Streptomyces hygroscopicus (Streptomyces hygroscopicus) WSH03-13, CCTCC NO:203062 (Chinese patent 03152956.9 2003 years .) is by the Environmental Biotechnology research department preservation of biotechnology institute of Southern Yangtze University with provide.
Substratum
Wheat juice slant medium (g/L): wheat juice 50, glucose 20, yeast extract paste 20, agar 20, pH 7.0.
Seed culture medium (g/L): glycerine 20, glucose 20, yeast extract paste 5, NaH
2PO
45, Na
2HPO
45, MgSO
45, pH7.0.
Fermention medium (g/L): starch 15, glucose 15, peptone 25, yeast extract paste 5, lime carbonate 5, NaH
2PO
45, Na
2HPO
45, MgSO
45, pH 7.0.
Cultural method
Sophisticated spore inoculating to wheat juice inclined-plane, is cultivated 7-8d down for 32 ℃.After treating the spore maturation, scrape the spore of getting on the inclined-plane, make spore suspension with sterilized water and be inoculated in the seed culture medium, 32 ℃ of shaking tables are cultured to the logarithmic growth later stage.Draw seed culture fluid, be inoculated in the fermention medium by the inoculum size of 2-4%, 32 ℃ of shaking tables are cultivated 48h.
The measuring method of Transglutaminase EC2.3.2.13 vigor
The mensuration that is used for vigor after 5 times of the clear liquid dilution of fermented liquid after centrifugal.
The Transglutaminase EC2.3.2.13 vigor adopts spectrophotometry to measure down at 37 ℃.Draw the 0.4ml dilute sample in test tube, add the 1ml reagent A, 37 ℃ of water bath heat preservation 10min add 0.4ml reagent B, termination reaction, 525nm colorimetric.Reagent A: 100mg substrate CBZ-GLN-GLY (Nalpha-Carbobenzyloxy-Glutamine Glycine) is dissolved among the NaOH of 2ml 0.2N fully, and adds: 4ml a, 2mlb, 2ml c, transferring pH is 6.0.(a:0.2M Tutofusin tris hydrochloric acid (Tris-HCl) damping fluid, pH6.0; The b:1M azanol; C:0.01M GSH (reduced form glutathione)).Reagent B:3N HCl, 12%TCA (trichoroacetic acid(TCA)), 5%FeCl
36H
2O (being dissolved among the 0.1N HCl) was by 1: 1: 1 mixed preparing.A unit Transglutaminase EC2.3.2.13 enzyme is lived and defined (u/mL): per minute catalysis forms the enzyme amount of 1 μ mol L-L-glutamic acid-γ-single Hydroxylamine HCL in the time of 37 ℃.
Improve the training method of glutamine transaminase output
The measuring principle of Transglutaminase EC2.3.2.13 is that azanol and substrate N α-CBZ-GLN-GLY reaction generate L-L-glutamic acid-γ-single hydroxamic acid, combines with iron ion and forms the complex compound colour developing, uses colorimetric method for determining.Therefore think that azanol is very likely is Transglutaminase EC2.3.2.13 synthetic inductor.The existence of inductor can make enzyme in running order in advance in the system, also is in induction state promotion product enzyme thereby make it produce enzyme.
During the fermentation, add an amount of azanol in good time can promote to produce enzyme.By adding azanol during the fermentation: add the 0.3-0.7mmol/L azanol fermenting to 36-40h, promoted system to produce enzyme, the enzyme running water of Transglutaminase EC2.3.2.13 is flat in the fermented liquid at the end of fermenting is 5.1U/mL.
Beneficial effect of the present invention: the streptomyces hygroscopicus that the present invention selects for use (Streptomyceshygroscopicus) WSH03-13 synthetic Transglutaminase EC2.3.2.13 is an extracellular enzyme, and be heat, alkaline stability and the best Transglutaminase EC2.3.2.13 of having reported up to now of protein cross characteristic, be used for food-processing and textile industry and have very big potentiality.Used azanol is cheap, and consumption is few, has significantly improved the output of Transglutaminase EC2.3.2.13, and the experimental level of shake-flask culture reaches leading domestic level.
Embodiment
Embodiment 1
At the azanol of the 38h of fermentation interpolation 0.5mmol/L, during fermentation ends, the output of Transglutaminase EC2.3.2.13 is 5.3U/mL in the fermented liquid, has improved 32.5% than the reference examples that does not add azanol.The output of not adding Transglutaminase EC2.3.2.13 in the fermented liquid of azanol is 4.0U/mL.
Embodiment 2
At the azanol of the 36h of fermentation interpolation 0.3mmol/L, during fermentation ends, the output of Transglutaminase EC2.3.2.13 is 4.6U/mL in the fermented liquid.
Embodiment 3
At the azanol of the 40h of fermentation interpolation 0.7mmol/L, during fermentation ends, the output of Transglutaminase EC2.3.2.13 is 5.1U/mL in the fermented liquid.
Claims (2)
1. one kind is improved the method that fermentation method prepares glutamine transaminase output, it is characterized in that adopting streptomyces hygroscopicus (Streptomyces hygroscopicus) WSHO3-13, culture presevation number is CCTCCNO:203062, as starting strain, prepare Transglutaminase EC2.3.2.13 through liquid fermenting, by adding azanol during the fermentation, realized the high expression level of Transglutaminase EC2.3.2.13.
2. method according to claim 1 is characterized in that adding during the fermentation azanol, ferments to add the azanol of 0.3-0.7mmol/L to 36-40h in fermented liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041693 CN1249222C (en) | 2004-08-12 | 2004-08-12 | Process for raising yield of glutamine transaminase prepared by fermination process by adding amine hydrocarbon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041693 CN1249222C (en) | 2004-08-12 | 2004-08-12 | Process for raising yield of glutamine transaminase prepared by fermination process by adding amine hydrocarbon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1597928A CN1597928A (en) | 2005-03-23 |
| CN1249222C true CN1249222C (en) | 2006-04-05 |
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|---|---|---|---|
| CN 200410041693 Expired - Fee Related CN1249222C (en) | 2004-08-12 | 2004-08-12 | Process for raising yield of glutamine transaminase prepared by fermination process by adding amine hydrocarbon |
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Families Citing this family (2)
| 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 |
| CN112695018B (en) * | 2019-10-23 | 2022-10-25 | 华东师范大学 | Method for improving output of transglutaminase produced by fermentation |
-
2004
- 2004-08-12 CN CN 200410041693 patent/CN1249222C/en not_active Expired - Fee Related
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