CN108102997A - A kind of bacillus subtilis for Pullulanase high efficient expression and High Density Cultivation - Google Patents
A kind of bacillus subtilis for Pullulanase high efficient expression and High Density Cultivation Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilises for Pullulanase high efficient expression and High Density Cultivation, belong to technical field of bioengineering.The present invention uses CRISPR/Cas9 gene editing systems, nprB, bpr, mpr, vpr, epr and wprA of bacillus subtilis WS5 this 6 genes are knocked out successively, respectively obtain WSH6, WSH7, WSH8, WSH9, WSH10 and WSH11 this 6 kinds of bacillus subtilis strains.Bacillus subtilis WS5 and above-mentioned six kinds of bacterium competences are prepared, Pullulanase expression plasmid is converted, obtains seven kinds of Pullulanase recombinant bacteriums, is screened by tank on shaking flask screening and 3 L tanks.Screening, which is drawn, produces Pullulanase enzyme activity highest when host strain is WSH9,3 L tanks fermentation 78h, Pullulanase enzyme activity is the extracellular highest level of the Pullulanase reported at present up to 2449.6U/mL.
Description
Technical field
The present invention relates to a kind of bacillus subtilises for Pullulanase high efficient expression and High Density Cultivation, belong to fermentation
Field of engineering technology.
Background technology
Bacillus subtilis (Bacillus subtilis) belongs to gram-positive bacteria, because it is easily isolated culture, has
More visible genetic background and good secretory, but the features such as no pathogenicity, it has also become important industrial strain, by increasingly
It is more for producing antibiotic, pharmaceutical protein and industrial enzyme preparation etc..The wild bacillus subtilis reported at present can at least produce
Raw eight kinds of extracellular proteases：Neutral proteinase (nprE and nprB), alkali protease (aprE), serine protease (epr, bpr
And vpr), metalloproteinases (mpr) and cell wall protein enzyme (wprA).Extracellular protease plays nutrition suction in bacillus subtilis
It receives, the important function such as normal physiological metabolism and wrong protein degradation.Correct folded protein due to its protease resistant conformation, it is difficult to
It is easily degraded by proteases.Folding is slower when non-homogeneous albumen is due to its cross-film, easier than homologous protein to be easily degraded by proteases.Protease
The quality of secreted albumen ensure that a certain extent to the degradation of foreign protein, but also reduce protein expression level,
The main function finally risen according to the folded state of secreted albumen is different.For not allowing the albumen of foldable, protease can
With incorrect folded protein of degrading, ensure its quality on folded.And for folding easier albumen, folded state is relatively good, egg
The albumen that white enzyme degradation correctly folds, influences protein expression level.
Pullulanase can be with α -1 in hydrolysis starch polysaccharide, 6 glycosidic bonds.In saccharifying, Pullulanase can coordinate
Starch is resolved into small molecule reduced sugar, cyclodextrin and amylose etc. by other amylase.Pullulanase is widely used in wine
In the industrial production of smart fuel, resistant starch and maltotriose slurry etc..Pullulanase in many hosts such as：Escherichia coli,
It is expressed in Brevibacillus brevis, bacillus subtilis, thermophilic aspergillus flavus and pichia pastoris yeast etc..Extracellular table in Escherichia coli
It is up to 1567.9U/mL up to amount, but there is food-safety problems.Extracellular expression amount is 1005.8U/ in Brevibacillus brevis
ML, but there is fermentation instability problems.Yield is all relatively low at present in other hosts, cannot still meet industrial requirement.Withered grass bud
Bacterial strain of the spore bacillus as food security, becomes the preferred of industrial applications, however the bacterial strain industrially used is such as
B.subtilis WB600 and B.subtilis WB800 are derived from B.subtilis 168, are transformed through protease knockout
Come.The fermented and cultured for the above-mentioned type strain reported at present cannot all reach higher cell density, and type strain compared with
The exogenous protein expression amount of some wild strains is low.Therefore, bacillus subtilis possessed into the characteristic of high density fermentation for general
The industrialized production of Shandong orchid enzyme plays the role of vital.
