CN106701736A - Composite mutagenesis method of aspergillus niger - Google Patents
Composite mutagenesis method of aspergillus niger Download PDFInfo
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Abstract
The invention discloses a composite mutagenesis method of aspergillus niger. The method comprises the following steps of: (1) pre-culturing spores; (2) carrying out enzymolysis on thalli and removing cell walls; (3) carrying out normal-pressure and room-temperature plasma mutagenesis; (4) carrying out regeneration culture on protoplasts; (5) screening by a 24-pore plate; and (6) fermenting by a shake flask and screening again. According to the method disclosed by the invention, the protoplasts without the cell walls are adopted and a mutagenic agent more easily enters cell nucleuses without the blocking of the cell walls and can rapidly act on hereditary substances; and the probability that the hereditary substances are mutated is greater and the efficiency is higher; and the mutagenesis effect is more obvious than a predicated effect.
Description
Technical field
The present invention relates to the mutagenic and breeding technical field of microorganism fungus kind, more particularly, to a kind of protoplast with it is normal
The method of the compound seed selection aspergillus niger citric acid superior strain of pressure chamber isothermal plasma mutagenesis.
Background technology
Citric acid has broad application prospects and the market demand.Consider for economic aspect, part research is existing
Citric acid is produced on the basis of strain using the less expensive sugar materials of price and agriculture waste residue, and another part research is then
It is devoted to improveing strain.Produce strain transformation and seed selection be citric acid fermentation industry basis, determine fermentation process into
Lose the value with fermented product industrialized production.China is Citric Acid Production big country, although citric acid conversion ratio is already close to reason
By level, yield can reach 170g/L, but fermentation period is partially long, and industrial production fermentation period is commonly 72h, industrial fermentation water
It is flat to need to be improved.
At present, domestic aspergillus niger production bacterial strain is obtained by mutation breeding, and conventional method of mutagenesis is mutagenesis (sulphur
Diethyl phthalate, nitrosoguanidine and lithium chloride etc.) and physical mutagenesis (ultraviolet,60Co etc.).Chemical mutagen mainly by with base knot
Conjunction forms adduct and causes DNA to be alkylated and introduces variation, ultraviolet, and cyclobutane pyrimidine dimer is formed by making DNA molecular
The normal pairing between base is hindered to cause variance.There are security risks in these mutagenesis operation, efficiency of inducing mutation is controllable in addition
Property is limited.Atmospheric pressure at room plasma mutagenesis is the technology that latest developments are got up, and electronics obtains energy under rf electric field effect,
By collision, there is energy exchange with neutral particle around, it decomposed, excited or ionize, under the effect of two step voltages, gas
It is breakdown to produce electric discharge, form the plasma with certain degree of ionization.Can be caused carefully by the neutral active particle of high concentration
The sublethal effect of born of the same parents, makes nucleic acid open loop, fracture, broken existing picture occur, recycles the DNA repair mechanisms of cell itself, realizes
The change of intracellular inhereditary material.Additionally, the active oxygen species (reactive oxygenspecies, ROS) that plasma is produced
Enter cell because permeability of cell membrane strengthens, also further result in DNA damage.Therefore plasma mutagenesis can be caused simultaneously
Gene mutation and chromosomal variation, are expected to obtain high yield aspergillus niger Citric Acid Production bacterial strain.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of composite mutagenesis method of aspergillus niger.This
Inventive method is simple and convenient to operate using equipment, efficiency of inducing mutation is high.
Technical scheme is as follows：
A kind of composite mutagenesis method of aspergillus niger, methods described be carried out on aspergillus niger protoplast atmospheric pressure at room etc. from
Daughter mutagenesis, specifically includes following steps：
(1) spore preculture；(2) bacterial enzyme frees wall；(3) atmospheric pressure at room plasma mutagenesis；(4) protoplast regeneration
Culture；(5) 24 orifice plates are screened；(6) shake flask fermentation secondary screening.
