CN104862299A - Bacillus subtilis heterologous protein expression quantity improving method - Google Patents
Bacillus subtilis heterologous protein expression quantity improving method Download PDFInfo
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- CN104862299A CN104862299A CN201510200528.8A CN201510200528A CN104862299A CN 104862299 A CN104862299 A CN 104862299A CN 201510200528 A CN201510200528 A CN 201510200528A CN 104862299 A CN104862299 A CN 104862299A
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Abstract
The present invention provides a bacillus subtilis heterologous protein expression quantity improving method. Recombinant plasmid-contained B.s ubtilis is directly induced for mutation by ARTP and starch-trypan blue panel prescreening, 96 orifice plate high-throughput screening and shaking flask fermentation test to obtain a high enzyme activity mutant strain, and the enzyme activity is increased by 31.4%. The ARTP technology used in the method is a biological mutagenesis technology very wide in application range; during induced mutation, the recombinant plasmid-contained B.s ubtilis strain is directly induced for mutation, the recombinant plasmid conversion step can be eliminated after the induced mutation, efficiency is high; at the same time, and by combination with a 96 orifice plate for high-throughput screening, the efficiency for screening of direct mutation strains can be greatly improved, so that the method can rapidly screen a heterologous protein high yield B.s ubtilis strain, and has important economic value.
Description
Technical field
The present invention relates to microorganism mutation breeding, fermentation engineering and zymetology field.Be specifically related to one improves subtilis (Bacillus subtilis) exogenous protein expression amount method in conjunction with ARTP selection by mutation.
Background technology
B.subtilis is a kind of sporiferous gram-positive microorganism, has genetic background, good protein excretion ability and a series of efficient signal peptide more clearly.Compared with conventional prokaryotic expression host E.coli, B.subtilis does not contain intracellular toxin, has non-virulent, is a kind of food-grade microorganisms, can be used for the production of food fermentation and important enzyme preparation.In addition, B.subtilis is used to produce proteolytic enzyme and amylase for a long time, and the culture condition needed for it is very simple.Therefore, B.subtilis is the desirable host of secreting, expressing foreign protein in current prokaryotic expression system.
The recombinant host mutagenesis screening of high expression builds mutated library after often adopting empty host (not containing recombinant plasmid) to carry out mutagenesis.By recombinant plasmid transformed mutated library, build the mutated library containing recombinant plasmid, then carry out next step screening.But, often there is the shortcomings (particularly for B.subtilis host) such as transformation efficiency is on the low side, the loss of the mutant in a large number with superperformance can be caused in this method.For solving the problem, the present invention adopts the direct mutagenesis of B.subtilis bacterial strain containing recombinant plasmid when mutagenesis, eliminates the step of converting (B.subtilis bacterial strain recombinant plasmid transformed difficulty) of recombinant plasmid after mutagenesis, has high efficiency.
At present, improve the method for recombinant bacterium exogenous protein expression amount, except the optimization of traditional physics and chemistry behavior, optimizing fermentation and recombinant plasmid controlling element, grew up a kind of novel biological induced-mutation breeding technique--atmospheric pressure at room plasma body (ARTP) induced-mutation technique in recent years, the core component of this selection by mutation system is the normal atmosphere radio frequency glow discharge plasma generator of bare metal electrode structure, and the active particle produced has electronics, photon, ion, is in the neutral particle of excited state and some free radicals.These active particles can act on nucleic acid and the protein of cell to a certain extent, thus cause the change of gene and microorganism cells structure and permeability, cause microorganism to be undergone mutation.Compared with traditional physics and chemistry behavior and ion beam mutation technology, it is simple that this induced-mutation technique has equipment, easy and simple to handle, safety, the advantages such as mutation rate is high, mutated library capacity is large.In addition, the active particle temperature near room temperature that plasma source produces, action condition is gentle, avoids the thermal damage that some is subject to heat sensitive microorganism.
