CN106047708A - Screening method for mutated strains capable of realizing high yield of amylase - Google Patents
Screening method for mutated strains capable of realizing high yield of amylase Download PDFInfo
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Abstract
The invention relates to a screening method for mutated strains capable of realizing high yield of amylase. The method comprises a step of screening of mutated strains, i.e., screening of first-generation strains having undergone mutation; the step comprises a substep of primary screening and a substep of secondary screening; the secondary screening is carried out in a manner the same as the manner used in primary screening so as to obtain second-generation mutated starting strains; then mutation and screening are carried out again; and the screening method for the generation II and generation III to generation n is same, and mutation and screening are carried out until high-amylase-yield stains meeting requirements are bred. With the screening method, the starting strains having undergone mutation treatment undergoes primary screening and secondary screening again so as to obtain the second-generation starting strains; the screening method is simple and easily practicable, and can obtain the high-amylase-yield stains in a short time; the whole screening process is low in investment and high in targeting performance; and orientated production of the amylase strains with high enzyme activity can be realized, and the method has large-scale promotion value.
Description
Technical field
The present invention relates to field of biology, particularly relate to bacterial strain screening method, specifically refer to high yield amylase strain after a kind of mutation
Screening technique.
Background technology
Amylase refers to hydrolyze starch, glycogen and the enzyme about the O-glucose key in polysaccharide.Generally refer to do for
α-Isosorbide-5-Nitrae-glucosan and the enzymes of hydrolyzing alpha-Isosorbide-5-Nitrae-glycosidic bond such as soluble starch, amylose, glycogen.Amylase is according to work
Two kinds can be divided into: α-amylase (EC3.2.1.1.) and beta amylase (EC3.2.1.2.) by mode.α-amylase is extensive
It is distributed in animal (saliva, pancreas etc.), plant (Fructus Hordei Germinatus, mountain dish) and microorganism.Wherein the enzyme of microorganism is the most all point
Secreting property.Amylase is with Ca2+For its necessary factor with as stable factor, both acted on amylose, and also acted on side chain and form sediment
Powder, the cut-out α-Isosorbide-5-Nitrae-chain of congenerous.Therefore, its reaction has an obvious phenomenon: for substrate solution viscosity rapid decrease and
Iod R disappears.China is a large agricultural country, and starch resource is the abundantest, and amylase has wide range of applications, but China
Amylase production status but allows of no optimist.
Screening high yield amylase strain from nature is the key solving this difficult problem, and the strong part of this method is to implement
Getting up convenient and simple, capital input is less.But its shortcoming is that randomness is very big, many times can not filter out preferably
Thalline.Flat-plate bacterial colony transparent circle method is existing screening conventional method of high yield amylase strain after mutation, but transparent circle diameter/
The degree of association specificity that colony diameter is lived with its shaking flask enzyme is poor, and this method also wastes time and energy.Therefore, exploring one can
The method realizing simplicity screening high yield amylase strain after mutation is the most necessary.
Summary of the invention
It is an object of the invention to the shortcoming overcoming above-mentioned prior art, it is provided that one can quick high yield amylase bacterium after mutation
The screening technique of strain.
To achieve these goals, after the mutation of the present invention, the screening technique of high yield amylase strain has a following composition:
The screening technique of high yield amylase strain after this mutation, it is mainly characterized by, and described screening technique comprises the steps:
Bacterial strain screening step after mutation: carry out the first generation bacterial strain through mutagenic treatment screening step, including primary dcreening operation sub-step
And answer sieve step, wherein:
(1) primary dcreening operation sub-step includes: be placed on starch agar plate by the round filter paper of diameter 3～6mm, every plate 4～8,
Respectively draws equal amounts culture fluid point is on filter paper, and 65 DEG C of dry 2h, with the detection of iodine liquid the diameter of more transparent circle, select
30～50 strains that bright loop diameter is big carry out multiple sieve；
(2) multiple sieve step includes: by 30～50 strain primary dcreening operation bacterial strains, every kind of bacterial strain 3～5 bottles, again carries out with (1.1) just
The screening of sieve mode, takes 3～5 strains starting strain as second filial generation mutation, again carries out mutation and screens step, the second filial generation,
The third generation is to the same first generation of screening technique in the n-th generation, to selecting high yield starch bacterial strain.