The method of step (1) the miospore preculture is：Collecting spore from the good inclined-plane of activation culture (will -80 DEG C of jelly
The aspergillus niger glycerol tube kind deposited is transferred to slant medium and carries out activation culture, and 34-37 DEG C is cultivated 6 days, is activated 2 times), and with
106The inoculum concentration of individual/mL is inoculated into the 250mL triangular flasks equipped with 50mL culture mediums, 30 DEG C, 200rpm cultures 16h, Ran Houshou
Collection bacterium ball.
The inclined-plane culture based formulas：Malt extract 30g/L, tryptone 5g/L, agar 1.5%, 121 DEG C of sterilizings
15min。
The specific method of the step (2) is：Filtered after bacterium ball is washed into 2~3 times with KMC liquid, be subsequently adding 50mL molten
Wall enzyme and chitinase solution, mix, and after being placed in 37 DEG C of water-bath isothermal holding 2h, 4 DEG C of 2000g are centrifuged 10min, abandon supernatant,
2 removing cell membrane enzymolysis liquids are washed with KMC liquid, protoplast is suspended in standby in 100 μ L KMC liquid.
The KMC liquid：0.7M KCl, 50mM CaCl2, 5.8,121 DEG C of 20mM Mes/NaOH, pH sterilizing 15min, 4 DEG C
Save backup；
The cell membrane enzymolysis liquid：5mg/mL lywallzymes, 0.2U/mL chitinases, 0.22 μM of sterilizing filter are degerming.
The specific method of the step (3) is：By protoplast suspension draw 10 μ L be placed on slide glass, atmospheric pressure at room etc. from
Daughter mutagenic treatment 120s, condition of work is：Gas flow 10slm, irradiation power 100W.
The specific method of the step (4) is：By protoplast KMC liquid graded series 10-105Times, draw 100 μ L former
Raw plastid dilution carries out regeneration culture, 35 DEG C of culture 36h of condition of culture in regenerated solids culture medium.
The protoplast regeneration culture medium prescription：Malt extract 30g/L, tryptone 5g/L, 1.2M sorbierite,
1% agar, 121 DEG C of sterilizing 15min.
The method of 24 orifice plates screening is in the step (5)：The list that will be grown on protoplast regeneration culture medium
Bacterium colony is waited to grow dense spore, and primary dcreening operation is carried out with 24 orifice plates；Specific steps include：
1. seed culture：Each hole fills 3mL seed culture mediums, is inoculated with 1 bacterial strain, 35 DEG C, 180rpm cultures 24h；It is described
Seed culture medium is that corn clear liquid and mixed liquid mix in proportion, total sugar content 10%, total nitrogen content 0.2%；
2. fermented and cultured：Each hole fills 3mL fermentation mediums, the μ L of inoculum concentration 300,35 DEG C, 180rpm cultures 60h；It is described
Fermentation medium：Corn clear liquid and mixed liquid mix in proportion, total sugar content 15%, total nitrogen content 0.08%, 121 DEG C of sterilizings
15min；
3. screen：Screening superior strain by detecting the lemon acid yield in each duct carries out preservation.
Step (6) the shake flask fermentation secondary screening is by two processes of seed culture and fermented and cultured, wherein seed culture：
The bottled 50mL seed culture mediums of 250mL triangles, 35 DEG C of temperature, 250rpm cultivates 24h；Fermented and cultured is that 250mL triangles are bottled
50mL zymotic fluids, seed inoculum concentration is 10%, 35 DEG C of temperature, and rotating speed 250rpm cultivates 72h.
