CN109536483A - Production collagen saccharomycete method of mutagenesis based on highfield gravity environment - Google Patents
Production collagen saccharomycete method of mutagenesis based on highfield gravity environment Download PDFInfo
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- CN109536483A CN109536483A CN201710859961.1A CN201710859961A CN109536483A CN 109536483 A CN109536483 A CN 109536483A CN 201710859961 A CN201710859961 A CN 201710859961A CN 109536483 A CN109536483 A CN 109536483A
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Abstract
The invention discloses a kind of production collagen saccharomycete method of mutagenesis based on highfield gravity environment, the highfield gravity environment generated using ground simulator carry out mutagenesis to collagen saccharomycete is produced.The production collagen yeast strain of logarithmic growth phase is first placed under the gravity environment of highfield by the method, magnetic induction density is 12~16T, gravity horizontal is 0~2g, mutation time is 24~72h, mutagenic strain is obtained, then by mutagenic strain applying solid culture medium, the eugonic single colonie of picking is inoculated in fluid nutrient medium shake flask fermentation, using collagen production as examination criteria, high collagen production bacterial strain is obtained.The highfield gravity environment method of mutagenesis generated using ground simulator of the invention, it is easy to operate, efficiency is higher, screening effect is preferable, no pollution to the environment, and bacterial strain is relieved to the fatigue effect of Typical physical and chemical mutagenesis method.
Description
Technical field
The present invention relates to the method for mutagenesis for producing collagen saccharomycete, and in particular to a kind of based on highfield gravity environment
Collagen saccharomycete method of mutagenesis is produced, mutation breeding technologies field is belonged to.
Technical background
Collagen is distributed widely in animal connective tissue, to maintenance cell, tissue, organ normal physiological function and
Injury repair plays an important role, because of its good physical property and biological characteristics, in medicine, organizational project, food, cosmetics
Equal fields also have a wide range of applications.Earliest, commercially available collagen product is mainly the side for passing through acid and alkali hydrolysis or enzymatic hydrolysis
Method is extracted from the tissue such as skin, bone of animal.Such preparation method is restricted the production scale of collagen,
And the inherent shortcoming for obtaining product is very restricted it in fields such as medicine, biomedical materials.Currently, both at home and abroad
Many enterprises and scientific research institutions to produce collagen recombinant strain building and fermentation technology optimization in terms of do a lot of work,
But its fermentation level is still in lower level.
Mutation breeding is, using factors such as physics, chemistry, to induce organism under conditions of artificial and generate mutation, accelerate
The process of its natural evolution obtains excellent character by screening later.In the past, people are obtained perhaps by classic mutagenesis techniques
The plant and microorganism of more merits, but classic mutagenesis breeding has favorable variation few, must largely handle material, the spectrum of mutation
It is narrow, improvement quantitative character effect is poor, the disadvantages of mutagenesis shape is unstable, (Yang little Chong, Chen Zhongjun novel physical induced-mutation technique existed
Application progress [J] food industry in Microbial Breeding, 2017 (3): 242-245.).Space Mutation Breeding is by space flight skill
A kind of new technology for synthesis that art is combined with original physics and chemistry behavior and molecular engineering, utilizes artificial satellite and high air
Ball carries, carries the Organism Samples such as microorganism, through special cosmic space condition (strong cosmic ray, high vacuum, microgravity
Deng) biological stain body is caused to distort, and then lead to organism hereditary variation, it, can be rapidly and efficiently after the selected test of ground
The new varieties (being) of biology are bred as, for producing and studying use.(such as radioinduction, chemistry lure relatively traditional method of mutagenesis
Become), space mutagenesis has many advantages, such as that variation amplitude is big, increases beneficial to variation, the easily stable passage of character.Therefore, educating in early period
In kind work, especially in the breeding work of Important Agricultural crop, some High quality agricultural products kinds are obtained by Space Mutation Breeding
, and carry out national popularization (Zhang Wentao, Du Jiuyuan, Bai Bin Crop spatial mutagenesis and its application [J] kind in genetic breeding
Son, 2014,33 (4): 48-52.).But Space Mutation Technique needs space carrying and carries the cost ten of laboratory sample
Divide valuableness, is not appropriate for general enterprises use.
Space ground simulator can not only save space carrying and carry laboratory sample cost, and can simulate spy
Different space environment, causes bio-genetic material to change, and then leads to organism hereditary variation, can be quick after breeding is tested
And the new varieties of biology are effectively brought out, for producing and studying use.Further, since operation relative ease, research staff can also
Enough single mutagens by control space ground simulator influence mutagenic processes, are produced to investigate different spaces factor
Raw different variation effects and its mechanism.Therefore, ground simulator mutation breeding in space combines Space Mutation Breeding and biography
The advantage of system breeding is a kind of effective, feasible mutation breeding means.There has been no carried out by method for mutation breeding at present
The improvement of collagen producing strains.
