CN108624503A - A kind of efficient high-throughput screening method of alpha-glucosidase superior strain - Google Patents
A kind of efficient high-throughput screening method of alpha-glucosidase superior strain Download PDFInfo
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Abstract
The invention discloses a kind of efficient high-throughput screening methods for the α glucuroide superior strains that screening flat board based on defined medium composition and 96 shallow bore hole tissue culture plates combine, and belong to microorganisms technical field.The present invention includes nitrosoguanidine (NTG) mutagenesis of bacterial strain；Add the selective tablet primary dcreening operation of α methyl glucosides (α MG) and maltose；High flux screening based on 96 shallow bore hole tissue culture plates；Shaking flask secondary screening and genetic stability are investigated.The present invention improves traditional screening technique, significantly reduces working strength and experimental cost, substantially increases the screening efficiency of superior strain.
Description
Technical field
The invention belongs to microorganism strains filtration technical fields, are related to a kind of efficient height of alpha-glucosidase superior strain
Thoroughput screening method.
Background technology
Alpha-glucosidase (EC.3.2.1.20, α-Glucosidases) is one kind in Starch Hydrolysis enzyme, is mainly existed
Extracellularly work.It hydrolyzes the phlorose glycosidic bond of substrate from the non-reducing end of polysaccharide, generates alpha-D-glucose, usually handle
They range the 3rd class of hydrolase, mainly hydrolyze disaccharides, oligosaccharide, fragrant glucosides, can be using sucrose and polysaccharide as substrate.Together
When, it also has transglucosidation, can be by oligosaccharide, and α-Isosorbide-5-Nitrae-glycosidic bond is converted to α -1,6- glycosidic bonds or other forms
Link, oligoisomaltose or sugar ester, glycopeptide etc. to obtain non-fermented.It is mainly used in oligoisomaltose
(IMO) production, and IMO is a kind of promotive factor of generally acknowledged effective bifidobacterium growth, has good moisture retention, resists
Saprodontia and difficult Fermented, it is industrial to be widely used to food, medicine, feed etc..
Alpha-glucosidase is extensive in distributed in nature, and type is various, distinct, is almost present in all organisms
It is interior.The alpha-glucosidase studied at present is in addition to minority is from plant and animal, and the overwhelming majority is both from micro-
In biology.It is wherein the most prominent with the alpha-glucosidase transglucosidation of Aspergillus niger origin.Commercially available major part alpha-glucosaccharase
Enzyme enzyme preparation product is originated from aspergillus niger, but the general yield of aspergillus niger wild strain is all very low, therefore with mutation breeding
Method increasingly increases to obtain the research of superior strain.And bacterial screening workload is huge after mutagenesis and efficiency is very low, be mesh
An important factor for preceding restriction industrial microorganism production fermentation.Guan Lizhong (the mutagenic and breedings of aspergillus niger alpha-glucosidase producing strains
The research Guangxi University master thesis produced with oligoisomaltose, 2007) it is carried out according to bacterial strain colonial morphology after mutagenesis
Shaking flask primary dcreening operation, but this screening technique randomness is higher, correlation is poor and heavy workload；(the A high- such as Chen Gui light
throughput method for screening of Aspergillus niger mutants with high
transglycosylation activity by detecting non-fermentable reducing sugar[J]
.World Journal of Microbiology and Biotechnology, 2011,27 (6)) it is blue using addition Qu Liben
It is screened with the tablet of starch, but the substrate that starch can simultaneously as a variety of enzymes, such as carbohydrase and alpha-glucosidase, institute
Specific aim is not high in this way, and is easily interfered by carbohydrase；(the selection and breeding of alpha-glucosidase superior strain HB-9-5 such as Xie Zhenrong
And the optimization biotechnologys notification of condition of enzyme production, 2010 (06):206-211.) according to bacterial strain added with enzyme activity determination substrate
Transparent circle size is screened on PNPG tablets, but PNPG prices are costly, and cost is excessively high in this approach for institute；And same conduct
It is more cheap that the methyl glucose of alpha-glucosidase enzyme activity determination substrate compares PNPG with maltose price, and Alpha-Methyl Portugal
Grape sugar and maltose also can be used as carbon source supply strain growth, so methyl glucose acts not only as screening flat board carbon source
Targetedly primary dcreening operation bacterial strain also can be used as fermentation medium carbon source and carry out secondary screening.Aspergillus niger mold colony diffusion velocity is fast, range
Extensively, it if do not inhibited the diffusion of bacterium colony, is difficult to select single bacterium colony.Rose-bengal can both inhibit the expansion of aspergillus niger single bacterium colony
It dissipates, and can easier discrimination and picking individual colonies.
The present invention is directed to establish a kind of high-throughput screening method efficiently quickly screening alpha-glucosidase superior strain.It is logical
Mutation breeding is crossed, alpha-glucosidase superior strain is screened in conjunction with the screening flat board primary dcreening operation and high throughput method of defined medium,
Both working efficiency can be improved, cost is reduced, shortens experimental period, and screening rate can be increased substantially.
