CN108977386A - A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain - Google Patents
A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain Download PDFInfo
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Abstract
The present invention relates to field of fermentation engineering, the method for specifically a kind of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain.Malic dehydrogenase is the key enzyme of cell centre oxidation access, and have relevant report its with bacterial flagellum movement has close association, the present invention carries out primary dcreening operation by bromocresol green, and acid stronger bacterial strain is produced in screening；Using the dynamic property of MTT detection bacterial strain, programmed screening is carried out, selects the stronger bacterial strain of dynamic property to survey enzyme activity, the low temperature bacterial strain of high yield MDHs is accurately filtered out by Double Selection, finally by detection enzyme activity, finally to determine superior strain.The present invention can easily and accurately screen the bacterium bacterial strain of low temperature high yield malic dehydrogenase intracellular in 20 days, compensate for the blank of malic dehydrogenase screening and culturing medium related fields.
Description
Technical field
The present invention relates to field of fermentation engineering, the side of specifically a kind of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain
Method.
Background technique
Key enzyme of the MDHs (low temperature malic dehydrogenase) as organism central metabolic pathway, in hereditary variation and individual
Development etc. has important researching value, and has in clinical diagnosis, industrial detection and be widely applied, and market demand is all with day
Increase, Huge value.From the fifties in last century, just there is scholar to isolate MDHs from animal cardiac muscle.At this stage, quotient in the market
The source MDHs of industry is mainly pig, rabbit, the cardiac muscle of ox, liver, extracts in skeletal muscle.Cost of material is low, source is wide, but extracts
Technique is cumbersome, and product enzyme activity is lower.Therefore, expanding new source is particularly important.And marine microorganism is huge resource
Treasure-house.Therefrom screening novel, low temperature, high activity purpose MDHs producing bacterial strain is particularly important.MDHs is widely present
In in nature biotechnology body, but high yield MDHs bacterial strain how is accurately filtered out, currently without specific method.
Due to there is no particularly relevant screening technique to the bacterial strain screening for producing MDHs at present, study its screening technique
It is very necessary to the research of high yield MDHs.
Summary of the invention
To filter out high yield MDHs bacterium bacterial strain, the present invention provides a kind of new screening technique, using bromocresol green-MTT
The screening mode of two steps filters out the strong bacterium bacterial strain of high-yield of low-temperature malate dehydrogenase activity.
For achieving the above object, the invention adopts the following technical scheme:
A method of screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain the described method comprises the following steps:
(1) collecting sample from deep-sea ooze, seawater；
(2) primary dcreening operation: the sample acquired in step (1) carries out gradient dilution, is coated on the beef extract egg containing bromocresol green
White peptone solid medium, 25 DEG C, constant temperature incubation 3-4 days；
(3) it purifies: growing vigorous, the biggish colony inoculation of yellow green transparent circle in picking step (2) in beef extract albumen
Peptone solid medium is isolated and purified, and 25 DEG C, constant temperature incubation 1-2 days, this step repeated 2-4 times；
(4) bacterium colony that step (3) purify secondary screening: is picked into single bacterium colony, percutaneous puncture-inoculation to MTT beef extract egg with transfer needle
White 1/3 depth of peptone semisolid culturemedium；
(5) it screens: choosing the culture medium position for being diffused into along puncture line and not puncturing in the nebulous bacterial strain of blue is diffused, connect
Kind is in beef extract-peptone solid medium, and 25 DEG C, constant temperature incubation 1-2 days；
(6) bacterial strain that step (5) obtains is inoculated in beef extract-peptone fluid nutrient medium with 5%, 150rad, 25 DEG C,
Shaking table culture 1-2 days, enzyme activity was surveyed in thallus ultrasonication, and the high bacterial strain of screening inulinase-producing activity obtains high-yield of low-temperature malate dehydrogenase
Enzyme bacterium bacterial strain.
A kind of above-mentioned method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, containing bromocresol green in step (2)
The bromocresol green content of beef extract-peptone solid medium is 0.1-0.2g/L.
A kind of above-mentioned method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, MTT beef extract albumen in step (3)
The content of the MTT of peptone semisolid culturemedium is 0.015g/L.
A kind of above-mentioned method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, used medium are matched by sea salt water
System.
The present invention can be catalyzed oxaloacetic acid using MDHs and generate this characteristic of L MALIC ACID, by bromocresol green, carry out
Primary dcreening operation (bromocresol green is in yellow in pH=3.8, is in blue-green when pH=5.4), acid stronger bacterial strain is produced in screening, is utilized
MTT selects the stronger bacterial strain of dynamic property to survey enzyme activity, passes through dual sieve to detect the dynamic property of bacterial strain to carry out programmed screening
Choosing, can reduce the scope from boundless and indistinct bacterial strain, more fast, accurately filter out the low temperature bacterial strain of high yield MDHs, finally by
Enzyme activity is detected, finally to determine superior strain.