Preferably, the described bacterial strain after mutation comes from following step:
The screening step of (a) first generation starting strain: include primary dcreening operation step and sieve step again, wherein primary dcreening operation step is that iodine contaminates method,
The bacterium colony choosing dropping iodine liquid generation transparent circle carries out multiple sieve step, and described multiple sieve step is bent profit benzene indigo plant development process, chooses product
The bacterium colony of raw transparent circle；
The mutagenesis steps of (b) first generation starting strain: the bacterium producing transparent circle picked out by described bent profit benzene indigo plant development process
Drop into row mutagenic treatment；
It is highly preferred that described (a) first generation is set out, the screening step of bacterium specifically includes following sub-step:
(a-a) weigh sample 0.5g and number, fully diluting dissolving with sterilized water, then taking equivalent and be forwarded to 200mL enrichment
Fluid medium, in 37 DEG C of incubators, 100～200r/min cultivate；
(a-b) after 24h, with sterilized water, each sample being carried out 10 times of gradient dilutions, drawing concentration is 10-6Culture fluid 100 μ L
It is spread evenly across on screening flat board, in 37 DEG C of incubators, cultivates 45～50h；
(a-c) add iodine liquid, observe the bacterium colony marking substantially hydrolysis circle, therefrom choose dyeing loop diameter and colony diameter ratio
Big single bacterium colony is numbered and is preserved；
(a-d) in 37 DEG C of incubators of the single bacterium colony that will be singled out, 100～200r/min cultivate 24h, carry out 10 successively2Again, 103
Again, 104Dilution again, takes 10～100 μ L respectively and is coated with flat boards, cultivate 24 hours for 37 DEG C, and addition song profit benzene is blue, observes measurement saturating
Bright circle size, the single bacterium colony therefrom choosing dyeing loop diameter maximum with colony diameter ratio is numbered and is preserved.
Further, described sample is taken from soil, particularly as follows: use five-spot to gather soil sample, chooses 2m × 2m's
Sample prescription carries out diagonal line, samples as sampling point at sampling point Chu Jiyu center, diagonal center equidistant four points of sampling point.
Closer, described sample also includes enrichment culture process, described enrichment culture process before carrying out primary dcreening operation after fetching
Use iodine dye method.
It is further preferred that described (1) primary dcreening operation sub-step specifically includes:
Every bacterial strain 1～2 bottles of bacterium solution are cultivated 50h at shaking table with 100～200r/min respectively, the round filter paper of diameter 4mm is placed
On starch agar plate, every plate 6, on difference draws equal amounts culture fluid point to filter paper, 65 DEG C of dry 2h, use iodine liquid
Detection the diameter of more transparent circle, 30～50 strains selecting transparent circle diameter big carry out multiple sieve.
Closer, described starch agar plate is 1.8% starch, 1.4% agar, the starch agar plate of 2 mm of thickness.
Also include processing sub-step it is further preferred that screen superior strain step after described mutation, particularly as follows:
The strains separation of mutation will be carried out to plate, beat agar block bacterium colony 1000～3000 pieces, picking 5%～10% on assaying table
The bacterium colony of transparent circle, is used for carrying out (1) primary dcreening operation sub-step.
Most preferably, described bacterium colony is cultivated the solid medium used and is the aseptic training poured out in 40～50 DEG CXia sterile workings
Support base.
Have employed the screening technique of high yield amylase strain after the mutation in this invention, it has technical effect that, adopts during sample primary dcreening operation
With the bacterial strain filtering out needs of rapid, high volume, and low cost can be screened by iodine dye method, use bent profit stupid indigo plant development process when multiple sieve,
Utilize the characteristic that bent profit benzene is blue, be possible not only to filter out the bacterium colony producing transparent circle, and itself the growth of microorganism do not pressed down
Make use, can accurately screen and target bacterium colony not damaged.Starting strain after mutagenic treatment is by primary dcreening operation again
With the starting strain that multiple sieve can obtain the second filial generation, screening process is simple and easy to operate, carries out permissible in the time period that enzymatic activity is the highest
Obtaining the high yield amylase strain of laminating production requirement at short notice, whole screening process invests little targeting height, can be directionally
Obtain the amylase strain that high enzyme is lived, there is large-scale promotional value.