Methods described also including step (7) mitotic stability detect, i.e.,：Spore is prepared into the cryopreservation tube containing 15% glycerine,
Freeze in -80 DEG C, activation culture and as the first generation；Spore liquid is scraped from first generation inclined-plane kind, solid is coated after dilution
Single bacterium colony is transferred to new inclined-plane culture by 35 DEG C of culture 24h of culture medium to single bacterium colony is grown, and this is the second generation；From the second generation
Inclined-plane scrapes spore liquid, and 35 DEG C of culture 24h of solid medium are coated after dilution to single bacterium colony is grown, and single bacterium colony is transferred to newly
Inclined-plane culture, this is the third generation；By that analogy, reached for the 20th generation, 20 generation bacterial strains are carried out into shake flask fermentation experiment, and detect hair
Zymotic fluid citric acid yield.
The present invention is beneficial to be had technical effect that：
The protoplast after cell membrane is sloughed in the inventive method, there is no the stop of cell membrane, mutagens are easier to enter
In nucleus, inhereditary material can be rapidly acted on, the probability that inhereditary material morphs is bigger, in hgher efficiency, mutagenesis effect
Fruit can become apparent from；
Atmospheric pressure at room plasma induced-mutation technique equipment is simple and convenient to operate, does not need vacuum condition in the inventive method,
Plasma jet temperature is low and active particle is evenly distributed, and environment and people are safe from harm；
The inventive method can obtain the mutagenic strain that citric acid yield is greatly improved, with preferable genetic stability, lemon
Lemon acid yield brings up to 140g/L, effect is significant from 120g/L.
Specific embodiment
With reference to embodiment, the present invention is specifically described.
Embodiment
A kind of composite mutagenesis method of aspergillus niger, methods described be carried out on aspergillus niger protoplast atmospheric pressure at room etc. from
Daughter mutagenesis, specifically includes following steps：
(1) spore preculture, the aspergillus niger glycerol tube kind that -80 DEG C are frozen is transferred to slant medium (malt extract
30g/L, tryptone 5g/L, agar 1.5%, 121 DEG C of sterilizing 15min) activation culture is carried out, 37 DEG C are cultivated 6 days, are activated 2 times
Afterwards, spore is scraped；The spore of scraping is with 106The inoculum concentration of individual/mL is inoculated into the 250mL triangular flasks equipped with 50mL culture mediums,
30 DEG C, then 200rpm culture 16h collect bacterium ball；
(2) bacterial enzyme frees wall：Take after bacterium ball washs 3 times with KMC liquid, be subsequently adding 50mL lywallzymes and chitinase is molten
Liquid (5mg/mL lywallzymes, 0.2U/mL chitinases, 0.22 μM of sterilizing filter are degerming), mixes, and is placed at 37 DEG C of water-bath insulations
After reason 2h, 2000g centrifugation 10min abandon supernatant, and 2 removing lywallzymes are washed with hypertonic KMC liquid, and protoplast is suspended in
100 μ L KMC liquid (0.7M KCl, 50mM CaCl2, 5.8,121 DEG C of 20mM Mes/NaOH, pH sterilizing 15min, 4 DEG C of preservations are standby
With) in it is standby；
(3) atmospheric pressure at room plasma mutagenesis：Protoplast suspension is drawn into 10 μ L to be placed on slide glass, atmospheric pressure at room etc.
Gas ions mutagenesis (gas flow 10slm, irradiation power 100W) processes 120s；
(4) protoplast regeneration culture：Take after mutagenesis and the KMC liquid gradient dilutions of the protoplast without mutagenesis (control)
10~105Times, 0.1mL protoplasts dilution is drawn in regenerated solids culture medium (malt extract 30g/L, tryptone 5g/
L, 1.2M sorbierite, 1% agar, 121 DEG C of sterilizing 15min) in carry out regeneration culture, 35 DEG C of culture 36h of condition of culture；
(5) 24 orifice plates are screened：The single bacterium colony grown on protoplast regeneration culture medium is waited to grow dense spore,
Primary dcreening operation is carried out with 24 orifice plates；Specific steps include：
1. seed culture：Each hole fills 3mL seed culture mediums, and (corn clear liquid and mixed liquid mix in proportion, total sugar content
10%, total nitrogen content 0.2%), it is inoculated with 1 bacterial strain, 35 DEG C, 180rpm cultures 24h；
2. fermented and cultured：Each hole fills 3mL fermentation mediums, and (corn clear liquid and mixed liquid mix in proportion, total sugar content
15%, total nitrogen content 0.08%, 121 DEG C of sterilizing 15min), the μ L of inoculum concentration 300,35 DEG C, 180rpm cultures 60h；
3. screen：Screening superior strain by detecting the lemon acid yield in each duct carries out preservation.