Invention content
The object of the present invention is to provide one kind being based on screening flat board and 96 shallow bore hole tissue culture plate combination alpha-glucosidases
Superior strain high-throughput screening method, can fast and effeciently improve Aspergillus Niger alpha-glucosidase enzyme activity.
The technical scheme is that：By starting strain produce alpha-glucosidase aspergillus niger, No. CGMCC 3.795, through Asia
It after nitroguanidine mutagenesis, is screened using methyl glucose (α-MG)-maltose-rose-bengal tablet, based on whether can be
Growth and the speed of growth carry out primary dcreening operation in screening flat board；Then it uses 96 shallow bore hole tissue culture plates to carry out high-throughput secondary screening, will sieve
Choosing obtains high yield alpha-glucosidase mutagenic strain and carries out shaking flask secondary screening verification, then carries out the secondary culture of five generations successively, finally
The mutagenic strain of high yield alpha-glucosidase shape inheritance stability is obtained, and is compared simultaneously with existing screening technique.
The present invention proposes a kind of height of the alpha-glucosidase combined based on screening flat board and 96 shallow bore hole tissue culture plates
The high-throughput screening method for producing bacterial strain, includes the nitrosoguanidine mutagenesis of bacterial strain；Selective tablet primary dcreening operation；It is trained based on 96 shallow bore hole cells
Support the high flux screening of plate；Shaking flask secondary screening and genetic stability are investigated.
It is as follows：
The first step, the nitrosoguanidine mutagenesis of bacterial strain：The starting strain spore that the maturity period is grown on the inclined-planes PDA is added
0.1%twenn80 solution is scraped into conical flask, is vibrated in shaking table, and it is outstanding to obtain monospore for removing mycelia after being filtered with absorbent cotton
Liquid；Then spore suspension is diluted, spore suspension is subjected to nitrosoguanidine mutagenesis, spore suspension after mutagenesis is centrifuged, is made
Mutagenesis bacterium solution is obtained with brine, resuspension；
Second step, selective tablet primary dcreening operation：It will be coated on methyl glucose (α-after spore suspension after mutagenesis suitably dilution
MG) and after cultivating in the incubator, it is flat in screening to observe mutagenic strain for maltose-rose-bengal screening flat board culture medium, inversion
Growing state and the speed of growth carry out primary dcreening operation on plate；
Third walks, the high flux screening based on 96 shallow bore hole tissue culture plates：Picking can grow and grow in screening flat board
The single bacterium of speed drops down onto in the 96 shallow bore hole plates equipped with screening fermentation medium, shaken cultivation, and centrifugation takes supernatant to survey α-Portugal
Polyglycoside enzyme enzyme activity, while the single bacterium colony of picking being crossed on PDA plate correspondingly, constant temperature incubation, with disc seal film
Sealing, refrigerator preserve；Bacterial strain, which is selected, according to enzyme activity determination result carries out shaking flask secondary screening；
4th step, shaking flask secondary screening and genetic stability are investigated：The bacterial strain that will be singled out shaking flask secondary screening to be carried out is trained from tablet
It supports in base in inoculating spores to the shaking flask containing fermentation medium, carries out shaking flask secondary screening；Inheritance stability is investigated in continuous passage culture
Property, screening obtains most stable of bacterial strain.
In the first step, the bacterial strain is aspergillus niger CGMCC 3.795.
In the first step, duration of oscillation is 20-40min in the shaking table；Preferably, it is 30min；
In the first step, the spore suspension miospore amount after the dilution is 106~107A/mL；Preferably, it is 106A/
mL。
In the first step, the nitrosoguanidine mutagenesis method is：With Tris- maleate buffers, nitroso guanidine solution is prepared,
The above nitroso guanidine solution and spore suspension equivalent is respectively taken to be added to mixing in sterile test tube, oscillation treatment；
Wherein, a concentration of 0.02~0.05mol/L of the Tris- maleate buffers；Preferably, it is 0.05mol/L.
Wherein, the pH of the Tris- maleate buffers is 5-6；Preferably, it is 6.0.
Wherein, a concentration of 1.0~5.0mg/mL of the nitroso guanidine solution；Preferably, it is 2.0mg/mL.
Wherein, the temperature of the oscillation treatment is room temperature；Preferably, it is 30 DEG C.
Wherein, the time of the oscillation treatment is 20~40min；Preferably, it is 30min.
In the first step, the rotating speed of the centrifugation is 4000~6000r/min；Preferably, it is 5000rpm
In the first step, the time of the centrifugation is 5~10min；Preferably, it is 6min.
In the first step, reaction can be terminated after the centrifugation.
In the first step, the brine 2~5 times；Preferably, it is 3 times.
In second step, the carbon source of the screening flat board is methyl glucose (α-MG) and maltose, and adds mould
Inhibitor rose-bengal.