Detailed description of the invention
In order to more clearly describe the technology contents of the present invention, conduct further description below in conjunction with specific embodiment.
After mutation in the present invention, the screening technique of high yield amylase strain has following preferably specific embodiment, does not wherein limit
It is as the criterion with existing microorganism routine operation with the operating procedure described.
Embodiment 1:
1 soil sampling
Apply five point sampling method collecting samples.Choosing representative soil in destination, choosing area is 2m × 2m.First
Using cornerwise midpoint as center sampling point, select the point of four Ge Yu center sampling point distances 0.5m as sample the most on the diagonal
Point.
2 strain primary dcreening operations and multiple sieve
By iodine dye method, the bacterium colony in sampling soil is carried out enrichment culture and Preliminary screening.
Amylase producing strains can produce amylase and excrete, at periphery of bacterial colonies starch-splitting.And iodine liquid meets the aobvious blueness of starch.
When sieved bacterium colony is amylase producing strains, around starch is decomposed, and dropping iodine liquid does not develops the color, and thus can produce in periphery of bacterial colonies
Raw obvious transparent circle.
Step:
(1) weigh each sample 0.5g respectively and number, fully diluting dissolving with sterilized water, then taking equivalent and be forwarded to 200mL
Enriched liquid culture medium, in 37 (± 0.5) DEG C incubator, 150r/min cultivates；
(2) after 24h, with sterilized water, each sample being carried out 10 times of gradient dilutions, drawing concentration is 10-6Culture fluid 100 μ L
It is spread evenly across on screening flat board, in 37 (± 0.5) DEG C incubator, cultivates 48h；
(3) add iodine liquid, observe the bacterium colony marking substantially hydrolysis circle, therefrom choose dyeing loop diameter big with colony diameter ratio
150 single bacterium colonies number and preserve.
Multiple sieve uses the stupid blue development process of bent profit.
When screening amylase producing strains, very directly perceived with iodine staining, but iodine liquid has bactericidal action, and hydrolysis is irised out,
Bacterium is likely to be killed, and experiment is produced impact.Bent profit benzene indigo plant does not has obvious inhibitory action to growth of microorganism, from this point
For surpassed with iodine liquid sieve bacterium.
Bent profit benzene indigo plant is a kind of non-specific dyestuff, is also trypan blue.Bent profit benzene is blue and starch is due to electrostatic non-specific adsorption knot
Making starch after conjunction is stable blueness, weakens because of the molecule absorption affinity that diminishes after starch is by amylase hydrolysis, and allows bent profit benzene indigo plant trip
Separating out, free bent profit benzene indigo plant is made color burn by the unhydrolysed starch adsorption of surrounding, Starch Hydrolysis district then formed colourless,
Transparent hydrolysis circle.
Step:
(1) take sterilized solid medium, culture dish, culture medium is dissolved, treats that temperature drops to 45 DEG C then in sterile working
Platform is down flat plate；
(2) in single bacterium colony 37 (± 0.5) DEG C incubator picked out 150r/min cultivate 24h, carry out successively 100 times, 1000
Times, 10000 times of dilutions, take respectively 10 μ l painting flat boards；
(3) 37 (± 0.5) DEG C are cultivated 24 hours, add bent profit benzene indigo plant, observe and measure transparent circle size, therefrom choose dyeing
Maximum 150 the single bacterium colonies of loop diameter and colony diameter ratio are numbered and are preserved.
Primary dcreening operation and multiple sieve after 3 starting strain mutagenic treatment
First generation starting strain is carried out mutagenic treatment, is separated on plate, beat agar block bacterium colony 1000 pieces, picking on assaying table
The bacterium colony of 10% transparent circle；Primary dcreening operation sub-step: by bacterial strain (1 bottle/bacterial strain) respectively shaking table 50 hours, little by diameter 4 millimeters
Circle filter paper, is put on starch agar plate (1.8% starch, 1.4% agar, 2 mm of thickness), every plate 6, inhales respectively
Take equivalent culture fluid point to filter paper, put in 65 (± 1) DEG C drying baker 2 hours (stability of the endoenzyme of this time is preferable,
Enzyme is lived and transparent circle size is proportionate), detect with iodine liquid, the diameter of more transparent circle, 30 strains selecting transparent circle diameter big are entered
The multiple sieve of row；30 strain primary dcreening operation bacterial strains (5 bottles/bacterial strain) are screened by multiple sieve step: the method utilizing primary dcreening operation sub-step,
Choose 3 strains starting strain as second filial generation mutation；The second filial generation, the third generation ... to the n-th same first generation of generation screening technique, directly
To selection-breeding to high yield amylase strain.