The content of the invention
The purpose of the present invention, which first consists in, provides a kind of bacillus subtilis suitable for High Density Cultivation, is with withered grass bud
Spore bacillus WS5 is starting strain, knocks out the recombinant bacillus bud of at least one gene in nprB, bpr, mpr, vpr, epr or wprA
Spore bacillus.
In one embodiment of the invention, the bacillus subtilis WS5 was preserved in China on 29th in September in 2016
Type Tissue Collection, deposit number are CCTCC NO:M 2016536, preservation address are Wuhan, China Wuhan University,
Disclosed in the patent application document of Publication No. CN106754466A.
In one embodiment of the invention, the recombined bacillus subtilis is using bacillus subtilis WS5 to go out
Bacterium germination strain knocks out the recombined bacillus subtilis of nprB, bpr, mpr, vpr, epr or wprA gene respectively.
In one embodiment of the invention, described knock out is knocked out with plasmid pHY300.
Second object of the present invention is to provide a kind of genetic engineering bacterium of efficient production Pullulanase, is applicable in described
Pullulanase gene is expressed in the bacillus subtilis of High Density Cultivation.
In one embodiment of the invention, Pullulanase gene is expressed by carrier of pHYPULd4；It is described
PHYPULd4 is the pHY300PLK for being connected with Pullulanase gene, and the construction method of the plasmid is in the paper of 2017
《High-level extracellular protein production in Bacillus subtilis using an
optimized dual-promoter expression system》Disclosed in.
Third object of the present invention is to provide a kind of production method of Pullulanase, and the method is that the restructuring is withered
Careless bacillus is seeded in fermentation medium, in 33~37 DEG C of cultures.
Fourth object of the present invention is to provide the fermentation process in high density of Pullulanase, and the method is by the restructuring
Hay bacillus is seeded in fermentation medium, controls pH 6.5-7.0,33-37 DEG C of cultivation temperature, and first dissolved oxygen maintains 28~
32% rises rapidly up to dissolved oxygen, starts the glucose feed supplement liquid of stream plus concentration for 500g/L, when Pullulanase enzyme activity declines
Terminate culture.
In one embodiment of the invention, the fermentation medium contains 15g/L dusty yeasts, 25g/L corn pulps,
15g/L lactose, 1g/L (NH4)2-H-citrate,2g/L Na2SO3,2.68g/L(NH4)2SO4,14.6g/L K2HPO4,4g/L
NaH2PO4·H2O,1g/L MgSO4·7H2O, 3mL/L trace element (TES).
In one embodiment of the invention, micro- (TES) contains：0.5g/L CaCl2,0.18g/
LZnSO4×7H2O,0.1g/L MnSO4×H2O,10.05g/L Na2-EDTA,8.35g/L FeCl3,0.16g/L CuSO4×
5H2O,and 0.18g/L CoCl2×6H2O。
In one embodiment of the invention, the lactose in culture medium is disappeared in recombined bacillus subtilis fermentation process
The phenomenon that there is dissolved oxygen rebound completely in consumption, dissolved oxygen rises rapidly.
In one embodiment of the invention, the recombined bacillus subtilis is followed by kind to fermented and cultured through overactivation
In base, the activation is containing 4g/L dusty yeasts, 12g/L peptones, 5g/L glycerine, 12.54g/L K2HPO4, 2.31g/
LKH2PO4Seed culture medium in cultivated.
In one embodiment of the invention, the method is that the recombined bacillus subtilis is inoculated with seed culture
In base, 37 DEG C, 200r/min culture 12h, the 3L fermentation tanks for being 0.9L by above-mentioned culture solution access liquid amount, with ammonium hydroxide and 20%
Phosphoric acid controls pH 6.5-7.0, and 33-37 DEG C of cultivation temperature is maintained dissolved oxygen by the way that throughput is coupled and adjusted with speed of agitator
30% or so, when dissolved oxygen rises rapidly, start stream plus glucose feed supplement liquid that concentration is 500g/L, when Pullulanase enzyme activity declines
When terminate to cultivate.