Compared with prior art, the invention has the benefit that the present invention provides a kind of high-yield of low-temperature malic dehydrogenase
The screening technique of bacterium, the present invention used in screening and culturing medium, be added to bromocresol green and MTT as Screening target, using bromine first
The green two step screening mode of-MTT of phenol screens the high microbes producing cellulase of low temperature, and low temperature high yield born of the same parents can be easily and accurately screened in 20 days
The bacterium bacterial strain of interior malic dehydrogenase compensates for the blank of malic dehydrogenase screening and culturing medium related fields.
Detailed description of the invention
Fig. 1 primary dcreening operation bacterial strain transparent circle；
Fig. 2 MTT secondary screening bacterial strain；A. MTT secondary screening bacterial strain in MTT secondary screening strain b- embodiment 2 in embodiment 1；
The colonial morphology of Fig. 3 colloid bacillus cereus (Bacillus mucilaginosus) W1；
Fig. 4 colloid bacillus cereus (Bacillus mucilaginosus) W1 individual morphology (10X10)；
The colonial morphology of Fig. 5 viscosity Serratieae (Serratia marcescens) Z5；
The individual morphology (10X10) of Fig. 6 viscosity Serratieae (Serratia marcescens) Z5.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.
Embodiment 1
The method that the present invention screens high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, specific as follows:
(1) bacterial strain primary dcreening operation: the ooze seawater sample near the Bohai Offshore of LiaoNing, China Dalian is taken, with the bromine containing 0.1g/L
It is diluted painting plate in the green beef extract-peptone solid medium of cresols, 25 DEG C, constant temperature incubation 3-4 days；According to bacterium colony week
Enclose the size of the partially yellow degree of the color of the yellowish green chromosphere of generation and color ring diameter ratio to judge the high vigor purpose bacterial strain of primary dcreening operation,
The method crossed using three innings is inoculated in beef extract-peptone solid medium (beef extract 3g/L, peptone 10g/L, agar
20g/L, seawater configuration, pH 7.2) purifying culture is carried out, 25 DEG C, constant temperature incubation 1-2 days, this step repeated 2-4 times；
(2) above-mentioned bacterium colony bacterial strain secondary screening: is picked into single bacterium colony, percutaneous puncture-inoculation to MTT semisolid culturemedium with transfer needle
1/3 depth, the content of MTT are 0.015g/L, and when percutaneous puncture-inoculation need to be protected from light operation；Selection is diffused into the training not punctured along puncture line
Base portion position is supported in the nebulous bacterial strain (see Fig. 2 a) of blue is diffused, is inoculated in beef extract-peptone solid medium, 25 DEG C, it is permanent
Temperature culture 1-2 days；Wherein, when preparing MTT beef extract-peptone Semi-solid cell culture, by beef extract-peptone semisolid culturemedium
121 DEG C, 0.1Mpa sterilizing 20min after, make cool to≤40 DEG C of culture medium, the 0.015g/L MTT solution after filtration sterilization added
Enter culture medium, dispenses inverted plate after shaking up.
(3) it surveys enzyme activity: the bacterial strain that step (2) obtains is inoculated in beef extract-peptone fluid nutrient medium, 150r/ with 5%
Min, 25 DEG C, shaking table culture 1-2 days, enzyme activity was surveyed in thallus ultrasonication, while NADH has maximum light absorption value, apple at 340nm
The enzyme activity of acidohydrogenase is by being measured with the reduction amount of UV spectrophotometer measuring NADH light absorption value at 340nm.
The high bacterial strain of inulinase-producing activity is screened, the bacterial strain of one plant of high-yield of low-temperature MDHs is obtained, by single colonie after purification, Gram's stain
For the positive, microscopically observation is elongated rod shape (see Fig. 1), and there is thicker polysaccharide shell in bacterium colony periphery, is not easy by picking, doubtful glue
Matter bacillus (Bacillus mucilaginosus), number W1 (see Fig. 3, Fig. 4).
