CN105820955B - A kind of culture medium of high flux screening high-yield glutamine transaminase bacterial strain - Google Patents
A kind of culture medium of high flux screening high-yield glutamine transaminase bacterial strain Download PDFInfo
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- CN105820955B CN105820955B CN201610168342.3A CN201610168342A CN105820955B CN 105820955 B CN105820955 B CN 105820955B CN 201610168342 A CN201610168342 A CN 201610168342A CN 105820955 B CN105820955 B CN 105820955B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
Abstract
The present invention relates to Microbial Breeding to screen field, discloses a kind of culture medium of novel high flux screening production glutamine transaminage bacterial strain, the raw material constituent including following weight proportion: glutamine transaminage substrate: 0.1%-2%；Agar: 1.5%-4%；Starch: 2%；Potassium nitrate: 1%；Dipotassium hydrogen phosphate: 0.5%；Sodium chloride: 0.5%；Epsom salt 0.5%；Green vitriol: 0.01%；Acid-base indicator: 0.0001%-0.01%；PH:6-8.When culture medium of the invention is used for glutamine transaminage bacterial strain screening, has many advantages, such as short cycle, a large amount of bacterial strains can be screened simultaneously, any label is not needed, can directly screen to obtain the bacterial strain of high-yield glutamine transaminase.Invention additionally discloses the application methods of the culture medium, on the culture medium, cultivate bacterial strain to be selected 3-5 days for screening superior strain.
Description
Technical field
The present invention relates to Microbial Breeding technical fields, are specifically related to a kind of novel high flux screening high-yield glutamine
The culture medium and preparation method thereof of transaminase bacterial strain.
Background technique
Glutamine transaminage is a kind of widely used biocatalyst, and commercial value has also obtained in the food industry
To fully demonstrating.The bacterial strain for obtaining high-yield glutamine transaminase is a Xiang Chongdian in terms of breeding, it may be said that a kind of high throughput
Screening and culturing medium has great advantage to superior strain is obtained.The screening of traditional high-yield glutamine transaminase bacterial strain all according to
Enzyme activity is surveyed after the fermentation of 96 orifice plates, but it is all not satisfactory in accuracy and efficiency.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of short cycle, the culture mediums of a large amount of bacterial strains can be screened simultaneously
With and preparation method thereof and application method.
In order to solve the above technical problems, the present invention is the following technical schemes are provided: novel high flux of the present invention screens high yield paddy
The culture medium of glutamine transaminase bacterial strain includes the raw material constituent of following weight proportion: glutamine transaminage substrate:
0.1%-2%；Agar: 1.5%-4%,；Starch: 2%；Potassium nitrate: 1%；Dipotassium hydrogen phosphate: 0.5%；Sodium chloride: 0.5%；
Epsom salt 0.5%；Green vitriol: 0.01%；Acid-base indicator: 0.0001%-0.01%；PH:6-8.
Preferably, the culture medium of novel high flux screening high-yield glutamine transaminase bacterial strain of the present invention comprising following
The raw material constituent of weight proportion: glutamine transaminage substrate: 1%；Agar: 2%；Starch: 2%；Potassium nitrate: 1%；Phosphorus
Sour hydrogen dipotassium: 0.5%；Sodium chloride: 0.5%；Epsom salt: 0.5%；Green vitriol: 0.01%；Soda acid instruction
Agent: 0.001%；PH:7.4.
Wherein, the glutamine transaminage substrate includes enzyme hydrolysis casein, gelatin.
Wherein, the acid-base indicator includes cresol red, phenol red.
The invention also provides the preparations of the culture medium of novel high flux screening high-yield glutamine transaminase bacterial strain
Method:
(1) 1% glutamine transaminage substrate, 2% agar, 2% starch, 1% potassium nitrate, 0.5% phosphoric acid hydrogen two are weighed
Potassium, 0.5% sodium chloride, 0.5% epsom salt, 0.01% green vitriol.
(2) after supplying volume with water, pH to 7.4 is adjusted, 121 DEG C of autoclave sterilizations after twenty minutes, are put into 65 DEG C of baking ovens
Constant temperature one hour, 0.001% acid-base indicator of filtration sterilization is added.
(3) it after mixing configured culture medium, is poured into each culture dish with the volume of 20mL/ ware and is cooled to room temperature.
The invention also provides a kind of uses of the culture medium of high flux screening high-yield glutamine transaminase bacterial strain
Method cultivates bacterial strain to be selected 3-5 days for screening superior strain on the culture medium.
Wherein, the bacterial strain includes streptomyces cyclopentadienyl source streptomycete.