About screening method mainly random screening method or the pre-sieve method of flat-plate bacterial colony of high yield heterologous protein mutant strain.After reducing screening scope, the acquisition probability adopt shaking flask primary dcreening operation to carry out fermentation culture one by one to mutant strain and measure amylase enzyme and live, this screening method cycle is long and workload is large, reducing high productive mutant.Based on the high mutation rate of ARTP induced-mutation technique, in conjunction with the high-throughout sieve method of 96 orifice plate, greatly can improve the efficiency obtaining high productive mutant, therefore this technology can be widely used in the selection by mutation of microorganism.
Summary of the invention
The object of the present invention is to provide a kind of method improving subtilis exogenous protein expression amount.
For achieving the above object, the technical scheme that the present invention is used is:
The present invention adopts ARTP mutation breeding technologies to carry out direct mutagenesis to the B.subtilis bacterial strain containing recombinant plasmid, obtains restructuring B.subtilis168 mutant library.Carry out pre-sifted by the transparent circle size that comparison starch-trypan blue nutrition flat board produces, reduce screening scope, then provide a kind of high-throughput screening method based on 96 orifice plates, obtain B.subtilis 168 mutant strain of high yield alkali starch enzyme efficiently.Sieve the enzymatic production ability verifying the high productive mutant screened finally by shaking flask again, sequence verification is carried out to the recombinant plasmid contained by superior strain and genetic stability checking is carried out to recombinant bacterial strain.
Specifically comprise the following steps:
To set out the activation of bacterium: the restructuring B.subtilis 168 containing recombinant plasmid is rule in fresh LB solid medium, 10 ~ 20h is cultivated at 25 ~ 37 DEG C, in order bacterium colony to fresh LB liquid nutrient medium, after 25 ~ 37 DEG C of cultivation 8 ~ 24h, by 20 ~ 60% inoculum size switching fresh LB, carry out synchronized culture;
ARTP mutagenesis: with resuspended thalline after stroke-physiological saline solution washing thalline 1 ~ 5 time, and suitable dilution cell concentration to 10
6 ~ 9individual/mL, gets 2 ~ 15 μ L and is evenly applied on aseptic slide glass, be placed in 2 ~ 6mm under process source, starts mutagenic treatment;
Cultivate after mutant strain: by the bacterium liquid after mutagenesis, put into fresh LB liquid nutrient medium immediately, cultivate after carrying out 1 ~ 4h, suitably dilution cell concentration to 10
6 ~ 9individual/mL, is spread evenly across cultivation 8 ~ 16h on fresh LB (Kana resistance) flat board;
Foreign protein high productive mutant screening (this patent is for alkali starch enzyme): by the mutant in restructuring B.subtilis 168 mutant library mutagenic obtained in step (3), with sterile toothpick one by one dibbling on starch-trypan blue nutrition flat board, on every block flat board, dibbling 2 ~ 4 strains of setting out contrast, observe transparent circle size after 25 ~ 37 DEG C of cultivation 6 ~ 12h, wherein larger than the little transparent circle of strain bacterium colony that sets out may be the strain of high yield alkali starch enzyme mutant; After reducing screening scope, carry out 96 orifice plate high flux screenings, finally carry out shake flask fermentation checking.
Recombinant plasmid checking in recombinant host: extract the recombinant plasmid containing object foreign protein, carry out gene sequencing, whether checking recombinant plasmid itself suddenlys change;
The checking of high productive mutant genetic stability: high productive mutant shaking flask checking obtained, continuous passage 3 ~ 10 times, surveys whether alkali starch enzyme enzyme is alive stablizes.
Thalline after washing described in step (2) is resuspended in physiological saline, then suitably dilutes, makes OD
600between 1.0 ~ 3.0, guarantee produce active particle can stepless action in thalline.In step (2), the optimum configurations of ARTP mutagenic treatment is: airshed 8 ~ 16slm, radio frequency power 80 ~ 140W, time 20 ~ 50s, distance process spacing are from 2 ~ 6mm.After plasma body mutagenesis, rear cultivation 1 ~ 4h, makes the sudden change of generation more stable.