In second step, the screening flat board culture medium prescription is：Methyl glucose (α-MG) 10~30g/L and malt
Sugar 0.01~0.1g/L, NaNO33~5g/L, MgSO4·7H2O 0.5~2g/L, KCl 0.5~2g/L, KH2PO41~3g/
L, FeSO4·7H20.01~0.03g/L of O, agar 15-20g/L, 0.03~0.09g/L of rose-bengal adjust pH 7.2-7.5；
Preferably, it is methyl glucose 20g/L, maltose 0.05g/L, NaNO33g/L, MgSO47H2O 1g/L, KCl 1g/L,
KH2PO42g/L, FeSO4·7H2O0.02g/L, agar 20g/L, rose-bengal 0.06g/L adjust pH 7.2.
In second step, the temperature of the incubator is 30-37 DEG C；Preferably, it is 30 DEG C.
In second step, the time of the culture is 3-5d；Preferably, it is 3d.
In third step, the 96 shallow bore hole screen selects the fermentative medium formula to be：30~50g/L of methyl glucose, yeast
Soak powder 10~30g/L, MgSO4·7H2O 0.5~2g/L, KH2PO45~7g/L adjusts pH5.5-6.0；Preferably, it is α-first
Base glucose 30g/L, yeast extract 20g/L, MgSO4·7H2O 1g/L, KH2PO45g/L, it is 5.6 to adjust pH.
In third step, the rotating speed of the shaken cultivation is 150~250r/min；Preferably, it is 200r/min.
In third step, 2~4d of time of the shaken cultivation；Preferably, it is 3d.
In third step, the time of the constant temperature incubation is 2~4d；Preferably, it is 4d.
In third step, the temperature of the constant temperature incubation is 30-37 DEG C；Preferably, it is 30 DEG C.
In third step, the temperature that the refrigerator preserves is 2~8 DEG C；Preferably, it is 4 DEG C.
In third step, the volume that fermentation medium is screened in the 96 shallow bore hole plate is 160~200 μ L；Preferably, it is 180 μ
L。
In 4th step, the formula of the fermentation medium is：30~50g/L of malt extract, 10~30g/ of yeast extract
L, MgSO4·7H2O 0.5~2g/L, KH2PO45~7g/L adjusts pH 5.5-6.0；Preferably, it is malt extract 40g/
L, yeast extract 20g/L, MgSO4·7H2O 1g/L, KH2PO45g/L, it is 5.6 to adjust pH.
In 4th step, the condition of the shaking flask secondary screening is：30~37 DEG C, 150~250r/min shaking table cultures, 3~5d；It is excellent
Selection of land is 30 DEG C, 200r/min shaking table cultures 5d.
In 4th step, the continuous passage culture is preferably 5 generation of continuous passage culture.
The invention also provides a kind of screening and culturing mediums, including：10~30g/L of carbon source methyl glucose and maltose
0.01~0.1g/L, NaNO33~5g/L, MgSO4·7H2O 0.5~2g/L, KCl 0.5~2g/L, KH2PO41~3g/L,
FeSO4·7H20.01~0.03g/L of O, agar 15-20g/L, 0.03~0.09g/L of rose-bengal adjust pH 7.2-7.5；It is excellent
Selection of land is methyl glucose 20g/L, maltose 0.05g/L, NaNO33g/L, MgSO47H2O 1g/L, KCl 1g/L,
KH2PO42g/L, FeSO4·7H2O 0.02g/L, agar 20g/L, rose-bengal 0.06g/L adjust pH 7.2.
The invention also provides application of the screening and culturing medium in strain culturing, bacterial strain selection.
The beneficial effects of the present invention are：
One, the present invention uses methyl glucose-maltose-rose-bengal screening flat board culture in primary dcreening operation tablet
Base.The screening technique of conventional alpha-glucosidase superior strain is to use PDA culture medium, not any selectivity, and bacterium
Silk extension is serious, and bacterium colony sprawling is unfavorable for screening；The present invention can inhibit mycelia to extend in view of rose-bengal, be conducive to screening,
Therefore rose-bengal is added in primary dcreening operation tablet, further, the present invention considers the most suitable substrate of alpha-glucosidase
For methyl glucose, if using methyl glucose as sole carbon source, the bacterial strain that can generate alpha-glucosidase can be with
Methyl glucose is decomposed, as carbon source for strain growth, the bacterial strain that can not generate alpha-glucosidase is then unable to decomposing alpha-
Methyl glucoside cannot be grown.Using this principle, only using methyl glucose as the primary dcreening operation of sole carbon source culture medium
On tablet, by for a long time, after general 6 days, having strain growth, but growing way is very weak, bacterial strain is seldom；Further, originally
Invention is being screened just it is considered that if adding another substrate maltose of micro alpha-glucosidase in the medium
Phase, bacterial strain are grown first with micro maltose, and after maltose consumption is net, the bacterial strain that can generate alpha-glucosidase then continues
Growth, can not generate the bacterial strain of alpha-glucosidase cannot then grow, and generating the high bacterial strain of alpha-glucosidase activity then has
Growth vigor, bacterium colony is larger, and then bacterium colony is smaller for the low bacterial strain of generation alpha-glucosidase activity on the contrary.This method of the present invention
Compared with the general PDA plate screening applied now, there is prodigious advantage.