Specifically, the application method of culture medium of the present invention, includes the following steps:
(1) bacterial strain to be selected is suspended in sterile saline, and is sufficiently diluted with sterile physiological saline, make to hang
Floating cell is no more than 500/mL.
(2) strain suspensions diluted are drawn 0.1mL to be added dropwise on culture medium of the present invention, and by the bacterial strain of dropwise addition
Suspension is uniformly coated on media surface.
(3) culture medium of the present invention for being coated with bacterium solution is placed on 28 DEG C constant temperature incubation 3-5 days.
(4) bacterial strain of high-yield glutamine transaminase is picked out according to the color of single colonie surrounding media.
The invention also provides the applications that culture medium of the invention is used for bacterial strain screening.
Wherein, the bacterial strain includes streptomyces cyclopentadienyl source streptomycete.
Culture medium of the present invention provides a kind of method of bacterial strain that can intuitively filter out high-yield glutamine transaminase.Shorten
The process of existing " breeding-culture-screening ", realizes the simplification process of " breeding-screening ".With auxotroph culture medium
It is entirely different, any label is not needed, can directly screen to obtain the bacterial strain of high-yield glutamine transaminase.Compare other simultaneously
By the method for selection markers screening, culture medium of the present invention avoids the disadvantage that superior strain is missed because marking not exclusively.
Beneficial effect of the present invention includes: the integral cycle that (1) shortens breeding screening；(2) the breeding screening period is reduced
Required cost；(3) can Effective selection obtain the bacterial strain of high-yield glutamine transaminase；(4) increasing can disposably screen
Flux.
The present invention is lauched the principle of solution release ammonia using glutamy transaminase in single substrate, in addition enzyme concentration and reaction rate
The principle being positively correlated characterizes enzyme concentration, directly producing enzyme efficiency of the measurement bacterial strain in ordinary culture medium using indicator.Simultaneously
Screening used is not utilized with substrate for bacterial strain itself, to exclude the influence of bacterial strain itself.
Detailed description of the invention
Fig. 1 is embodiment 1 using enzyme hydrolysis casein as matrix, of the present invention screening and identification of the cresol red as indicator
As a result.
Fig. 2 is embodiment 2 using gelatin as matrix, of the present invention screening and identification result of the phenol red as indicator.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention,
Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below,
There are no special restrictions to content by the present invention.
Embodiment 1 prepares the culture medium of novel high flux of the present invention screening high-yield glutamine transaminase bacterial strain, including with
The raw material of lower weight proportion forms:
Enzyme hydrolysis casein: 1%
Agar: 2%
Starch: 2%
Potassium nitrate: 1%
Dipotassium hydrogen phosphate: 0.5%
Sodium chloride: 0.5%
Epsom salt: 0.5%
Green vitriol: 0.01%
Cresol red: 0.001%
PH:7.4
The method for making the culture medium of novel high flux screening high-yield glutamine transaminase bacterial strain as described above:
1. weighing 1% enzyme hydrolysis casein, 2% agar, 2% starch, 1% potassium nitrate, 0.5% dipotassium hydrogen phosphate, 0.5%
Sodium chloride, 0.5% epsom salt, 0.01% green vitriol.
2. after supplying volume with water, adjusting pH to 7.4,121 DEG C of autoclave sterilizations after twenty minutes, are put into 65 DEG C of baking oven perseverances
Temperature one hour, is added 0.001% acid-base indicator of filtration sterilization.
3. being poured into each culture dish with the volume of 20mL/ ware after configured culture medium is mixed and being cooled to room temperature.
4. needing the production glutamine transaminage bacterial strain identified to cultivate for two kinds, culture medium is made using step (3)
Novel culture medium.
5. being judged according to the culture medium red depth around single colonie, as shown in Figure 1 after culture 3 days.It is shown in figure
Show, darker, the explanation according to the present invention of B ratio A bacterial strain, the producing enzyme of the producing enzyme ratio A of B bacterial strain is higher.Then to two kinds of need
Identification strain progress ferment in common fermentation culture medium, using N- α-benzyloxy hydroxyl-glutamine-glycine as
The colorimetric method of substrate carries out enzyme activity determination.It carries out enzyme activity and compares discovery, A bacterial strain enzyme activity in 30h fermentation medium is about 1.8U/
ML, B bacterial strain enzyme activity in 30h culture medium are about 2.4U/mL.
Screening and culturing is carried out using the novel screening and culturing medium of the present invention, 96 orifice plates that compare screening saves unicellular expansion
The time of single colonie is increased to, while diluting coated plate efficiency far and choosing bacterium screening higher than 96 orifice plates.The screening period is greatly shortened, together
When also improve screening efficiency.