Restructuring B.subtilis 168 mutant library containing alkali starch enzyme gene produced in step (4), dibbling carries out pre-sifted in starch-trypan blue nutrition flat board one by one, by the size screening high yield recombinase mutant strain of transparent circle, then 96 orifice plate high flux screenings are carried out, obtain a small amount of High yield Mutant, finally adopt shake flask fermentation checking enhanced variant.Starch used in this step-trypan blue nutrient medium ingredients and content are: Zulkovsky starch 0.5 ~ 1.5%, trypan blue 0.01 ~ 0.06%, NaCl 0.5 ~ 1.5%, Yeast Extract 0.2 ~ 0.8%, Tryptone 0.5 ~ 2.0%, agar 1.5 ~ 2.5%, 121 DEG C of sterilizing 15min.In screening flat board, the working concentration of kantlex is 30 ~ 60 μ g/mL.
In step (4), shake flask fermentation verifies that seed culture medium used is LB substratum, after 25 ~ 37 DEG C of cultivation 8 ~ 12h, is inoculated in fermention medium by the inoculum size of 1.0 ~ 4.0%.Medium of shaking flask fermentation composition is: Zulkovsky starch 0.1 ~ 1.5%, soy peptone 0.5 ~ 3.0%, soybean cake powder 0.5 ~ 3.0%, NaCl 0.5 ~ 1.5%, 121 DEG C of sterilizing 15min.Liquid amount is 15 ~ 60mL/250mL, and leavening temperature is 30 ~ 37 DEG C, and fermented incubation time is 48 ~ 80h.High flux screening adopts 96 orifice plates, and every hole liquid amount 400 ~ 1000 μ L, its composition of fermention medium used and preparation steps are: first join phosphoric acid buffer, take 1.5 ~ 3.0g KH
2pO
4with 10.0 ~ 15.0g K
2hPO
4, be settled to 100mL; Yeast Extract 18.0 ~ 30.0g, Tryptone 8.0 ~ 16.0g, glycerine 2.0 ~ 6.0mL, is dissolved in 900mL water; Sterilizing, when phosphoric acid buffer is cooled to 50 ~ 60 DEG C, adds in 900mL substratum, shakes up, stand-by.
The genetic stability of checking high productive mutant in step (5), by the high productive mutant that shaking flask checking obtains, whether continuous passage 3 ~ 10 times, survey alkali starch enzyme enzyme activity and stablize.
Accompanying drawing explanation
Figure 196 orifice plate high flux screening result
Fig. 2 mutant strain mut-16# compares with the strain enzyme activity that sets out
Embodiment
In order to have a better understanding the present invention, existing the present invention is described further with specific examples by reference to the accompanying drawings.
Embodiment 1: utilize atmospheric pressure at room plasma body mutagenesis B.subtilis 168
Strain of setting out restructuring B.subtilis 168 carries out ruling at fresh LB flat board, activation culture, and picking list bacterium colony is in fresh LB liquid nutrient medium, and 250mL Erlenmeyer flask is cultivated, and the working concentration of kantlex is 50 μ g/mL, rotating speed 200rpm.After cultivation terminates, fresh LB liquid nutrient medium of transferring, after 2 ~ 4h when thalline is in synchronous growth state, draws bacterium liquid, centrifugal rear stroke-physiological saline solution washing, suitable diluting cells concentration.Draw bacterium liquid to be evenly applied on aseptic slide glass.This breeding system is working gas with He, and radio frequency power is set to 100W, gas flow 10slm, treatment time 30s.
Carry out rear cultivation operation to the thalline after ARTP mutagenesis, the thalline (together with slide glass) mutagenic treatment crossed, puts into the centrifuge tube that fresh LB liquid nutrient medium is housed immediately, and after sealed membrane sealing, on oscillator, fully concussion is rear cultivates.
Embodiment 2: the screening method of alkali starch enzyme high yield B.subtilis 168 bacterial strain
Starch-trypan blue nutrition plate screening
Bacterium liquid after mutagenic treatment, gradient dilution to 10 after rear cultivation
-4, be then 10 by undiluted bacterium liquid and dilution
-1, 10
-2, 10
-3, 10
-4these 5 gradients are coated with 3 pieces of LB flat boards respectively, obtain restructuring B.subtilis 168 mutant library after cultivation, with sterile toothpick by the single bacterium colony on flat board one by one dibbling on starch-trypan blue nutrition flat board, after cultivation, pick out the bacterium colony that transparent circle is greater than the bacterium that sets out.