The 5th purpose of the present invention is to provide application of the method in field of food.
The present invention also provides the recombined bacillus subtilis to produce protein-contg product, prepare alcohol fuel, resistance
Application in terms of starch or maltotriose slurry.
Advantageous effect：Pullulanase enzyme activity is most in shaking flask level by recombined bacillus subtilis WSH5 provided by the invention
Height is 90.7U/mL, remaining is successively：WSH6 is 85.0U/mL, WSH9 84.9U/mL, WSH10 82.9U/mL, WSH8 are
76.7U/mL, WSH7 are 72.8U/mL and WSH11 is 62.2U/mL.Above-mentioned recombined bacillus subtilis 3-L tanks ferment 78h,
Pullulanase enzyme activity is the extracellular highest level of the Pullulanase reported at present up to 2449.6U/mL, at this time dry cell weight (DCW)
For 67.01g/L.Remaining is when WSH10 is host strain successively, Pullulanase enzyme activity in 72h up to 2177.1U/mL, DCW at this time
For 70.49g/L；When WS5 is host strain, Pullulanase enzyme activity is in 75h up to 2095U/mL, and DCW is 64.60g/L at this time；
When WSH11 is host strain, Pullulanase enzyme activity is in 51h up to 1047.6U/mL, and DCW is 56.12g/L at this time.
Description of the drawings
Fig. 1 is nprB, bpr, mpr, vpr, epr and wprA gene knockout Xho I digestion verification nucleic acid electrophoresis figures；Wherein,
WT represents wild type；MT represents saltant type.
Fig. 2 is that bacillus subtilis WSH9 is that host recombinantly expresses Pullulanase 3-L tank fermented and cultureds SDS-PAGE figures,
Middle digital representation different fermentations time (unit：h).
Specific embodiment
Pullulanase enzyme activity determination method：
1% pulullan polysaccharide substrates of 1mL and 0.9mL100mM pH4.5 acetic acid-sodium acetate buffer solutions are uniformly mixed, put
In preheating 10min in water-bath, appropriate diluted Propiram enzyme solution 0.1mL is then added in, after reacting 10min, adds in 3mLDNS
Developing solution is placed in ice water and terminates reaction, and 7min is then boiled in boiling water bath, then adds 10mL deionized waters, mixing, in 540nm
Lower measure light absorption value calculates enzyme activity.The enzyme amount of 1 μm of ol reduced sugar of generation per minute is defined as an enzyme activity unit.
The measure of biomass：
The fermented sample of 5mL is periodically taken, 4 DEG C, 12000rpm is centrifuged 10 minutes.The NaCl solution of 0.9% (w/v) of precipitation
It is resuspended, then again at 4 DEG C, 12000rpm is centrifuged 10 minutes.It is deposited in 105 DEG C of drying and draws unit sample to constant weight divided by 5mL
The dry cell weight (DCW) of product.
Embodiment 1：Knock out the structure of plasmid
According to the gene order of bacillus subtilis designed for selectively targeted nprB, bpr, mpr, vpr, epr and
The sgRNA of wprA the genes, (matter on the basis of the CRISPR/Cas9 of this laboratory structure early period knocks out plasmid pHY300dsrf1
The construction method of grain is disclosed in the paper of 2016《Multigene disruption in undomesticated
Bacillus subtilisATCC6051ausing the CRISPR/Cas9system》In), design primer PCR amplification mutation
Original sgRNA obtains the knockout plasmid of six modifieds；Then using bacillus subtilis WS5 genomes as template, PCR amplification
Homologous reparation arm pieces section with XbaI enzyme cutting site, is connected on pMD-18T cloning vectors, and conversion JM109 is expanded.XbaI
Digestion is connected with the cloning vector of homology arm and knocks out plasmid respectively, and then T4 ligases connection overnight, obtains six knockout plasmids
PHY300dnprB, pHY300dbpr, pHY300dmpr, pHY300dvpr, pHY300depr and pHY300dwpr.