Embodiment 2 prepares the culture medium of novel high flux of the present invention screening high-yield glutamine transaminase bacterial strain, including with
The raw material of lower weight proportion forms:
Gelatin: 2%
Agar: 2%
Starch: 2%
Potassium nitrate: 1%
Dipotassium hydrogen phosphate: 0.5%
Sodium chloride: 0.5%
Epsom salt: 0.5%
Green vitriol: 0.01%
Phenol red: 0.01%
PH:7.4
A kind of production method of the culture medium of novel high flux screening high-yield glutamine transaminase bacterial strain as described above:
1. weigh 2% gelatin, 2% agar, 2% starch, 1% potassium nitrate, 0.5% dipotassium hydrogen phosphate, 0.5% sodium chloride,
0.5% epsom salt, 0.01% green vitriol.
2. after supplying volume with water, adjusting pH to 7.4,121 DEG C of autoclave sterilizations after twenty minutes, are put into 65 DEG C of baking oven perseverances
Temperature one hour, is added 0.01% phenol red of filtration sterilization.
3. being poured into each culture dish with the volume of 20mL/ ware after configured culture medium is mixed and being cooled to room temperature.
4. needing the production glutamine transaminage bacterial strain identified to cultivate for two kinds, culture medium is made using step (3)
Novel culture medium.
5. being judged according to the culture medium red depth around single colonie, as shown in Figure 2 after culture 5 days.It is shown in figure
Show, darker, the explanation according to the present invention of B ratio A bacterial strain, the producing enzyme of the producing enzyme ratio A of B bacterial strain is higher.Then to two kinds of need
Identification strain progress ferment in common fermentation culture medium, using N- α-benzyloxy hydroxyl-glutamine-glycine as
The colorimetric method of substrate carries out enzyme activity determination.It carries out enzyme activity and compares discovery, A bacterial strain enzyme activity in 30h fermentation medium is about 1.8U/
ML, B bacterial strain enzyme activity in 30h culture medium are about 2.4U/mL.
Claims (4)
1. a kind of culture medium of high flux screening high-yield glutamine transaminase bacterial strain, which is characterized in that the culture medium by with
The raw material of lower weight proportion forms:
Glutamine transaminage substrate: 1%-2%；
Agar: 2%；
Starch: 2%；
Potassium nitrate: 1%；
Dipotassium hydrogen phosphate: 0.5%；
Sodium chloride: 0.5%；
Epsom salt 0.5%；
Green vitriol: 0.01%；
Acid-base indicator: 0.001%-0.01%；
PH:7.4；
Wherein, the glutamine transaminage substrate be enzyme hydrolysis casein or gelatin, the acid-base indicator be cresol red or
Phenol red.
2. culture medium according to claim 1, which is characterized in that its application method is cultivated to be selected on the culture medium
Bacterial strain 3-5 days for screening superior strain.
3. culture medium according to claim 2, which is characterized in that the bacterial strain includes that streptomyces production glutamine turns ammonia
The bacterial strain of enzyme.
4. culture medium described in claim 1 is used for answering in the luxuriant source streptomycete screening of high-yield glutamine transaminase
With.
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| CN201610168342.3A CN105820955B (en) | 2016-03-23 | 2016-03-23 | A kind of culture medium of high flux screening high-yield glutamine transaminase bacterial strain |
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| CN109593821A (en) * | 2019-01-05 | 2019-04-09 | 江苏鸣生物股份有限公司 | The method for screening glutamine transaminage superior strain |
| CN110616208A (en) * | 2019-11-04 | 2019-12-27 | 泰兴市东圣生物科技有限公司 | Optimized fermentation method of glutamine transaminase based on hydrodynamics |
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| JP4323147B2 (en) * | 2002-09-10 | 2009-09-02 | 天野エンザイム株式会社 | Transglutaminase producing bacteria |
| CN101735971A (en) * | 2010-01-12 | 2010-06-16 | 江南大学 | Method for screening high-yield glutamic acid strains in high flux |
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Address after: 200241 No. 500, Dongchuan Road, Shanghai, Minhang District Co-patentee after: TAIXING DONGSHENG FOOD TECHNOLOGY Co.,Ltd. Patentee after: EAST CHINA NORMAL University Address before: 200062 No. 3663, Putuo District, Shanghai, Zhongshan North Road Co-patentee before: TAIXING DONGSHENG FOOD TECHNOLOGY Co.,Ltd. Patentee before: EAST CHINA NORMAL University |