CN104164415A - Screening method of D-arabitol high-yield yeast mutant strain - Google Patents
Screening method of D-arabitol high-yield yeast mutant strain Download PDFInfo
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Abstract
The invention provides a screening method of a D-arabitol high-yield yeast mutant strain. By using a mutant strain obtained by carrying out mutagenesis on Kluyveromyces lactis by a low-energy N+ ion implantation technique as a screening object, a high-flux screening method, which is implemented by primary screening based on a high-sugar solid culture medium and secondary screening based on paper chromatography/high-performance liquid chromatography combination, is established. The method has the advantages of high screening efficiency, low cost, short screening period and the like, is easy to operate, and provides a new method for efficiently screening the D-arabitol high-yield strain by modifying the yeast with low-energy ion implantation.
Description
Technical field
The invention belongs to ion beam technology field, be specifically related to a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain.
Background technology
D-R alcohol is a kind of five-carbon sugar alcohol, and molecular formula is C
5h
12o
5, molecular weight is 152.12, is the isomers of Xylitol and ribitol.Sugar alcohol is through a class polyvalent alcohol of sugar reduction, has low heat value, anti-dental caries, does not affect the advantages such as insulin level, has important using value in fields such as medicine, food, chemical industry.At present, conventional method of producing D-R alcohol is mainly that chemical method is synthetic, but the method reaction process complexity, equipment requirements is high, environmental pollution is serious.For the shortcoming of chemical synthesis, people concentrate on sight by biotransformation method and produce D-R alcohol, and its reaction conditions gentleness, production technique are simple, thereby receive much concern.
The people such as Spencer study and find that osmophilic yeast oozes under condition and can produce polyvalent alcohol at height, the polyvalent alcohol that metabolizable glucose produces mainly contains glycerine, D-R alcohol, red liquor-saturated sugar alcohol etc., wherein five carbon polyols are taking D-R alcohol as main, can be used for up to now producing the bacterial strain of D-R alcohol, mainly comprise Eurotium (Aspergillus), mycocandida (Candida), Debaryomyces (Debaryomyces), the mould genus of robe (EndomycoPsis) in intending, little Cong stalk robe belongs to (Moniliella), Hansenula (Hansenula), finish red female belong to (Pichia), zygosaccharomyces belongs to (Zygosaccharomyces).But, the yeast strain of most of product D-R alcohol is lower to the transformation efficiency of substrate glucose, the transformation efficiency of the substrate glucose of the transforming glucose product D-R alcohol of most yeast strains is between 10%～40%, and ubiquity glycerine and other byproduct of polyhydric alcohol things.Kluyveromyces lactis is a kind of yeast that can be using lactic acid as its unique carbon source and the energy, have nutritional requirement simple, grow vigorous, biomass large, growth temperature wide accommodation (25～46 DEG C), secretory protein ability by force, do not produce intracellular toxin, to advantages such as human security, be therefore used to for a long time fermentation industry always.
Low energy ion N
+implantttion technique, as a kind of new mutation source, obtains remarkable effect in Microbial Breeding at present at home, and its reason is that low energy ion exists energy exchange, energy deposition, quality deposition and the large effect of charge-exchange four simultaneously while injecting organism.Different owing to injecting the combination of charge number, total mass number, energy and dosage of ion, numerous mutagenic conditions can be provided, make ion implantation mutagenesis there is sudden change spectrum width, the feature that mutation rate is high, thus can screen the mutant that meets production requirement, improves output.But low energy N
+the randomness that ion implantation technique transforms is larger, sets up a kind of high-throughout rapid screening method particularly important.
Summary of the invention
The object of the present invention is to provide a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain.
For achieving the above object, the present invention has adopted following technical scheme:
1) primary dcreening operation: the yeast strain that sets out through mutagenesis is inoculated in YPD liquid nutrient medium, then under 28～37 DEG C, 160～200r/min, shaking culture 24～28h obtains preculture liquid, preculture liquid is coated on high sugared solid medium, then in 28～37 DEG C of constant temperature culture 48～60h, the glucose that the sugared solid medium of described height contains 500～550g/L;
2) multiple sieve: after constant temperature culture, picking grows in the single bacterium colony on high sugared solid medium and is inoculated in YPD liquid nutrient medium, then in 28～37 DEG C, under 160～200r/min, shaking culture 24～28h obtains seed culture fluid, seed culture fluid is inoculated in YPD fermention medium, then in 160～200r/min, at 28～37 DEG C, shaking culture 96～144h obtains fermented liquid, fermented liquid is centrifugal, the centrifugal supernatant liquor obtaining is analyzed by paper chromatography, according to the higher mutant strain of colour developing spot screening D-R alcohol output that on paper chromatography, D-R alcohol is corresponding.