Two, according to the enzyme activity determination method of alpha-glucosidase, screening flat board is designed, using 96 shallow bore hole plate hole plate secondary screenings,
Shaking flask verifies the pattern of secondary screening；It, should compared with existing screening mode uses shaking flask to screen according to colonial morphology primary dcreening operation and directly
Method effectively reduces working strength, the use of alpha-glucosidase enzyme activity determination substrate methyl glucose and maltose is multiple
Carbon source is closed, further according to bacterial strain after mutagenesis in the speed of growth of screening flat board, the specific aim screening to superior strain, drop may be implemented
Low screening randomness, substantially increases the probability for screening superior strain, greatly increases the screening work of superior strain
Make efficiency；Increase can bacterium quantity, existing shaking flask screening technique can once screen 60 plants of mutagenic strains, and one 96
Shallow bore hole culture plate can screen at least 60 plants of bacterium, and to screen every time in terms of 8 96 orifice plates, mutagenic strain screening flux can reach existing
Methodical 8 times, to realize high flux screening；Experimental period is shortened, to screen 8 96 shallow bore hole tissue culture plates every time
Meter, until shaking flask verification is completed to need 11d, and existing screening Fang Fang screening same number bacterial strains need 124d；It is shallow using 96 simultaneously
Porocyte culture plates are screened, and reduce fermentation medium usage amount compared to shaking flask screening, to screen every time in terms of 8 96 orifice plates, hair
Ferment culture medium only needs 862mL, and original shaking flask screening technique then needs 3L fermentation mediums, greatly reduces experimental cost.
Description of the drawings
Fig. 1 is methyl glucose-maltose-rose-bengal screening flat board primary dcreening operation figure.
Fig. 2 is 96 orifice plate high flux screenings as a result, the corresponding abscissa of wherein each block diagram is from left to right followed successively by N-
2-1、N-2-2、N-2-3、……、N-2-65、N-2-66、N-2-67。
Fig. 3 is superior strain shaking flask verification result.
Fig. 4 is that superior strain passes on verification result.
Fig. 5 be methyl glucose tablet primary dcreening operation as a result, the corresponding abscissa of wherein each block diagram from left to right successively
For N-3-1, N-3-2, N-3-3 ..., N-3-65, N-3-66, N-3-67.
Fig. 6 is PDA plate primary dcreening operation as a result, the corresponding abscissa of wherein each block diagram is from left to right followed successively by N-4-1, N-
4-2、N-4-3、……、N-4-65、N-4-66、N-4-67。
Fig. 7 is PDA plate and methyl glucose-tablet and methyl glucose-maltose-rose-bengal screening flat board
Primary dcreening operation figure.
Fig. 8 is PDA plate and methyl glucose-tablet and methyl glucose-maltose-rose-bengal screening flat board
The comparison of primary dcreening operation result.
Fig. 9 is the comparison of high-throughput screening method and existing screening technique secondary screening result.
Specific implementation mode
In conjunction with following specific examples 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 domain.The process of the implementation present invention,
Condition, reagent, test method etc. are among the general principles and common general knowledge in the art in addition to the following content specially referred to,
Content is not particularly limited in the present invention.
In following embodiment, the enzyme activity determination method of alpha-glucosidase is as follows：
The sodium carbonate for preparing the PNPG solution of 5mmol/L, the acetic acid sodium-acetate buffer of 0.1M pH5.5 and 1mol/L is molten
Liquid is sequentially added according to following table in 96 orifice plates.
After having added sodium carbonate liquor, OD405nm is measured in microplate reader, experimental group light absorption value is denoted as A1, and control group is inhaled
Light value is denoted as A2；
Enzyme activity (U/mL)=[(k × (A1-A2)p1-b/1000×T×V1)]×N×V2
V1：Add enzyme liquid amount (mL), V2：Reaction system (mL), N:Extension rate, T:Reaction time, k, b are p-nitrophenyl
The slope and intercept of phenol standard curve；
Enzyme activity defines：The amount that enzyme solution catalysis substrate per minute generates 1 μm of olPNP at pH5.5,50 DEG C is an enzyme activity list
Position.
The high-throughput screening method of alpha-glucosidase superior strain of the present invention, comprises the concrete steps that：
It is prepared by spore suspension
3.795 inclined-planes Aspergillus niger strain CGMCC of 3d (30 DEG C) will be cultivated, 0.1%twenn80 solution is scraped to band glass
In the conical flask of glass pearl, 30min is vibrated in shaking table, and mycelia is removed after being filtered with absorbent cotton and obtains monospore suspension；
Nitrosoguanidine mutagenesis
Nitrosoguanidine mutagenesis method is the pH value 6.0 with the Tris- maleate buffers of 0.05mol/L, and compound concentration is
The nitroso guanidine solution of 2.0mg/mL respectively takes the above nitroso guanidine solution and spore suspension equivalent to be added in sterile test tube and mixes
Even, shaken at room temperature handles 30min, and 5000rpm centrifugations 5min terminates reaction after mutagenic treatment, removes supernatant, precipitation uses
It is resuspended after brine 3 times and obtains mutagenesis bacterium solution；
Bacterium solution selectivity tablet primary dcreening operation after mutagenesis
Methyl glucose-and/or maltose-rose-bengal screening will be coated on after spore suspension after mutagenesis suitably dilution
Plating medium is inverted, 30 DEG C of incubator temperature, after cultivating 3d, observes mutagenic strain growing state and life in screening flat board
Long speed carries out primary dcreening operation, i.e. selection is relatively large in diameter, and form is full, the fast single bacterium colony of the speed of growth.