Embodiment 2:
1 soil sampling
Apply five point sampling method collecting samples.Choosing representative soil in destination, choosing area is 2m × 2m.First
Using cornerwise midpoint as center sampling point, select the point of four Ge Yu center sampling point distances 1m as sample the most on the diagonal
Point.
2 strain primary dcreening operations and multiple sieve
By iodine dye method the bacterium colony in sampling soil carried out enrichment culture and Preliminary screening:
(1) weigh each sample 0.5g respectively and number, fully diluting dissolving with sterilized water, then taking equivalent and be forwarded to 200mL
Enriched liquid culture medium, in 37 DEG C of incubators, 200r/min cultivates；
(2) after 24h, with sterilized water, each sample being carried out 10 times of gradient dilutions, drawing concentration is 10-6Culture fluid 100 μ L
It is spread evenly across on screening flat board, in 37 DEG C of incubators, cultivates 45h；
(3) add iodine liquid, observe the bacterium colony marking substantially hydrolysis circle, therefrom choose dyeing loop diameter big with colony diameter ratio
200 single bacterium colonies number and preserve.
The multiple stupid blue development process of the bent profit of sieve employing:
(1) take sterilized solid medium, culture dish, culture medium is dissolved, treats that temperature drops to 42 DEG C then in sterile working
Platform is down flat plate；
(2) in 37 DEG C of incubators of the single bacterium colony picked out 200r/min cultivate 24h, carry out successively 200 times, 2000 times, 20000
Dilution again, takes 20 μ l respectively and is coated with flat boards；
Cultivate 24 (± 1) hour for (3) 37 DEG C, add bent profit benzene indigo plant, observe and measure transparent circle size, therefrom choose dyeing circle
Maximum 200 the single bacterium colonies of diameter and colony diameter ratio are numbered and are preserved.
Primary dcreening operation and multiple sieve after 3 starting strain mutagenic treatment
First generation starting strain is carried out mutagenic treatment, is separated on plate, beat agar block bacterium colony 2000 pieces, picking on assaying table
The bacterium colony of 8% transparent circle；Primary dcreening operation sub-step: by bacterial strain (2 bottles/bacterial strain) respectively shaking table 50 hours, little by diameter 3 millimeters
Circle filter paper, is put on starch agar plate (1.8% starch, 1.4% agar, 2 mm of thickness), every plate 8, inhales respectively
Take equivalent culture fluid point to filter paper, put in 65 DEG C of drying baker 2 hours, detect with iodine liquid, the diameter of more transparent circle, select
40 strains that bright loop diameter is big carry out multiple sieve；Multiple sieve step: the method utilizing primary dcreening operation sub-step, by 40 strain primary dcreening operation bacterial strains (4
Bottle/bacterial strain) screen, choose 4 strains starting strain as second filial generation mutation；The second filial generation, the third generation ... to the n-th generation sieve
The same first generation of choosing method, until selection-breeding is to high yield amylase strain.
Embodiment 3:
1 soil sampling
Apply five point sampling method collecting samples.Choosing representative soil in destination, choosing area is 2m × 2m.First
Using cornerwise midpoint as center sampling point, select the point of four Ge Yu center sampling point distances 2m as sample the most on the diagonal
Point.
2 strain primary dcreening operations and multiple sieve
By iodine dye method the bacterium colony in sampling soil carried out enrichment culture and Preliminary screening:
(1) weigh each sample 0.5g respectively and number, fully diluting dissolving with sterilized water, then taking equivalent and be forwarded to 200mL
Enriched liquid culture medium, in 37 (± 1) DEG C incubator, 100r/min cultivates；
(2) after 24h, with sterilized water, each sample being carried out 10 times of gradient dilutions, drawing concentration is 10-6Culture fluid 100 μ L
It is spread evenly across on screening flat board, in 37 (± 1) DEG C incubator, cultivates 50h；
(3) add iodine liquid, observe the bacterium colony marking substantially hydrolysis circle, therefrom choose dyeing loop diameter big with colony diameter ratio
100 single bacterium colonies number and preserve.