(6) shake flask fermentation secondary screening：By two processes of seed culture and fermented and cultured, wherein seed culture is：250mL tri-
The bottled 50mL seed culture mediums in angle, 35 DEG C of temperature, 250rpm cultivates 24h；Fermented and cultured is the bottled 50mL fermentations of 250mL triangles
Liquid, seed inoculum concentration is 10%, 35 DEG C of temperature, and rotating speed 250rpm cultivates 72h；
(7) mitotic stability detection：Spore is prepared into the cryopreservation tube containing 15% glycerine, is frozen in -80 DEG C, activation culture is simultaneously
As the first generation；Spore liquid is scraped from first generation inclined-plane kind, 35 DEG C of solid medium culture 24h are coated after dilution to growing
Single bacterium colony, new inclined-plane culture is transferred to by single bacterium colony, and this is the second generation；Spore liquid is scraped from second generation inclined-plane, is applied after dilution
35 DEG C of culture 24h of solid medium are distributed in single bacterium colony is grown, single bacterium colony is transferred to new inclined-plane culture, this is the third generation；
By that analogy, reached for the 20th generation, 20 generation bacterial strains are carried out into shake flask fermentation experiment, and detect zymotic fluid citric acid yield.
Test case：
(1) measure of lemon acid yield
Citric acid determination of yield method is 24 orifice plate screening AASs, shake flask fermentation secondary screening HPLC methods.
Described AAS is nutrient solution in 24 orifice plates, after taking 100 times of supernatant dilution, by 143 μ L acetic anhydride, 32
μ L pyridines and the zymotic fluid of 25 μ L dilutions are mixed, and 96 orifice plates are placed in into 28 DEG C of constant incubator reaction 30min, ELIASA detection
OD420.Test result is as shown in table 1.
Table 1
Described HPLC detection methods are fermentation broth samples collects supernatant through 13000rpm centrifugations 5min, and with 0.45 μm of filter membrane
Filtering, freezes in -20 DEG C；Using Amethyst C18-H chromatographic columns (Sepax technoligies, Inc.) with 0.01%
H3PO4It is mobile phase, flow velocity 0.8mLmin-1, temperature keep 30 DEG C, detected at 210nm using UV-detector.Survey
Test result result is as shown in table 2.
Table 2
| Bacterial strain | A427 | B122 | C38 | H915-1 |
| Citric acid content (g/L) | 157 | 163 | 152 | 145 |
(2) genetic stability experiment
Spore is prepared into the cryopreservation tube containing 15% glycerine, is frozen in -80 DEG C, activation culture and as the first generation；From
Generation inclined-plane kind scraping spore liquid, coats 35 DEG C of culture 24h of solid medium to single bacterium colony is grown after dilution, single bacterium colony is turned
New inclined-plane culture is connected to, this is the second generation；Spore liquid is scraped from second generation inclined-plane, 35 DEG C of solid medium is coated after dilution
24h is to single bacterium colony is grown for culture, and single bacterium colony is transferred into new inclined-plane culture, and this is the third generation；By that analogy, the 20th is reached
In generation, 20 generation bacterial strains are carried out into shake flask fermentation experiment, and detect zymotic fluid citric acid yield.
The 7th generation bacterial strain for choosing superior strain carries out shake flask fermentation experiment, detects that zymotic fluid citric acid contains with HPLC methods
Amount, the citric acid yield of bacterial strain is in 150g/L or so.