Methyl glucose-maltose-rose-bengal screening flat board culture medium prescription is：Methyl glucose 20g/L, and
Maltose 0.05g/L, NaNO33g/L, MgSO4.7H2O 1g/L, KCl1g/L, KH2PO41g/L, FeSO47H2O 0.02g/
L, agar 20g/L, rose-bengal 0.06g/L adjust pH7.2.
High flux screening based on 96 shallow bore hole tissue culture plates
Picking can be grown in screening flat board and the faster single bacterium of the speed of growth drops down onto and screens fermentation medium equipped with 180 μ L
96 shallow bore hole plates in, at 30 DEG C, 200r/min, shaken cultivation 3d, centrifugation, take supernatant survey alpha-glucosidase enzyme activity, together
When the single bacterium colony of picking is crossed on PDA plate correspondingly, in 30 DEG C of constant temperature incubation 3d, sealed with disc seal film, 4 DEG C
Refrigerator preserves；Bacterial strain, which is selected, according to enzyme activity determination result carries out shaking flask secondary screening；
96 shallow bore hole tissue culture plates screen fermentative medium formula：Methyl glucose 30g/L, yeast extract 20g/L,
MgSO4.7H2O 1g/L, KH2PO45g/L adjusts pH5.6.
Shaking flask secondary screening and genetic stability are investigated
Will be singled out the bacterial strain of shaking flask secondary screening to be carried out from plating medium inoculating spores to shaking containing fermentation medium
In bottle, shaking flask secondary screening is carried out；The condition of shaking flask secondary screening is：30 DEG C, 200r/min shaking table cultures 5d；It in 5 generation of continuous passage culture, examines
Genetic stability is examined, screening obtains most stable of bacterial strain；
Shaking flask secondary screening culture medium prescription is：Malt extract 40g/L, yeast extract 20g/L, MgSO4.7H2O1g/L,
KH2PO45g/L adjusts pH6.0.
Embodiment 1:Mutagenesis is carried out to Aspergillus niger strain CGMCC 3.795 in the way of nitrosoguanidine mutagenesis
3.795 inclined-planes Aspergillus niger strain CGMCC of 3d (30 DEG C) will be cultivated, 0.1%twenn80 solution is scraped to band glass
In the conical flask of glass pearl, 30min is vibrated in shaking table, and mycelia is removed after being filtered with absorbent cotton and obtains monospore suspension；With
The Tris- maleate buffers of 0.05mol/L, pH value 6.0, compound concentration be 2.0mg/mL nitroso guanidine solution, respectively take with
Upper nitroso guanidine solution and spore suspension equivalent are added to mixing in sterile test tube, and shaken at room temperature handles 30min, mutagenic treatment knot
5000rpm centrifuges 5min and terminates reaction after beam, removes supernatant, and precipitation obtains mutagenic bacteria using being resuspended after brine 3 times
Liquid；
Embodiment 2：Screening-methyl glucose (α-MG)-maltose-of alpha-glucosidase enzyme superior strain after mutagenesis
Rose-bengal screening flat board method
(1) selective tablet primary dcreening operation
It will be coated on methyl glucose (α-MG)-maltose-rose-bengal sieve after spore suspension after mutagenesis suitably dilution
Plating medium is selected, is inverted, 30 DEG C of incubator temperature, after cultivating 2d, since observation mutagenic strain grows feelings in screening flat board
Condition and the speed of growth carry out primary dcreening operation (Fig. 1) such as Fig. 1 arrows meaning single bacterium colony, other opposite single bacterium colonies are relatively large in diameter, and form is more
To be full, the speed of growth is faster, thus it is speculated that the bacterium colony may more excel at leveraging methyl glucose, so as to higher production
Enzyme ability；
(2) 96 orifice plate high flux screenings
Picking can grow and be relatively large in diameter in screening flat board, and form is more full, the faster single bacterium of the speed of growth drops down onto
In the 96 shallow bore hole plates for screening fermentation medium equipped with 180 μ L, at 200r/min, shaken cultivation 3d, centrifugation takes supernatant to survey α-
Glucuroide enzyme activity, while the single bacterium colony of picking being crossed on PDA plate correspondingly, in 30 DEG C of constant temperature incubation 2d,
It is sealed with disc seal film, 4 DEG C of refrigerators preserve；Bacterial strain, which is selected, according to enzyme activity determination result (Fig. 2) carries out shaking flask secondary screening；
It is 100% mapping with 3.795 enzyme activity of starting strain CGMCC, the results are shown in Figure 2 by 96 shallow bore hole plate primary dcreening operations：
Compared with starting strain CGMCC 3.795 (0.15U/mL), the mutagenic strain that producing enzyme improves 20% or more has 43 plants, accounts for total screening
The 64% of bacterial strain number；The mutagenic strain that producing enzyme improves 60% or more has 17 plants, wherein there is the raising of two plants (N-2-39, N-2-64)
Amount carries out it shaking flask secondary screening verification, and investigate genetic stability 100% or more.