96 orifice plate high flux screenings
Select bacterium colony in above-mentioned steps (1) is connected to respectively in seed culture medium (LB liquid nutrient medium), high flux screening is carried out in the substratum of transferring after cultivation in 96 orifice plates, every strain is done 3 and parallelly to be fermented, measure alkali starch enzyme enzyme activity, enzyme activity determination result is as Fig. 1.Wherein, No. 16 bacterium enzyme activities improve the most remarkable, and the bacterium that comparatively sets out improves 33.2%.
Shake flask fermentation screening verification
No. 16 mutant strains obtained by high flux screening carry out shake flask fermentation, and the inoculum size with 2% is transferred in fermention medium, 250mL triangular flask, rotating speed 200rpm, survey amylase enzyme activity by DNS method.Result shows, and shake flask results is similar to 96 orifice plate the selection result, and No. 16 mutant strain (16#) enzyme strains (the wild-type) of comparatively setting out alive improve 31.4% (see Fig. 2).
Embodiment 3: the genetic stability checking of alkali starch enzyme high productive mutant B.subtilis 168 mut-16#
By high productive mutant B.subtilis 168 mut-16# that is preserved in glycerine pipe on solid LB flat board, line activation, after picking list colony inoculation is cultivated in fresh liquid LB substratum, transfer in another bottle of fresh LB, similarity condition cultivates 12h, after switching repeatedly like this 5 times, be seeded in fermention medium after fermentation, measure alkali starch enzyme enzyme activity by DNS method.Result shows, and the high productive mutant B.subtilis 168-mut16 bacterium enzymatic productivity that comparatively sets out improves 30.1%, and the mutant strain genetic stability that visible the method obtains is good.
Claims (8)
1. improve a method for subtilis exogenous protein expression amount, it is characterized in that, comprise the following steps:
(1) starting strain activation: the restructuring B.subtilis (this patent is for B.subtilis 168) containing recombinant plasmid be preserved in glycerine pipe is rule on LB solid medium, 10 ~ 20h is cultivated at 25 ~ 37 DEG C, picking list bacterium colony is in fresh LB liquid nutrient medium, after 25 ~ 37 DEG C of cultivation 8 ~ 24h, by 20 ~ 60% inoculum size switching fresh LB, carry out synchronized culture;
(2) ARTP mutagenic treatment: with resuspended thalline again after stroke-physiological saline solution washing thalline 1 ~ 5 time, suitably dilution cell concentration to 106 ~ 9/mL, gets 2 ~ 15 μ L and is evenly applied on aseptic slide glass, be placed in 2 ~ 6mm under process source, start mutagenic treatment;
(3) the rear cultivation of mutant strain: by the bacterium liquid after mutagenesis, put into fresh LB liquid nutrient medium immediately, after rear cultivation 1 ~ 4h, suitably dilution cell concentration is 106 ~ 9/mL, is spread evenly across cultivation 4 ~ 16h on fresh LB (Kana resistance) flat board;
(4) foreign protein high productive mutant screening (this patent is for alkali starch enzyme): by the mutant in restructuring B.subtilis 168 mutant library mutagenic obtained in step (3), with sterile toothpick one by one dibbling on starch-trypan blue nutrition flat board, on every block flat board, dibbling 2 ~ 4 strains of setting out contrast, observe transparent circle size after 25 ~ 37 DEG C of cultivation 6 ~ 12h, wherein larger than the little transparent circle of strain bacterium colony that sets out may be the strain of high yield alkali starch enzyme mutant; After reducing screening scope, carry out 96 orifice plate high flux screenings, finally carry out shake flask fermentation checking.
(5) recombinant plasmid checking in recombinant host: extract the recombinant plasmid containing object foreign protein, carry out gene sequencing, whether checking recombinant plasmid itself suddenlys change;
(6) checking of high productive mutant genetic stability: high productive mutant shaking flask checking obtained, whether continuous passage 3 ~ 10 times, survey the amount of producing alkali starch enzyme and stablize.