Utilize low energy ion implantttion technique to carry out mutagenesis to the yeast strain that sets out.
The described yeast strain that sets out is Kluyveromyces lactis.
Described mutagenesis specifically comprises the following steps:
A) picking sets out the single colony inoculation of yeast strain in YPD liquid nutrient medium, and then under 28～37 DEG C, 160～200r/min, shaking culture 18～24h obtains bacterium liquid;
B) with sterilized water, bacterium liquid is diluted to 0.5～1.0 × 10
7cFU/mL obtains mycelium dilution liquid, and 100～150 μ L mycelium dilution liquid are evenly coated to aseptic plate central authorities, then dries up to obtain mycoderm with sterile wind;
C) mycoderm is placed on the aseptic target platform of ion implanter, and by following condition, mycoderm is carried out to low energy ion injection: injection ion is N
+, implantation dosage is 1.5 × 10
15～2.5 × 10
16ions/cm
2, Implantation Energy is 15～25KeV, and the burst length is 5～10s, and be 5～10s interval time, and vacuum tightness is 1.5～2.0 × 10
-4pa.
The composition of the sugared solid medium of described height comprises the glucose of 500.0g/L, peptone, the yeast extract paste of 10.0g/L and the agar of 20.0g/L of 20.0g/L.
The composition of described YPD fermention medium comprises glucose, the peptone of 20.0g/L and the yeast extract paste of 10.0g/L of 200.0g/L.
The developping agent that described paper chromatography adopts is the mixture of propyl carbinol, pyridine and water, propyl carbinol: pyridine: the volume ratio of water is 14:3:3; The developer that paper chromatography adopts is saturated Silver Nitrate-acetone soln and saturated sodium hydroxide-ethanol solution, the preparation method of saturated Silver Nitrate-acetone soln is: saturated 0.1mL silver nitrate aqueous solution is joined in 40mL acetone to obtain to mixture, after adding 1mL water in mixture, by stirring, the Silver Nitrate of crystallization is dissolved completely, the preparation method of saturated sodium hydroxide-ethanol solution is: saturated 10mL aqueous sodium hydroxide solution is mixed with 520mL dehydrated alcohol; Paper chromatography accepted standard product are the equal-volume mixed solution of D/W, the D-R alcohol solution of mass concentration 30g/L and the aqueous glycerin solution of mass concentration 30g/L of mass concentration 30g/L.
Described screening method is further comprising the steps of:
Checking: utilize high performance liquid chromatography to analyze the fermented liquid of the higher mutant strain of D-R alcohol output.
Beneficial effect of the present invention is embodied in:
The present invention is to utilize low energy ion (N
+) implantttion technique is carried out mutagenesis to the yeast strain that sets out (Kluyveromyces lactis) and the mutant strain that obtains is screening object, has set up high glucose medium primary dcreening operation, the high-throughput screening method that paper chromatography is sieved again.
The present invention has carried out a large amount of experiments to the condition of the low energy ion implantttion technique of utilizing and has groped, and the conditions such as determined implantation dosage can ensure stable screening efficiency.Composition and the developer of the present invention to standard substance is optimized, and can accelerate breakneck acceleration, improves the reliability of the selection result.The present invention is by integrating the high efficiency of high glucose medium primary dcreening operation, the high accuracy of the simplicity that paper chromatography is sieved again and high performance liquid chromatography checking and the high-flux fast screening method set up can ensure stable screening efficiency.
Brief description of the drawings
Fig. 1 is the mitotic stability test of superior strain.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
(1) substratum
Consisting of of high sugared solid medium: glucose 500.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, agar 20.0g/L, pH is 6.5;
Consisting of of YPD liquid nutrient medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, pH is 6.5;
Consisting of of YPD fermention medium: glucose 200.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, pH is 6.5.
Consisting of of slant medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract paste 10.0g/L, agar 20g/L, pH is 6.5;
(2) method of screening institute foundation
(1) resistance to high sugar experiment: the starting strain injecting through low energy ion is inoculated on high sugared solid medium, in 37 DEG C of constant temperature culture 48h, observes thalli growth situation.