(3) the 7th generation Aspergillus niger strain B122, prior art obtained in embodiment are often inoculated in respectively with aspergillus niger Co82
PDA culture medium (malt extract 30g/L, tryptone 5g/L) upper 35 DEG C raw spores culture 7 days, scrapes spore, with 106/ mL's
Inoculum concentration is inoculated in seed culture medium (cornstarch culture medium, total sugar content 10%, total nitrogen content 0.2%), 37 DEG C, 250r/
Min cultivates 24h, and with 1/10 inoculum concentration switching fermentation medium, 35 DEG C, 250r/min fermentation 72h, zymotic fluid centrifugation is gone degerming
Body, dilutes 10 times, and citric acid content is detected with HPLC with after membrane filtration.Test data is as shown in table 3.
Table 3
| Citric acid content (g/100mL) | Conversion ratio (%) | Fermentation period (hour) | |
| Embodiment | 16.1 | 95 | 55 |
| Aspergillus niger Co82 | 13 | 92 | 60 |
| Aspergillus niger TN-A09 | 12.5 | 92 | 60 |
(4) the 7th generation Aspergillus niger strain B122 of embodiment gained, prior art are often inoculated in respectively with aspergillus niger zjs-8
ME culture mediums (malt extract 30g/L, tryptone 5g/L) upper 35 DEG C raw spores culture 7 days, scrapes spore, with 106/ mL's connects
The amount of kind is inoculated in seed culture medium (cornstarch culture medium, total sugar content 10%, total nitrogen content 0.2%), 37 DEG C, 250r/min
Culture 24h, with 1/10 inoculum concentration switching fermentation medium, 42 DEG C, 250r/min fermentations 72h.Zymotic fluid centrifugation removal thalline is dilute
10 times are released, citric acid content is detected with HPLC with after membrane filtration.Test result is as shown in table 4.
Table 4
| Citric acid content (g/100mL) | Conversion ratio (%) | Fermentation period (hour) | |
| Embodiment | 13.2 | 78.22 | 50 |
| Aspergillus niger zjs-8 | 10 | 61.83 | 60 |
Can be seen that the prepared mutagenic strain of the embodiment of the present invention by the data of table 4 has good heat-resisting quantity, is carried in temperature
Lemon acid yield and conversion ratio remain above existing aspergillus niger zjs-8 in the case of height.
(5) the 7th generation Aspergillus niger strain B122 and aspergillus niger Co82 of embodiment gained is inoculated in ME culture medium (malt respectively
Extract 30g/L, tryptone 5g/L) upper 35 DEG C raw spores culture 7 days, spore is scraped, with 106The inoculum concentration of/mL is inoculated in kind
Sub- culture medium (cornstarch culture medium, total sugar content 10%, total nitrogen content 0.2%, pH 3.5), 37 DEG C, 250r/min cultures
24h, with 1/10 inoculum concentration switching fermentation medium (pH 2.0), 42 DEG C, 250r/min fermentations 72h.Zymotic fluid centrifugation is gone degerming
Body, dilutes 10 times, and citric acid content is detected with HPLC with after membrane filtration.Test result is as shown in table 5.
Table 5
| Citric acid content (g/100mL) | Conversion ratio (%) | Fermentation period (hour) | |
| Embodiment | 16.5 | 95 | 60 |
| Aspergillus niger Co82 | 13 | 93 | 65 |
As can be seen from Table 5, embodiment of the present invention gained strain is under more harsh acid condition, still with preferable
Lemon acid yield and conversion ratio, relatively short fermentation period, strain of the present invention have more preferable acid resistance.
Claims (7)
1. a kind of composite mutagenesis method of aspergillus niger, it is characterised in that methods described is that normal pressure is carried out on aspergillus niger protoplast
Room-temperature plasma mutagenesis, specifically includes following steps：
(1) spore preculture；(2) bacterial enzyme frees wall；(3) atmospheric pressure at room plasma mutagenesis；(4) protoplast regeneration training
Support；(5) 24 orifice plates are screened；(6) shake flask fermentation secondary screening.