Summary of the invention
The present invention is directed to the deficiency of existing mutation breeding technologies, provides a kind of production collagen egg based on highfield gravity environment
White saccharomycete method of mutagenesis, the highfield gravity environment that the method is generated based on space ground simulator, can be effectively
It improves and produces collagen saccharomycete efficiency of inducing mutation under the gravity environment of magnetic field, and obtain corresponding direct mutation bacterial strain, be subsequent bacterial strain
Screening and scale fermenting and producing provide basis.
Technical scheme is as follows:
Production collagen saccharomycete method of mutagenesis based on highfield gravity environment, the specific steps are as follows:
Step 1, the production collagen yeast strain of logarithmic growth phase is placed under the gravity environment of highfield, magnetic induction is close
Degree is 12~16T, and gravity horizontal is 0~2g, and mutation time is 24~72h, obtains mutagenic strain；
Step 2, by mutagenic strain applying solid culture medium, the eugonic single colonie of picking is inoculated in fluid nutrient medium and shakes
Bottle fermentation, using collagen production as examination criteria, obtains high collagen production bacterial strain.
Preferably, in step 1, the production collagen saccharomycete is pichia pastoris CGMCC NO.5021.
Preferably, in step 1, magnetic induction density 12T, gravity horizontal 2g, mutagenesis 48h.
Compared with prior art, the invention has the following advantages that
(1) mutagenesis is carried out using highfield gravity environment, is standard as screening technique using collagen production, operation is simple
Just, high-efficient, screening effect is good, relieve bacterial strain to the fatigue effect of Typical physical and chemical mutagenesis；
It (2) is 12T in magnetic field condition, under the conditions of gravity 2g, mutagenesis 48h, to the collagen producing strains of active growth
The Mutagenic Effect of pichia pastoris CGMCC NO.5021 is best, and expressing quantity 0.724g/L is improved compared with control group
5.9%, collagen producing strains are improved, and high-yield character can stablize heredity.
Specific embodiment
The present invention will be further explained with reference to the examples below.
Each culture medium used in embodiment is as follows:
Solid medium: 1% yeast extract, 2% peptone, 2% glucose, 1.5% agar.
Seed culture medium: 100mM phosphate buffer (pH6.0), 1.34%YNB, 4 × 10-5%Biotin, 1% glycerol (V/
V)。
Fermentation medium: 85%H3PO426.7mL/L；CaSO40.93g/L；K2SO418.2g/L；MgSO4·7H2O
14.9g/L；KOH 4.13g/L；Glycerol 40.0g/L adds PTM after sterilizing1Microelement (CuSO4·5H2O 6.0g/L；
NaI 0.08g/L；MnSO4·H2O 3.0g/L；NaMoO4·2H2O 0.2g/L；H3BO30.02g/L；CoCl20.5g/L；
ZnCl220.0g/L；FeSO4·7H2O 65.0g/L；Biotin 0.2g/L；H2SO45.0mL/L, with 0.22 μm of membrane filtration
Degerming, room temperature preservation).
Induced medium: 100% methanol (PTM containing 12mL/L1)。
Strain: pichia pastoris CGMCC NO.5021.
Embodiment 1
(1) strain culturing:
By strain inoculated in 30mL seed culture medium, shaking table culture to logarithmic growth phase (48h-72h), the dense OD value of bacterium is about
It is 3 or so, revolving speed 180r/min, 28 DEG C of cultivation temperature.
(2) highfield gravity environment mutagenesis:
Mutagenesis is carried out to bacterial strain using superconducting magnet sample stage, tests and divides three groups: a 0-g group (0g, 12T), 1-g group (1g,
16T), 2-g group (2g, 12T).The bacteria suspension 9mL in logarithmic growth phase is taken, (each 3mL) is gone in three culture dishes.By three
A culture dish is respectively placed in three specific positions of sample stage, and apparent gravitation level is respectively 0g, 1g and 2g.Mutagenesis temperature
It is 28 DEG C.Each culture dish seals up preservation with sealed membrane.
In highfield when gravity environment mutagenesis, different time samplings is chosen.Sample time: for 24 hours, 48h, 72h.It takes every time
Sample 0.8mL, and add 0.5mL fresh culture, sample is filled with the microcentrifugal tube of 1.5mL, is stored in 4 DEG C of refrigerators.Initial strains take
Sample 0.8mL, and add 0.5mL fresh culture, sample is filled with the microcentrifugal tube of 1.5mL, 4 DEG C of refrigerators is stored in, compares use.
(3) primary dcreening operation:
9 samples are obtained due to the difference of mutation time in above-mentioned three groups of samples through highfield gravity environment mutagenesis.