(2) paper chromatography is sieved again: respectively by 5 μ L supernatant liquors (fermented liquid is centrifugal) and standard substance point sample in chromatography filter paper, at developping agent propyl carbinol-pyridine-water (V/V/V, chromatography 24h (room temperature) 14:3:3), dry that rear (1. getting the saturated silver nitrate solution of 0.1mL joins in 40mL acetone with developer, then dropwise add 1mL water and stir the Silver Nitrate dissolving extremely completely that makes crystallization, be mixed with saturated Silver Nitrate-acetone soln, wherein saturated silver nitrate solution is that mass concentration is the silver nitrate aqueous solution of 2500g/L, 2. getting the saturated sodium hydroxide solution of 10mL joins in 520mL dehydrated alcohol, be mixed with saturated sodium hydroxide-ethanol solution, wherein saturated sodium hydroxide solution is that mass concentration is the aqueous sodium hydroxide solution of 530g/L) colour developing, development step is: under room temperature, on the filter paper after drying, first spray the saturated Silver Nitrate-acetone soln of one deck, after filter paper seasoning, on filter paper, spray the saturated sodium hydroxide-ethanol solution of one deck again, standard substance are the equal-volume mixed solution of following three kinds of solution: the D/W of mass concentration 30g/L, the D-R alcohol solution of mass concentration 30g/L and the aqueous glycerin solution of mass concentration 30g/L.In standard substance, glucose, D-R alcohol and the sequence of positions of glycerine on filter paper are: below for glucose, centre is D-R alcohol, is glycerine above, the spot size on observation D-R alcohol correspondence position.
(3) high performance liquid chromatography checking: detect the outer D-R alcohol content of born of the same parents in fermented liquid.Chromatographic column: SH1011; Moving phase: 0.01mol/L H
2sO
4; Flow velocity: 0.8mL/min; Sample size: 5 μ L; Column temperature: 50 DEG C; Detector: differential detector (RID).
(3) structure of mutant strain
Use low energy N
+ion implantation technique is carried out mutagenesis to Kluyveromyces lactis, builds D-R alcohol high production bacteria.
(1) preparation of bacteria suspension
By the Kluyveromyces lactis of this laboratory preservation (Kluyveromyces lactis, ATCC12426, purchased from Chinese industrial microbial strains preservation administrative center) as starting strain, starting strain is rule on slant medium, be placed in after 37 DEG C of constant temperature culture 24h, picking list colony inoculation, in YPD liquid nutrient medium, obtains bacterium liquid in 37 DEG C, 160r/min shaking culture 18h.
(2) mycoderm preparation
Bacterium liquid is diluted to 1.0 × 10 with sterilized water
7cFU/mL obtains mycelium dilution liquid, gets 100 μ L mycelium dilution liquid and evenly coats aseptic plate central authorities, dries up and makes mycoderm with sterile wind.
(3) low energy ion injects optimum parameter
Be 20KeV, burst length to be that 5s, interval time are 10s at energy, vacuum tightness is 1.5 × 10
-4under Pa condition, by various dose (0 × 10
14, 15 × 10
14, 35 × 10
14, 55 × 10
14, 75 × 10
14, 95 × 10
14, 115 × 10
14, 135 × 10
14ions/cm
2) Kluyveromyces lactis is carried out to mutagenic treatment, lethality rate situation in conjunction with low energy ion to cell, draw the curve of lethality rate and implantation dosage, lethality rate reduces along with implantation dosage increase is first to increase " saddle " type curve increasing again afterwards, obtaining best mutagenesis injection parameter is: energy is 20KeV, burst length to be that 5s, interval time are 10s, and vacuum tightness is 1.5 × 10
-4pa, implantation dosage is 75 × 10
14ions/cm
2.
(4) low energy ion injects mycoderm
To be placed in the aseptic plate of described mycoderm on the aseptic target platform of ion implanter, be that 20keV, implantation dosage are 75 × 10 with energy
14ions/cm
2, the burst length is 5s, interval time to be that 10s, vacuum tightness are 1.5 × 10
-4pa, carries out low energy ion injection.
(4) mutant strain screening process
Kluyveromyces lactis after low energy ion injects is inoculated on high sugared solid medium and carries out preliminary screening; The bacterial strain that primary dcreening operation is obtained further carries out YPD liquid fermentation and culture, utilizes paper chromatography and liquid chromatography analysis fermentation gained fermented liquid, thereby to the further Screening and Identification of mutant strain.Described fermentation culture comprises the following steps: being transferred to YPD fermention medium, is to cultivate 96～144h under 160～200r/min condition in 28～37 DEG C, rotating speed.