2. method according to claim 1, it is characterised in that the method for step (1) the miospore preculture is：From work
Change cultured inclined-plane and collect spore, and with 106The inoculum concentration of individual/mL is inoculated into the 250mL triangular flasks equipped with 50mL culture mediums
In, 30 DEG C, then 200rpm culture 16h collect bacterium ball.
3. method according to claim 1, it is characterised in that the specific method of the step (2) is：By bacterium ball KMC liquid
Filtered after washing 2~3 times, be subsequently adding 50mL lywallzymes and chitinase solution, mixed, be placed in 37 DEG C of water-bath isothermal holding 2h
Afterwards, 4 DEG C of 2000g centrifugation 10min, abandon supernatant, and 2 removing cell membrane enzymolysis liquids are washed with KMC liquid, and protoplast is suspended in
It is standby in 100 μ L KMC liquid.
4. method according to claim 1, it is characterised in that the specific method of the step (3) is：Protoplast is hanged
Liquid is drawn 10 μ L and is placed on slide glass, atmospheric pressure at room plasma mutagenic treatment 120s, and condition of work is：Gas flow 10slm, shines
Penetrate power 100W.
5. method according to claim 1, it is characterised in that the specific method of the step (4) is：Protoplast is used
KMC liquid graded series 10-105Times, draw 100 μ L protoplasts dilutions carries out regeneration culture in regenerated solids culture medium,
35 DEG C of culture 36h of condition of culture.
6. method according to claim 1, it is characterised in that the method for 24 orifice plates screening is in the step (5)：Will be
The single bacterium colony grown on protoplast regeneration culture medium is waited to grow dense spore, and primary dcreening operation is carried out with 24 orifice plates.
7. method according to claim 1, it is characterised in that methods described also detects including step (7) mitotic stability,
I.e.：Spore is prepared into the cryopreservation tube containing 15% glycerine, is frozen in -80 DEG C, activation culture and as the first generation；It is oblique from the first generation
Face kind scraping spore liquid, coats 35 DEG C of culture 24h of solid medium to single bacterium colony is grown after dilution, single bacterium colony is transferred to newly
Inclined-plane culture, this is the second generation；Spore liquid is scraped from second generation inclined-plane, 35 DEG C of cultures of solid medium are coated after dilution
Single bacterium colony is transferred to new inclined-plane culture by 24h to single bacterium colony is grown, and this is the third generation；By that analogy, reached for the 20th generation, will
20 generation bacterial strains carry out shake flask fermentation experiment, and detect zymotic fluid citric acid yield.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586101A (en) * | 2009-07-10 | 2009-11-25 | 徐州工程学院 | The preparation method of lithium chloride mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains |
| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN106222094A (en) * | 2016-07-27 | 2016-12-14 | 烟台大学 | The rich soy sauce brewing bacterial strain producing succinic acid and mutagenic breeding method thereof and application |
-
2017
- 2017-01-12 CN CN201710022793.0A patent/CN106701736A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586101A (en) * | 2009-07-10 | 2009-11-25 | 徐州工程学院 | The preparation method of lithium chloride mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains |
| CN103215250A (en) * | 2013-03-30 | 2013-07-24 | 徐州鸿宇农业科技有限公司 | Protoplast mutation breeding method for improving cordycepin content of cordyceps militaris |
| CN106222094A (en) * | 2016-07-27 | 2016-12-14 | 烟台大学 | The rich soy sauce brewing bacterial strain producing succinic acid and mutagenic breeding method thereof and application |
Non-Patent Citations (1)
| Title |
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| 范新蕾: "产葡萄糖氧化酶菌株的诱变选育及发酵条件优化", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
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Application publication date: 20170524 |