Methyl glucose (α-MG)-maltose-rose-bengal screening flat board culture medium prescription is：Methyl glucose
20g/L, maltose 0.05g/L, NaNO33g/L, MgSO4.7H2O 1g/L, KCl1g/L, KH2PO42g/L, FeSO4·7H2O
0.02g/L, agar 20g/L, rose-bengal 0.06g/L adjust pH7.3.
96 shallow bore hole screens select the fermentative medium formula to be：Methyl glucose 40g/L, yeast extract 20g/L,
MgSO4.7H2O 1g/L, KH2PO45g/L adjusts pH 6.0.
(3) shaking flask secondary screening and genetic stability
Corresponding bacterium colony (N-2-39, N-2-64) in picking PDA plate culture medium, inoculating spores to fermented and cultured containing 50mL
In the 250mL shaking flasks of base, 30 DEG C, after the shaking table culture 5d of 200r/min, its enzyme activity is measured, as a result sees Fig. 3, the results show that shaking
Bottle verification result is similar to 96 orifice plate secondary screening results, and the enzyme activity of bacterial strain N-64 is improved to going out bacterium germination (CGMCC 3.795)
209%, enzyme activity reaches 0.31U/mL；The enzyme activity of bacterial strain N-39 is improved to going out the 185% of bacterium germination (CGMCC 3.795), this explanation
96 shallow bore hole tissue culture plate the selection results and shaking flask secondary screening results relevance are preferable.By bacterial strain N-64 and N-39 continuous passage culture
In 5 generations, investigated genetic stability.As a result see Fig. 4, the results show that bacterial strain N-64 maintains high yield level substantially, there is good something lost
Stability is passed, after 5 generation of continuous passage, alpha-glucosidase enzymatic productivity is improved to the 201% of starting strain；Bacterial strain N-39 is same
It is horizontal to maintain good high yield, after 5 generation of continuous passage, enzymatic productivity is improved to the 188% of starting strain.This illustrates this screening
Method, that is, methyl glucose (α-MG)-maltose-rose-bengal screening flat board is combined screening with 96 shallow bore hole tissue culture plates
The method of alpha-glucosidase superior strain is feasible and effective.
Embodiment 3：The screening of alpha-glucosidase enzyme superior strain after mutagenesis:Methyl glucose (α-MG)-Bangladesh
Red screening flat board method
Selective tablet primary dcreening operation：3.795 spore suspensions of starting strain CGMCC after mutagenesis are applied respectively after appropriate dilution
It is distributed in methyl glucose (α-MG)-rose-bengal screening flat board, after 30 DEG C of constant temperature are inverted culture 6d, in screening flat board picking bacterium
It falls and is relatively large in diameter, form is full, the faster single bacterium colony of the speed of growth 67 plants (Fig. 7).It will be from methyl glucose-rose-bengal sieve
The single bacterium colony of picking on tablet is selected to cross to fresh PDA tablet, 30 DEG C of constant temperature are directly seeded to after being inverted culture 2d equipped with 50mL
In the 250mL shaking flasks of liquid fermentation medium, 30 DEG C, after 200r/min shaking table cultures 5d, its enzyme activity, primary dcreening operation enzyme activity determination are measured
The results are shown in Figure 5.
As seen from Figure 5, with starting strain enzyme activity be 100%, methyl glucose (α-MG)-rose-bengal screening flat board with
What the screening technique primary dcreening operation that shaking flask screening combines obtained being higher than in bacterial strain 20% or more bacterium germination enzyme activity shares 10 plants, accounts for total sieve bacterium
Several 15% is higher than only 1 plant, bacterial strain N-3-50 of 40% or more bacterium germination, is 142%；
Methyl glucose-rose-bengal screening flat board culture medium prescription is：Methyl glucose 20g/L, NaNO3 3g/
L, MgSO4.7H2O 1g/L, KCL1g/L, KH2PO42g/L, FeSO4·7H2O 0.02g/L, agar 20g/L, rose-bengal
0.06g/L adjusts pH 7.3.
Shaking flask primary dcreening operation culture medium prescription is：Malt extract 40g/L, yeast extract 20g/L, MgSO4.7H2O 1g/L,
KH2PO45g/L adjusts pH6.0.
Embodiment 4 (reference examples)：The screening of alpha-glucosidase enzyme superior strain after mutagenesis:PDA plate screening technique
PDA plate primary dcreening operation：3.795 spore suspensions of starting strain CGMCC after mutagenesis are respectively coated on after appropriate dilution
PDA plate is relatively large in diameter, form is full, and the speed of growth is very fast after 30 DEG C of constant temperature are inverted culture 2d in screening flat board picking colony
Single bacterium colony 67 plants (Fig. 7).By from PDA plate the single bacterium colony of picking cross to fresh PDA tablet, 30 DEG C of constant temperature are inverted culture
It is directly seeded to after 2d in the 250mL shaking flasks equipped with 50mL liquid fermentation mediums, 30 DEG C, after 200r/min shaking table cultures 5d,
Its enzyme activity is measured, the results are shown in Figure 6 for primary dcreening operation enzyme activity determination.