By 9 sample applying solid culture mediums, after growing fine (3d), first round screening is carried out.The plate being coated with from each sample
In three eugonic single colonies of each picking be inoculated in fluid nutrient medium respectively and carry out shake flask fermentation, it is another to be arranged without mutagenesis
The original strain of processing is as a control group.Second wheel screening, expression quantity is higher in preferred three groups of shake flask fermentations of previous round respectively
One sample, totally 9 shaking flasks are fermented, while setting a control group, final preferably to go out the highest bacterial strain preservation of one plant of yield.Just
Result is sieved as shown in table 1, table 2, table 3:
Table 1. (0g, 12T) condition primary dcreening operation result
Table 2. (1g, 16T) condition primary dcreening operation result
Table 3. (2g, 12T) condition primary dcreening operation result
(4) secondary screening:
According to the first round screening as a result, will under each mutagenic condition gained sample in the higher bacterium of destination protein expression quantity
Strain summarizes, and is respectively as follows: (1) No.3 for 24 hours 0-g 12T；0-g 12T 48h No.4(1)；0-g 12T 72h No.9(1)；1-g
16T 24h No.1(2)；1-g 16T 48h No.4(2)；1-g 16T 72h No.7(2)；2-g 12T 24h No.1(3)；
2-g 12T 48h No.6(3)；2-g 12T 72h No.8(3)；The second wheel screening is carried out later, and purpose is in identical fermentation item
Under part, the destination protein expression quantity for investigating each bacterial strain is horizontal.Totally 9 groups of experimental group, control group is the 10th group.Secondary screening result such as table 4
It is shown:
4. secondary screening result of table
The selection result of the first round is compareed with the selection result of the second wheel, can be determined substantially through 2-g 12T
No. 6 bacterial strain of 48h condition mutagenesis, destination protein expression quantity are 0.724g/L, improve 5.9% compared with blank control group, tool
There are the value further studied and large-scale production value, genetic stability experiment shows that its high yield characteristics has inheritance stability special
Property.
Claims (3)
1. the production collagen saccharomycete method of mutagenesis based on highfield gravity environment, which is characterized in that specific step is as follows:
Step 1, the production collagen yeast strain of logarithmic growth phase is placed under the gravity environment of highfield, magnetic induction density is
12~16T, gravity horizontal are 0~2g, and mutation time is 24~72h, obtain mutagenic strain；
Step 2, by mutagenic strain applying solid culture medium, the eugonic single colonie of picking is inoculated in fluid nutrient medium shaking flask hair
Ferment obtains high collagen production bacterial strain using collagen production as examination criteria.
2. method of mutagenesis according to claim 1, which is characterized in that in step 1, the production collagen saccharomycete is
Pichia pastoris CGMCC NO.5021.
3. method of mutagenesis according to claim 1, which is characterized in that in step 1, magnetic induction density 12T, gravity horizontal
For 2g, mutagenesis 48h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115368995A (en) * | 2022-09-21 | 2022-11-22 | 合肥大圩葡萄酒有限公司 | Preparation method of mulberry fruit wine |
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|---|---|---|---|---|
| CN102443057A (en) * | 2011-10-26 | 2012-05-09 | 南京理工大学 | Recombinant humanized collagen and its preparation method |
| CN103382466A (en) * | 2013-06-28 | 2013-11-06 | 安徽农业大学 | Method of breeding high-slime-yield mutant strains of lactobacillus by microwave mutagenesis |
| CN104004741A (en) * | 2014-05-29 | 2014-08-27 | 东北农业大学 | Method for preparing plasmin by mutagenizing fermentation of compound mold |
| CN106434457A (en) * | 2016-10-11 | 2017-02-22 | 天津科技大学 | Alkaline protease high-producing strain and alkaline protease produced by same |
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2017
- 2017-09-21 CN CN201710859961.1A patent/CN109536483A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443057A (en) * | 2011-10-26 | 2012-05-09 | 南京理工大学 | Recombinant humanized collagen and its preparation method |
| CN103382466A (en) * | 2013-06-28 | 2013-11-06 | 安徽农业大学 | Method of breeding high-slime-yield mutant strains of lactobacillus by microwave mutagenesis |
| CN104004741A (en) * | 2014-05-29 | 2014-08-27 | 东北农业大学 | Method for preparing plasmin by mutagenizing fermentation of compound mold |
| CN106434457A (en) * | 2016-10-11 | 2017-02-22 | 天津科技大学 | Alkaline protease high-producing strain and alkaline protease produced by same |
Non-Patent Citations (2)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115368995A (en) * | 2022-09-21 | 2022-11-22 | 合肥大圩葡萄酒有限公司 | Preparation method of mulberry fruit wine |
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Application publication date: 20190329 |