The multiple stupid blue development process of the bent profit of sieve employing:
(1) take sterilized solid medium, culture dish, culture medium is dissolved, treats that temperature drops to 50 DEG C then in sterile working
Platform is down flat plate；
(2) in single bacterium colony 37 (± 1) DEG C incubator picked out 100r/min cultivate 24h, carry out successively 500 times, 5000
Times, 50000 times of dilutions, take respectively 100 μ l painting flat boards；
24 (± 2) hour are cultivated in (3) 37 (± 1) DEG C, add bent profit benzene indigo plant, observe and measure transparent circle size, Cong Zhongxuan
100 the single bacterium colonies taking dyeing loop diameter maximum with colony diameter ratio are numbered and are preserved.
Primary dcreening operation and multiple sieve after 3 starting strain mutagenic treatment
First generation starting strain is carried out mutagenic treatment, is separated on plate, beat agar block bacterium colony 3000 pieces, picking on assaying table
The bacterium colony of 5%% transparent circle；Primary dcreening operation sub-step: by bacterial strain (2 bottles/bacterial strain) respectively shaking table 50 hours, little by diameter 6 millimeters
Circle filter paper, is put on starch agar plate (1.8% starch, 1.4% agar, 2 mm of thickness), every plate 4, inhales respectively
Take equivalent culture fluid point to filter paper, put in 65 DEG C of drying baker 2 hours, detect with iodine liquid, the diameter of more transparent circle, select
50 strains that bright loop diameter is big carry out multiple sieve；Multiple sieve step: the method utilizing primary dcreening operation sub-step, by 50 strain primary dcreening operation bacterial strains (3
Bottle/bacterial strain) screen, choose 5 strains starting strain as second filial generation mutation；The second filial generation, the third generation ... to the n-th generation sieve
The same first generation of choosing method, until selection-breeding is to high yield amylase strain.
Have employed the screening technique of high yield amylase strain after the mutation in this invention, it has technical effect that, adopts during sample primary dcreening operation
With the bacterial strain filtering out needs of rapid, high volume, and low cost can be screened by iodine dye method, use bent profit stupid indigo plant development process when multiple sieve,
Utilize the characteristic that bent profit benzene is blue, be possible not only to filter out the bacterium colony producing transparent circle, and itself the growth of microorganism do not pressed down
Make use, can accurately screen and target bacterium colony not damaged.Starting strain after mutagenic treatment is by primary dcreening operation again
With the starting strain that multiple sieve can obtain the second filial generation, screening process is simple and easy to operate, screens in the time period that enzymatic activity is the highest
Can obtain the high yield amylase strain of laminating production requirement at short notice, whole screening process invests little targeting height, Ke Yiding
Obtain the amylase strain that high enzyme is lived to ground, there is large-scale promotional value.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still may be made that various
Amendment and conversion are without departing from the spirit and scope of the present invention.Therefore, description is regarded in an illustrative, rather than a restrictive.
Claims (10)
1. the screening technique of high yield amylase strain after a mutation, it is characterised in that described screening technique comprises the steps:
Bacterial strain screening step after mutation: carry out the first generation bacterial strain through mutagenic treatment screening step, including primary dcreening operation sub-step and multiple sieve step, wherein:
(1) primary dcreening operation sub-step includes: be placed on starch agar plate by the round filter paper of diameter 3～6mm, every plate 4～8, distinguish draws equal amounts culture fluid point to filter paper, 65 DEG C of dry 2h, with the detection of iodine liquid the diameter of more transparent circle, 30～50 strains selecting transparent circle diameter big carry out multiple sieve；
(2) multiple sieve step includes: by 30～50 strain primary dcreening operation bacterial strains, every kind of bacterial strain 3～5 bottles, again carry out the screening with (1) primary dcreening operation mode, take 3～5 strains starting strain as second filial generation mutation, again carry out mutation and screening step, the second filial generation, the third generation are to the same first generation of screening technique in the n-th generation, to selecting high yield starch bacterial strain.