As seen from Figure 6, with starting strain alpha-glucosidase enzyme activity for 100%, directly PDA plate is used to be screened with shaking flask
In conjunction with screening technique primary dcreening operation obtain being higher than 10% or more bacterium germination enzyme activity share 2 plants, account for the 2.9% of total sieve bacterium number, bacterial strain
N-4-15 and N-4-45, respectively 117% and 112%, without the bacterial strain for being higher than 20% or more bacterium germination.
Shaking flask primary dcreening operation culture medium prescription is：Malt extract 40g/L, yeast extract 20g/L, MgSO4.7H2O 1g/L,
KH2PO4 5g/L adjust pH6.0.
Embodiment 5：PDA plate is screened with methyl glucose-tablet and methyl glucose-maltose-rose-bengal
The shaking flask secondary screening of superior strain obtained by tablet primary dcreening operation
The primary dcreening operation superior strain that embodiment 2,3,4 obtains is lined into fresh PDA tablet, 30 DEG C of constant temperature are inverted culture 2d
Afterwards, in inoculating spores to the 250mL shaking flasks of the fermentation medium containing 50mL, 30 DEG C, after the shaking table culture 5d of 200r/min, it is measured
As a result enzyme activity is shown in Fig. 9, as seen from Figure 9, the shaking flask verification enzyme activity of bacterial strain N-2-39 and N-2-64 are starting strain enzyme activity
210% and 185%, it is consistent with primary dcreening operation result；The enzyme activity of bacterial strain N-3-50 is the 130% of starting strain, compared with primary dcreening operation result
It is declined slightly；And bacterial strain N-4-15 and N-4-45 enzyme activity is only the 118% and 113% of starting strain.This result illustrates Alpha-Methyl
The high-throughput screening method that glucose-maltose-rose-bengal screening flat board is combined with 96 shallow bore hole tissue culture plates with it is existing
Screening technique is compared, and is greatly improved the probability for screening superior strain, is shortened experimental period and period, reduces work
Intensity realizes screening in a short time and obtains the target of stable, high-yielding alpha-glucosidase bacterial strain.
Embodiment 2,3,4 kind of three kinds of tablet primary dcreening operation compares figure as shown in fig. 7, the wherein (methyl glucose (α-of embodiment 2
MG)-maltose-rose-bengal screening flat board) due to adding rose-bengal, it is seen that its flat single bacterium colony is more disperseed, and is not spread,
Simultaneously as adding maltose, compared with addition methyl glucose screening flat board merely, grown in identical incubation time
Effect is more preferable, colony diameter bigger, more full；And the growth of embodiment 4 (common PDA plate) bacterium colony is normal, and single bacterium colony bacterium
The more sprawlings of silk are a piece of, it is difficult to picking single bacterium colony；Primary dcreening operation tablet in other two embodiments is compared, 3 (Alpha-Methyl of embodiment
The screening flat board of glucose (α-MG)-rose-bengal) then bacterium colony it is smaller, mycelia is relatively thin, it is more difficult to observe single bacterium colony, Alpha-Methyl Portugal
Grape sugar-rose-bengal screening flat board at least need 30 DEG C of constant temperature be inverted culture 6d just can picking single bacterium colony, and methyl glucose-wheat
Bud sugar-rose-bengal screening flat board only needs 2d.
To go out bacterium germination enzyme activity for 100%, 2,3,4 primary dcreening operation bacterial strain of embodiment is drawn with respect to enzyme activity result figure, as shown in figure 8,
It can be seen that there are 43 plants using the mutagenic strain that producing enzyme in high-throughput screening method primary dcreening operation bacterial strain improves 20% or more, total bacterium is accounted for
Several 64%, the mutagenic strain that producing enzyme improves 60% or more has 17 plants, wherein there is the raising amount of two plants (N-2-39, N-2-64) to exist
100% or more；Screening flat board using only addition methyl glucose is higher than bacterium germination with shaking flask primary dcreening operation combination primary dcreening operation bacterial strain
20% or more enzyme activity shares 10 plants, accounts for the 15% of total sieve bacterium number, is higher than only 1 plant of 40% or more bacterium germination, is 142%；
And the screening technique primary dcreening operation directly combined using PDA plate and shaking flask screening obtains being higher than the shared of 10% or more bacterium germination enzyme activity
2 plants, the 2.9%, respectively 117% and 112% of total sieve bacterium number is accounted for, without the bacterial strain for being higher than 20% or more bacterium germination.Three kinds of screenings
Method is compared, the high pass combined with 96 shallow bore hole tissue culture plates using methyl glucose-maltose-rose-bengal screening flat board
The probability that amount screening technique screens superior strain will be far above using only addition methyl glucose screening flat board and PDA plate
In the screening mode that shaking flask screening combines.
Above-described embodiment simply to illustrate that the present invention technical concepts and features, its object is to allow the common of this field
Technical staff cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.Every
According to the equivalent change or modification that the essence of the content of present invention is made, should all cover within the scope of the present invention.