1 sgRNA sequences of table
2 XbaI enzyme cutting system of table is：
37 DEG C of endonuclease reaction 2h.
Embodiment 2：Hay bacillus method for transformation
The bacillus subtilis frozen is picked with oese, then in the flat lining outs of LB, 37 DEG C of overnight incubation activation.It chooses
Single bacterium colony is taken to be inoculated in 5mL LB fluid nutrient mediums, 37 DEG C of overnight incubation culture 18h.A certain amount of overnight culture is taken to arrive
In the GM I of 4.5mL, OD600 values is made to reach 0.1-0.2, leave 4.5mL mixed bacteria liquids.37 DEG C of 200rpm shaken cultivations, each
20min surveys an OD600, when OD600 reaches 0.4-0.6 (taking around 60-90min)；Continue shaken cultivation 90min, draw
0.05mL bacterium solutions are into the sterile test tube for the GM II for having 0.45mL preheatings；37 DEG C of vibration 90min, have many in culture at this time
Competent cell is formed；Add the plasmid (15-20 μ l) of 1 μ g, 37 DEG C of shaken cultivation 30min；Centrifugation removes most supernatant,
Cell is resuspended, is coated in the screening flat board containing corresponding antibiotic, 37 DEG C of overnight incubations.
Culture medium prescription：
(1) 10 × minimum salting liquid：K2HPO414g(K2HPO4·3H2O 18.34g), KH2PO46g, (NH4)2SO42g, lemon
Lemon acid sodium (Na3C6H5O7·2H2O) 1g, MgSO4·7H2O 0.2g, dissolve successively in distilled water, add water to 100mL.
(2) L-trp solution, 2mg/mL are stored in brown bottle, and 113 DEG C of sterilizing 30min are wrapped up in black paper bag.
(3) I solution of GM：1 × minimum salting liquid 95mL, 50% glucose 1mL, 5% caseinhydrolysate 0.4mL, 10% ferment
Female juice 1mL, 2mg/mL L-trp 2.5ml.
(4) II solution of GM：1 × minimum salting liquid 97.5mL, 50% glucose 1mL, 5% caseinhydrolysate 0.08mL,
10% yeast juice 0.04mL, 0.5M MgCl20.5mL (2.5mM), 0.1M CaCl20.5mL (0.5mM), 2mg/mL L-
trp0.5mL(5ug/mL)。
Embodiment 3：B.subtilis WS5 gene knockouts
Respectively with plasmid pHY300dnprB, pHY300dbpr, pHY300dmpr, pHY300dvpr, pHY300depr and
PHY300dwpr knocks out six genes of nprB, bpr, mpr, vpr, epr and wprA in B.subtilis WS5 genomes.Using
Method in embodiment 2 will knock out plasmid and be transformed into bacillus subtilis bacterium competence cell, is applied to LB solid mediums
On (containing 20 μ g/mL tetracyclines), 37 DEG C are incubated overnight.Picking positive colony extracts genome, using genome as template bacterium colony
Then the homologous reparation segment of PCR amplification carries out Xho I digestion verifications at 37 DEG C.Since the bacterial strain for knocking out gene carries out genome
Xho I restriction enzyme sites are inserted during homologous reparation at gene break, it is possible to it is cut by Xho I restriction endonucleases, and wild type
Will not then be cut open, the correct clpp gene degerming of digestion verification carried out at 51 DEG C knock out plasmid elimination (Fig. 1).It knocks out successively
Respectively obtained after six genes nprB, bpr, mpr, vpr, epr and wprA 6 kinds of clpp gene degerming B.subtilis WSH6,
B.subtilis WSH7, B.subtilis WSH8, B.subtilis WSH9, B.subtilis WSH10 and B.subtilis
WSH11
3 XhoI digestion systems of table are：
37 DEG C of endonuclease reaction 2h.
Embodiment 4：The structure of Bacillus deramificans sources Pullulanase genetic engineering bacterium
Plasmid for building bacillus subtilis expression vector is pHY300PLK, with double-promoter PHpaII-PamyQ’。
With plasmid pHY300PLK and plasmid pNCMO2/pulA-d2, (the Bacillus deramificans of this Laboratories Accession come respectively
Expression plasmid of the source Pullulanase in Escherichia coli) it is template, it is amplified with primer P1/P2 and P3/P4PCR same with 15bp
The carrier segments and genetic fragment of source sequence, then connected with In-Fusion HD Cloning Plus kit ligases, connection production
Object Transformed E .coli JM109 competent cells through 37 DEG C of culture 8h, choose transformant containing 100mg/L ampicillin liquids
LB in shaken cultivation, extract plasmid, sequence verification obtains expression plasmid pHYPULd4.