The screening technique of high yield amylase strain after mutation the most according to claim 1, it is characterised in that the described bacterial strain after mutation comes from following step:
The screening step of (a) first generation starting strain: include primary dcreening operation step and sieve step again, wherein primary dcreening operation step is that iodine contaminates method, the bacterium colony choosing dropping iodine liquid generation transparent circle carries out multiple sieve step, and described multiple sieve step is bent profit benzene indigo plant development process, chooses the bacterium colony producing transparent circle；
The mutagenesis steps of (b) first generation starting strain: the bacterium colony producing transparent circle picked out by described bent profit benzene indigo plant development process carries out mutagenic treatment.
The screening technique of high yield amylase strain after mutation the most according to claim 2, it is characterised in that the set out screening step of bacterium of described (a) first generation specifically includes following sub-step:
(a-a) weighing sample 0.5g and number, fully diluting dissolving with sterilized water, then taking equivalent and be forwarded to 200mL enriched liquid culture medium, in 37 DEG C of incubators, 100～200r/min cultivate；
(a-b) after 24h, with sterilized water, each sample being carried out 10 times of gradient dilutions, drawing concentration is 10-6Culture fluid 100 μ L be spread evenly across screening flat board on, in 37 DEG C of incubators cultivate 45～50h；
(a-c) adding iodine liquid, observe the bacterium colony marking substantially hydrolysis circle, the single bacterium colony therefrom choosing dyeing loop diameter big with colony diameter ratio is numbered and is preserved；
(a-d) in 37 DEG C of incubators of the single bacterium colony that will be singled out, 100～200r/min cultivate 24h, carry out 10 successively2Again, 103Again, 104Dilution again, takes 10～100 μ L respectively and is coated with flat boards, cultivate 24 hours for 37 DEG C, and addition song profit benzene is blue, observes measurement transparent circle size, and the single bacterium colony therefrom choosing the loop diameter that dyes maximum with colony diameter ratio is numbered and preserved.
The screening technique of high yield amylase strain after mutation the most according to claim 3, it is characterised in that described sample is taken from soil.
The screening technique of high yield amylase strain after mutation the most according to claim 4, it is characterized in that, described sample from soil sampling particularly as follows: use five-spot gather soil sample, the sample prescription choosing 2m × 2m carries out diagonal line, samples as sampling point at sampling point Chu Jiyu center, diagonal center equidistant four points of sampling point.
The screening technique of high yield amylase strain after mutation the most according to claim 4, it is characterised in that described sample also includes before carrying out primary dcreening operation after fetching that enrichment culture process, described enrichment culture process use iodine dye method.
The screening technique of high yield amylase strain after mutation the most according to claim 1, it is characterised in that described (1) primary dcreening operation sub-step specifically includes:
Every bacterial strain 1～2 bottles of bacterium solution are cultivated 50h at shaking table with 100～200r/min respectively, the round filter paper of diameter 4mm is placed on starch agar plate, every plate 6, distinguish draws equal amounts culture fluid point to filter paper, 65 DEG C of dry 2h, with the detection of iodine liquid the diameter of more transparent circle, 30～50 strains selecting transparent circle diameter big carry out multiple sieve.
The screening technique of high yield amylase strain after mutation the most according to claim 6, it is characterised in that described starch agar plate is 1.8% starch, 1.4% agar, the starch agar plate of 2 mm of thickness.
The screening technique of high yield amylase strain after mutation the most according to claim 1, it is characterised in that screen superior strain step after described (1) mutation and also include processing sub-step, particularly as follows:
The strains separation of mutation will be carried out to plate, and beat agar block bacterium colony 1000～3000 pieces, the bacterium colony of picking 5%～10% transparent circle on assaying table, be used for carrying out (1) primary dcreening operation sub-step.
10. according to the screening technique of high yield amylase strain after the mutation described in any one of claim 1～9, it is characterised in that described bacterium colony is cultivated the solid medium used and is the aseptic culture medium poured out in 40～50 DEG CXia sterile workings.
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| CN111040953A (en) * | 2019-12-12 | 2020-04-21 | 南昌大学 | Method for rapidly screening variant microorganisms based on PET imaging system |
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