Be described as follows:
(1) preculture
After above-mentioned steps (4) low energy ion injects mycoderm, with 2mL YPD liquid nutrient medium immersion mycoderm, 37 DEG C of incubation 2h, with the mycoderm on aseptic glass spatula wash-out plate, obtain elutriant; The contrast mycoderm not injecting is also done same processing.With liquid-transfering gun, elutriant is transferred in the test tube that 5mL YPD liquid nutrient medium is housed, obtains preculture bacterium liquid in 37 DEG C, 160r/min shaking culture 24h.
(2) high sugared solid medium primary dcreening operation result
Gained preculture bacterium liquid is coated on high sugared solid medium, and after 37 DEG C of constant temperature culture 48h, the good bacterium colony of picking growing state sieves again.
(3) the tentatively multiple sieve of paper chromatography
From step (2), in the good bacterium colony of growing state, random choose goes out 50 larger single bacterium colonies and is inoculated in respectively in the test tube containing 5mL YPD liquid nutrient medium, and under 37 DEG C, 160r/min, cultivate 24h and obtain seed culture fluid, taking volume ratio as 5% inoculum size, seed culture fluid is inoculated in the 250mL triangular flask containing 100mL YPD fermention medium, then at 160r/min, 37 DEG C, cultivate 96h, then in the centrifugal 10min of 10000r/min, the centrifugal supernatant liquor obtaining is analyzed by paper chromatography.
(4) high performance liquid chromatography confirmation
The fermented liquid of the 5 strain bacterial strains that colour developing spot that step (3) is obtained is larger concentrated (by fermented liquid 70 DEG C of constant temperature be concentrated into original volume 1/50) after with filtering with microporous membrane, it is verified by high performance liquid chromatography.Test-results shows that in the larger corresponding fermented liquid of spot of colour developing, D-R alcohol content is higher, reaches as high as 88.23g/L.
(5) repeatability of screening method
The present invention has carried out the starting strain mutagenic treatment (injection) of multiple (1000 batches) batch, then respectively the bacterial strain after each batch processed is carried out to primary dcreening operation and multiple sieve, through statistics, each batch all can filter out 5～6 strain superior strains in 30～50 clones from preliminary screening, and in fermented liquid, D-R alcohol content is at least 81.35g/L.
(6) genetic stability of mutant strain
To after the mutant strain activation screening, be inoculated in the triangular flask containing 200mLYPD liquid nutrient medium and cultivate (37 DEG C, 160r/min), the genetic stability result that 8 generations of cultivation of going down to posterity are measured mutant strain be referring to Fig. 1.The output of D-R alcohol slightly fluctuates, but variation is not remarkable, illustrates that bacterial strain genetic stability is high.
The present invention uses low energy N
+ion implantation technique is carried out mutagenesis to Kluyveromyces lactis, builds D-R alcohol enhanced variant.The character that has resistance to height to ooze according to superior strain, has set up the method with high sugared solid medium primary dcreening operation.Combine to use in conjunction with paper chromatography and high performance liquid chromatography again and combine multiple sieve, obtain compared with superior strain according to the size screening of paper chromatography colour developing spot, recycling high performance liquid chromatography is further verified the selection result of paper chromatography.Experimental result shows, the height sugar solid medium that the present invention sets up carries out screening method that primary dcreening operation and combination paper chromatography and high performance liquid chromatography coupling sieve again, and to have the cost of screening low, easy to operate, can from mutagenic strain in enormous quantities, filter out superior strain rapidly.
Claims (8)
1. a screening method for D-R alcohol high yield yeast mutation bacterial strain, is characterized in that: this screening method comprises the following steps:
1) primary dcreening operation: the yeast strain that sets out through mutagenesis is inoculated in YPD liquid nutrient medium, then under 28～37 DEG C, 160～200r/min, shaking culture 24～28h obtains preculture liquid, preculture liquid is coated on high sugared solid medium, then in 28～37 DEG C of constant temperature culture 48～60h, the glucose that the sugared solid medium of described height contains 500～550g/L;
2) multiple sieve: after constant temperature culture, picking grows in the single bacterium colony on high sugared solid medium and is inoculated in YPD liquid nutrient medium, then in 28～37 DEG C, under 160～200r/min, shaking culture 24～28h obtains seed culture fluid, seed culture fluid is inoculated in YPD fermention medium, then in 160～200r/min, at 28～37 DEG C, shaking culture 96～144h obtains fermented liquid, fermented liquid is centrifugal, the centrifugal supernatant liquor obtaining is analyzed by paper chromatography, according to the higher mutant strain of colour developing spot screening D-R alcohol output that on paper chromatography, D-R alcohol is corresponding.
2. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 1, is characterized in that: utilize low energy ion implantttion technique to carry out mutagenesis to the yeast strain that sets out.
3. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 2, is characterized in that: described in the yeast strain that sets out be Kluyveromyces lactis.
4. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 2, is characterized in that: described mutagenesis specifically comprises the following steps:
A) picking sets out the single colony inoculation of yeast strain in YPD liquid nutrient medium, and then under 28～37 DEG C, 160～200r/min, shaking culture 18～24h obtains bacterium liquid;
B) with sterilized water, bacterium liquid is diluted to 0.5～1.0 × 10
7cFU/mL obtains mycelium dilution liquid, and 100～150 μ L mycelium dilution liquid are evenly coated to aseptic plate central authorities, then dries up to obtain mycoderm with sterile wind;
C) mycoderm is placed on the aseptic target platform of ion implanter, and by following condition, mycoderm is carried out to low energy ion injection: injection ion is N
+, implantation dosage is 1.5 × 10
15～2.5 × 10
16ions/cm
2, Implantation Energy is 15～25KeV, and the burst length is 5～10s, and be 5～10s interval time, and vacuum tightness is 1.5～2.0 × 10
-4pa.
5. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 1, is characterized in that: the composition of the sugared solid medium of described height comprises peptone, the yeast extract paste of 10.0g/L and the agar of 20.0g/L of the glucose of 500.0g/L, 20.0g/L.
6. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 1, is characterized in that: the composition of described YPD fermention medium comprises the peptone of the glucose of 200.0g/L, 20.0g/L and the yeast extract paste of 10.0g/L.
7. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 1, is characterized in that: the developping agent that described paper chromatography adopts is the mixture of propyl carbinol, pyridine and water, propyl carbinol: pyridine: the volume ratio of water is 14:3:3; The developer that paper chromatography adopts is saturated Silver Nitrate-acetone soln and saturated sodium hydroxide-ethanol solution, the preparation method of saturated Silver Nitrate-acetone soln is: saturated 0.1mL silver nitrate aqueous solution is joined in 40mL acetone to obtain to mixture, after adding 1mL water in mixture, by stirring, the Silver Nitrate of crystallization is dissolved completely, the preparation method of saturated sodium hydroxide-ethanol solution is: saturated 10mL aqueous sodium hydroxide solution is mixed with 520mL dehydrated alcohol; Paper chromatography accepted standard product are the equal-volume mixed solution of D/W, the D-R alcohol solution of mass concentration 30g/L and the aqueous glycerin solution of mass concentration 30g/L of mass concentration 30g/L.
8. a kind of screening method of D-R alcohol high yield yeast mutation bacterial strain according to claim 1, is characterized in that: described screening method is further comprising the steps of:
Checking: utilize high performance liquid chromatography to analyze the fermented liquid of the higher mutant strain of D-R alcohol output.
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Cited By (3)
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| CN112730694A (en) * | 2021-01-28 | 2021-04-30 | 成都第一制药有限公司 | A herba Leonuri injection with controlled D-arabitol content and its quality control method |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
| CN113444750A (en) * | 2021-08-19 | 2021-09-28 | 河北工程大学 | Method for producing D-arabitol by seawater non-sterilization high-permeability fermentation glycerol under stress of constant-current weak electric field |
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| CN103333881A (en) * | 2013-07-11 | 2013-10-02 | 西安工业大学 | Method for improving degradation capability of Pseudomonas sp on petroleum hydrocarbons |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112730694A (en) * | 2021-01-28 | 2021-04-30 | 成都第一制药有限公司 | A herba Leonuri injection with controlled D-arabitol content and its quality control method |
| CN113030359A (en) * | 2021-01-28 | 2021-06-25 | 成都第一制药有限公司 | Detection method for various index components in motherwort injection and quality control method of motherwort injection |
| CN113444750A (en) * | 2021-08-19 | 2021-09-28 | 河北工程大学 | Method for producing D-arabitol by seawater non-sterilization high-permeability fermentation glycerol under stress of constant-current weak electric field |
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