4 PCR reaction systems of table are：
Response procedures are as follows：94 DEG C of pre-degeneration 4min；98 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 1.5min carry out 30 Xun Huans；
72 DEG C of extension 10min, are cooled to 4 DEG C.
5 primer sequence of table
By plasmid pHYPULd4 convert B.subtilis WS5, B.subtilis WSH6, B.subtilis WSH7,
Seven kinds of B.subtilis WSH8, B.subtilis WSH9, B.subtilis WSH10 and B.subtilis WSH11 host strains,
On LB tablets of the coating containing tetracycline (20mg/L), 37 DEG C of culture 8h.It chooses single bacterium to drop down onto in liquid LB, 37 DEG C of overnight incubations, protect
Glycerol tube is deposited, finally obtains seven kinds of Pullulanase genetic engineering bacteriums.
Embodiment 5：Seven kinds of Pullulanase genetic engineering bacterium shaking flask screenings
Shake flask fermentation tank culture is carried out to the seven kinds of Pullulanase genetic engineering bacteriums built in above-described embodiment 4, is examined
Pullulanase expression, incubation are as follows：The glycerol tube bacterium solution for drawing 20 μ l is inoculated in equipped with 10mL seed culture mediums
In 50mL triangular flasks, 37 DEG C, 200r/min cultures 8-10h.By above-mentioned culture using the inoculum concentration of 5% (v/v) access liquid amount as
Fermentation tank in the 250mL triangular flasks of 50mL, 37 DEG C, 200r/min cultures 2h.Then 33 DEG C, 200r/min cultures 48h.Measure born of the same parents
Outer Pullulanase enzyme activity.Enzyme activity is up to 90.7U/mL when being drawn by shaking flask screening using WSH5 as host strain, remaining is successively
WSH6 is 85.0U/mL, WSH9 84.9U/mL, WSH10 82.9U/mL, WSH8 76.7U/mL, WSH7 be 72.8U/mL and
WSH11 is 62.2U/mL.
Culture medium prescription：
(1) seed culture medium：Peptone 20g/L, dusty yeast 10g/L, glucose 20g/L.
(2) fermentation medium：4g/L dusty yeasts, 12g/L peptones, 5g/L glycerine, 12.54g/L K2HPO4, 2.31g/L
KH2PO4。
Embodiment 6：The fermentation screening of Pullulanase genetic engineering bacterium 3-L tanks
Four kinds of Pullulanase recombinant bacteriums that host strain in above-described embodiment 4 is WS5, WSH9, WSH10 and WSH11 are carried out
3-L fermented and cultureds examine Pullulanase expression.Incubation is as follows：The glycerol tube bacterium solution for drawing 200 μ l is inoculated in and is equipped with
In the 500mL triangular flasks of 100mL seed culture mediums, 37 DEG C, 200r/min culture 12h, it is by above-mentioned culture solution access liquid amount
The 3L fermentation tanks of 0.9L, with ammonium hydroxide and 20% phosphoric acid control pH 6.5-7.0,33-37 DEG C of cultivation temperature, by with speed of agitator
It coupling and adjusts throughput dissolved oxygen is maintained 30% or so, when dissolved oxygen rises rapidly, start stream plus Portugal that concentration is 500g/L
Grape sugar feed supplement liquid terminates to cultivate when Pullulanase enzyme activity declines.
Culture medium prescription：
(1) seed culture medium：20g/L peptones, 10g/L dusty yeasts, 20g/L glucose.