Embodiment 2
The method that the present invention screens high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, specific as follows:
(1) bacterial strain primary dcreening operation: the ooze seawater sample near the Yellow Sea of Shandong Province of China Qingdao is taken, containing 0.15g/L's
It is diluted painting plate in the beef extract-peptone solid medium of bromocresol green, 25 DEG C, constant temperature incubation 3-4 days；According to bacterium colony
Around generate the partially yellow degree of color of yellowish green chromosphere and the size of color ring diameter ratio judge the high vigor purpose of primary dcreening operation
Bacterial strain, the method crossed using three innings are inoculated in beef extract-peptone solid medium and carry out purifying culture, and 25 DEG C, constant temperature incubation
1-2 days, this step repeated 2-4 times；
(2) above-mentioned bacterium colony bacterial strain secondary screening: is picked into single bacterium colony, percutaneous puncture-inoculation to MTT semisolid culturemedium with transfer needle
1/3 depth, the content of MTT are 0.015g/L；Choosing the culture medium position for being diffused into along puncture line and not puncturing is in diffuse blue cloud
Misty bacterial strain (see Fig. 2 b), is inoculated in beef extract-peptone solid medium, 25 DEG C, constant temperature incubation 1-2 days；
(3) it surveys enzyme activity: the bacterial strain that step (2) obtains is inoculated in beef extract-peptone fluid nutrient medium with 5%,
150rad, 25 DEG C, shaking table culture 1-2 days, enzyme activity was surveyed in thallus ultrasonication, and the high bacterial strain of screening inulinase-producing activity obtains a plant height
The bacterial strain for producing low temperature MDHs, by single colonie after purification, Gram's stain is feminine gender, and microscopically observation is subsphaeroidal quarter butt
Bacterium (see Fig. 2), bacterium colony is sticky, and easily by picking, doubtful viscosity Serratieae (Serratia marcescens), number Z5 is (see figure
5、6)。
Claims (4)
1. a kind of method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain, which is characterized in that the method includes following
Step:
(1) collecting sample from deep-sea ooze, seawater；
(2) primary dcreening operation: the sample acquired in step (1) carries out gradient dilution, is coated on the beef extract-peptone containing bromocresol green
Solid medium, 25 DEG C, constant temperature incubation 3-4 days；
(3) it purifies: growing vigorous, the biggish bacterium colony of yellow green transparent circle in picking step (2) and isolated and purified, using three innings
The method of scribing line is inoculated in beef extract-peptone solid medium；25 DEG C, constant temperature incubation 1-2 days, this step repeated 2-4 times；
(4) bacterium colony that step (3) purify secondary screening: is picked into single bacterium colony, percutaneous puncture-inoculation to MTT beef extract-peptone with transfer needle
1/3 depth of semisolid culturemedium；
(5) it screens: choosing the culture medium position for being diffused into along puncture line and not puncturing in the nebulous bacterial strain of blue is diffused, be inoculated in
In beef extract-peptone solid medium, 25 DEG C, constant temperature incubation 1-2 days；
(6) bacterial strain that step (5) obtains is inoculated in beef extract-peptone fluid nutrient medium with 5%, 150rad, 25 DEG C, shaking table
Enzyme activity is surveyed in culture 1-2 days, thallus ultrasonication, and it is thin to obtain high-yield of low-temperature malic dehydrogenase for the high bacterial strain of screening inulinase-producing activity
Bacteria strain.
2. a kind of method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain as described in claim 1, which is characterized in that
The bromocresol green content range of beef extract-peptone solid medium in step (2) containing bromocresol green is 0.1~0.2g/L.
3. a kind of method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain as described in claim 1, which is characterized in that
The content of the MTT of MTT beef extract-peptone semisolid culturemedium is 0.015g/L in step (4).
4. a kind of method for screening high-yield of low-temperature malic dehydrogenase bacterium bacterial strain as described in claim 1, which is characterized in that
Used medium is by extra large saline.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109897805A (en) * | 2019-04-04 | 2019-06-18 | 大连大学 | A kind of screening technique producing sarcosine oxidase bacterial strain |
| CN113450354A (en) * | 2021-08-30 | 2021-09-28 | 山东仕达思生物产业有限公司 | Trichomonas detection method based on convolutional neural network and swing activity characteristics |
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| US20060046288A1 (en) * | 2004-08-27 | 2006-03-02 | San Ka-Yiu | Mutant E. coli strain with increased succinic acid production |
| CN105648036A (en) * | 2014-11-17 | 2016-06-08 | 山东国际生物科技园发展有限公司 | A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897805A (en) * | 2019-04-04 | 2019-06-18 | 大连大学 | A kind of screening technique producing sarcosine oxidase bacterial strain |
| CN113450354A (en) * | 2021-08-30 | 2021-09-28 | 山东仕达思生物产业有限公司 | Trichomonas detection method based on convolutional neural network and swing activity characteristics |
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Application publication date: 20181211 |