Claims (11)
1. the alpha-glucosidase that a kind of screening flat board based on defined medium composition and 96 shallow bore hole tissue culture plates combine
The high-throughput screening method of superior strain, which is characterized in that the nitrosoguanidine mutagenesis including bacterial strain；Add methyl glucose α-
The selective tablet primary dcreening operation of MG and maltose；High flux screening based on 96 shallow bore hole tissue culture plates；Shaking flask secondary screening and heredity are steady
Qualitative investigation.
2. screening technique according to claim 1, which is characterized in that the specific steps are：
The first step, the nitrosoguanidine mutagenesis of bacterial strain：The starting strain spore that the maturity period is grown on the inclined-planes PDA is added
0.1%twenn80 solution is scraped into conical flask, is vibrated in shaking table, and it is outstanding to obtain monospore for removing mycelia after being filtered with absorbent cotton
Liquid；Then spore suspension is diluted, spore suspension is subjected to nitrosoguanidine mutagenesis, spore suspension after mutagenesis is centrifuged, is made
Mutagenesis bacterium solution is obtained with brine, resuspension；
Second step, selective tablet primary dcreening operation：Will spore suspension after mutagenesis suitably dilution after be coated on methyl glucose α-MG and
Maltose-rose-bengal screening flat board culture medium is inverted, and after cultivating in the incubator, observation mutagenic strain is in screening flat board
Growing state and the speed of growth carry out primary dcreening operation；
Third walks, the high flux screening based on 96 shallow bore hole tissue culture plates：Picking can be grown in screening flat board and the speed of growth
Faster single bacterium drops down onto in the 96 shallow bore hole plates equipped with screening fermentation medium, shaken cultivation, and centrifugation takes supernatant to survey phlorose
Glycosides enzyme enzyme activity, while the single bacterium colony of picking being crossed on PDA plate correspondingly, constant temperature incubation is sealed with disc seal film,
Refrigerator preserves；Bacterial strain, which is selected, according to enzyme activity determination result carries out shaking flask secondary screening；
4th step, shaking flask secondary screening and genetic stability are investigated：The bacterial strain of shaking flask secondary screening to be carried out be will be singled out from plating medium
In middle inoculating spores to the shaking flask containing fermentation medium, shaking flask secondary screening is carried out；Genetic stability, sieve are investigated in continuous passage culture
Choosing obtains most stable of bacterial strain.
3. screening technique according to claim 1 or 2, which is characterized in that the bacterial strain is aspergillus niger CGMCC 3.795.
4. screening technique according to claim 2, which is characterized in that duration of oscillation is 20-40min in the shaking table；With/
Or, the spore suspension miospore amount after the dilution is 106~107A/mL.
5. screening technique according to claim 2, which is characterized in that in second step, the screening flat board culture medium prescription
For：10~30g/L of methyl glucose, maltose 0.01~0.1g/L, NaNO33~5g/L, MgSO4·7H20.5~2g/ of O
L, KCL0.5~2g/L, KH2PO41~3g/L, FeSO4·7H20.01~0.03g/L of O, agar 15-20g/L, rose-bengal
0.03~0.09g/L adjusts pH 7.2-7.5.
6. screening technique according to claim 2, which is characterized in that in third step, the 96 shallow bore hole screen publishes ferment training
Foster based formulas is：30~50g/L of methyl glucose, yeast extract 10~30g/L, MgSO4·7H20.5~2g/L of O,
KH2PO45~7g/L adjusts pH 5.5-6.0.
7. screening technique according to claim 2, which is characterized in that in third step, the rotating speed of the shaken cultivation is 150
~250r/min；And/or 2~4d of time of the shaken cultivation；And/or the time of the constant temperature incubation is 2~4d；With/
Or, the temperature of the constant temperature incubation is 30-37 DEG C；And/or it is 160 that the volume of fermentation medium is screened in the 96 shallow bore hole plate
~200 μ L.
8. screening technique according to claim 2, which is characterized in that in the 4th step, the fermentative medium formula is：Wheat
Bud 30~50g/L of extract, yeast extract 10~30g/L, MgSO4·7H2O 0.5~2g/L, KH2PO45~7g/L is adjusted
pH5.5-6.0。
9. screening technique according to claim 2, which is characterized in that in the 4th step, the condition of the shaking flask secondary screening is：30
~37 DEG C, 150~250r/min shaking table cultures, 3~5d；And/or the continuous passage culture is 5 generation of continuous passage culture.
10. a kind of screening and culturing medium, which is characterized in that it includes：10~30g/L of methyl glucose, maltose 0.01~
0.1g/L, NaNO33~5g/L, MgSO4·7H2O 0.5~2g/L, KCl 0.5~2g/L, KH2PO41~3g/L, FeSO4·
7H20.01~0.03g/L of O, agar 15-20g/L, 0.03~0.09g/L of rose-bengal adjust pH 7.2-7.5.
11. a kind of application of screening and culturing medium as claimed in claim 10 in strain culturing, bacterial strain selection.
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