(2) fermentation medium：15g/L dusty yeasts, 25g/L corn pulps, 15g/L lactose, 1g/L (NH4)2-H-citrate,
2g/L Na2SO3,2.68g/L(NH4)2SO4,14.6g/L K2HPO4,4g/L NaH2PO4·H2O,1g/L MgSO4·7H2O,
3mL/L trace elements (TES).
Micro- (TES)：0.5g/L CaCl2,0.18g/LZnSO4×7H2O,0.1g/LMnSO4×H2O,10.05g/
L Na2-EDTA,8.35g/L FeCl3,0.16g/L CuSO4×5H2O,and 0.18g/L CoCl2×6H2O。
Fermented and cultured 78h during using WSH9 as host strain, Pullulanase enzyme activity up to 2449.6U/mL, be report at present it is general
The extracellular highest level of Shandong orchid enzyme (Fig. 2), dry cell weight (DCW) is 67.01g/L at this time.WSH10 is 2177.1U/mL successively for remaining
It is 2095U/mL in 75h (DCW is 64.60g/L at this time) and WSH11 in 72h (DCW is 70.49g/L at this time), control strain WS5
It is 1047.6U/mL 51h (DCW is 56.12g/L at this time).
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of bacillus subtilis for Pullulanase high efficient expression and High Density Cultivation
<160> 10
<170> PatentIn version 3.3
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Claims (10)
1. a kind of bacillus subtilis suitable for High Density Cultivation, which is characterized in that using bacillus subtilis as starting strain,
Knock out at least one gene in nprB, bpr, mpr, vpr, epr or wprA.
2. the bacillus subtilis according to claim 1 suitable for High Density Cultivation, which is characterized in that it is described go out bacterium germination
Strain is bacillus subtilis WS5, is preserved in China typical culture collection center within 29th in September in 2016, deposit number is
CCTCC NO:M 2016536, preservation address are Wuhan, China Wuhan University.
3. the bacillus subtilis according to claim 1 suitable for High Density Cultivation, which is characterized in that with withered grass gemma
Bacillus WS5 is starting strain, knocks out nprB, bpr, mpr, vpr, epr or wprA gene.
4. a kind of genetic engineering bacterium of efficient production Pullulanase, which is characterized in that be to be suitable for height described in claim 3
Pullulanase gene is expressed in the bacillus subtilis of density culture.
5. a kind of production method of Pullulanase, which is characterized in that the genetic engineering bacterium described in claim 4 is seeded to fermentation
In culture medium, in 33~37 DEG C of cultures.
6. a kind of fermentation process in high density of Pullulanase, which is characterized in that be inoculated with the genetic engineering bacterium described in claim 4
Into fermentation medium, control pH 6.5-7.0, dissolved oxygen is first maintained 28~32% by 33-37 DEG C of cultivation temperature, fermentation until
Dissolved oxygen rises rapidly, and stream plus glucose terminate to ferment when Pullulanase enzyme activity declines.
7. according to the method described in claim 6, it is characterized in that, the genetic engineering bacterium passes through before fermentation medium is seeded to
Overactivation, the activation is containing 4~6g/L dusty yeasts, 10~15g/L peptones, 3~8g/L glycerine, 10~15g/
LK2HPO4, 1~5g/L KH2PO4Seed culture medium in cultivated.
8. the method according to the description of claim 7 is characterized in that genetic engineering bacterium is seeded in seed culture medium, 35~
37 DEG C, cultivate 10~16h, be forwarded in fermentation medium, control pH 6.5-7.0,33-37 DEG C of cultivation temperature, by with stirring
Rotating speed is coupled and adjusts throughput and dissolved oxygen is maintained 28~32%, when dissolved oxygen rises rapidly, start stream plus concentration for 400~
The glucose feed supplement liquid of 500g/L terminates to cultivate when Pullulanase enzyme activity declines.
9. any bacillus subtilises suitable for High Density Cultivation of claim 1-3 answering being prepared extracellular protein
With.
10. the answering in terms of alcohol fuel, resistant starch or maltotriose slurry is prepared of the genetic engineering bacterium described in claim 4
With.
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