2. the method in conjunction with ARTP mutagenesis and high flux screening alkali starch enzyme high yield B.subtilis 168 mutant strain according to claim 1, is characterized in that: described (2) middle stroke-physiological saline solution is resuspended to be prepared bacteria suspension and suitably dilutes OD
600between 1.0 ~ 3.0; Mutagenic treatment optimum configurations is: airshed 8 ~ 16slm, radio frequency power 80 ~ 140W, time 20 ~ 50s.
3. the method in conjunction with ARTP mutagenesis and high flux screening alkali starch enzyme high yield B.subtilis 168 mutant strain according to claim 1, it is characterized in that: in described (3), cultivate after carrying out 1 ~ 4h, thus repair the DNA of mutant damage in library, make sudden change more stable.
4. the method in conjunction with ARTP mutagenesis and high flux screening alkali starch enzyme high yield B.subtilis 168 mutant strain according to claim 1, it is characterized in that: in described (4), obtain possibility high productive mutant according to bacterium colony and transparent circle size around starch-trypan blue nutrition flat board, nutrient medium ingredients used is: Zulkovsky starch 0.5 ~ 1.5%, trypan blue 0.01 ~ 0.06%, NaCl 0.5 ~ 1.5%, Yeast Extract 0.2 ~ 0.8%, Tryptone 0.5 ~ 2.0%, agar 1.5 ~ 2.5%, 121 DEG C of sterilizing 15min.In flat board, the working concentration of kantlex is 30 ~ 60 μ g/mL.
5. the method in conjunction with ARTP mutagenesis and high flux screening alkali starch enzyme high yield B.subtilis 168 mutant strain according to claim 1, it is characterized in that: the high flux screening completing alkali starch enzyme High yield Mutant in described (4) based on 96 orifice plates, fermentation medium components used and preparation steps are: first join phosphoric acid buffer, take 1.5 ~ 3.0g KH respectively
2pO
4with 10.0 ~ 15.0g K
2hPO
4, be settled to 100mL; Yeast Extract 18.0 ~ 30.0g, Tryptone 8.0 ~ 16.0g, glycerine 2.0 ~ 6.0mL, is dissolved in 900mL water.Under 121 DEG C of conditions, sterilizing 15min, when phosphoric acid buffer is cooled to about 50 ~ 60 DEG C, adds in 900mL substratum, shakes up respectively, stand-by.
6. the method in conjunction with ARTP mutagenesis and high flux screening alkali starch enzyme high yield B.subtilis 168 mutant strain according to claim 1, it is characterized in that: in described (4), shake flask fermentation verifies that nutrient media components used is: Zulkovsky starch 0.1 ~ 1.5%, soy peptone 0.5 ~ 3.0%, soybean cake powder 0.5 ~ 3.0%, NaCl 0.5 ~ 1.5%, 121 DEG C of sterilizing 15min.
7. recombinant plasmid checking in recombinant host according to claim 1, is characterized in that: extract the recombinant plasmid containing object foreign protein, carry out gene sequencing, whether checking recombinant plasmid itself suddenlys change.
8. according to claim 1 to the checking of high productive mutant genetic stability, it is characterized in that: the high productive mutant that shaking flask checking is obtained, continuous passage 3 ~ 10 times, survey alkali starch enzyme enzyme activity and whether stablize.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762762A (en) * | 2019-01-11 | 2019-05-17 | 江苏大学 | A kind of superior strain of lipopeptid and its preparation method and application |
| CN112409464A (en) * | 2020-11-23 | 2021-02-26 | 江南大学 | Signal peptide mutant for improving extracellular production level of bacillus subtilis recombinant protein and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762762A (en) * | 2019-01-11 | 2019-05-17 | 江苏大学 | A kind of superior strain of lipopeptid and its preparation method and application |
| CN112409464A (en) * | 2020-11-23 | 2021-02-26 | 江南大学 | Signal peptide mutant for improving extracellular production level of bacillus subtilis recombinant protein and application thereof |
| CN112409464B (en) * | 2020-11-23 | 2022-04-22 | 江南大学 | Signal peptide mutant for improving extracellular production level of bacillus subtilis recombinant protein and application thereof |
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Application publication date: 20150826 |