CN106497857B - One plant can be with the bacillus licheniformis engineering bacteria of high yield Polyurethane-epoxy resin - Google Patents
One plant can be with the bacillus licheniformis engineering bacteria of high yield Polyurethane-epoxy resin Download PDFInfo
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Abstract
The present invention provides the bacillus licheniformis engineering bacterias that a plant height produces Polyurethane-epoxy resin, its deposit number is CCTCC No:M2016439, it is named as bacillus licheniformis Bacillus licheniformis (WX-02)/pHY-zwf, and further provides the method for the construction method and high yield Polyurethane-epoxy resin of bacillus licheniformis WX-02/pHY-zwf.The synthesis for increasing NADPH intracellular by genetic engineering transformation is horizontal, improves the yield of γ-PGA.It by constructing glucose-6-phosphate dehydrogenase over-express vector pHY-zwf, and converts into the previously stored production γ-PGA bacillus licheniformis WX-02 in laboratory, has rebuild bacillus licheniformis and produced γ-PGA engineering bacteria WX-02/pHY-zwf.The bacterial strain can significantly improve the synthesis of γ-PGA under Optimal Medium and condition of culture.Its Polyurethane-epoxy resin yield is up to 51.13g/L；Polyurethane-epoxy resin yield significantly improves 49% compared with original strain.
Description
Technical field
The invention belongs to genetic engineerings and microbial metabolism field of engineering technology, can be poly- with high yield more particularly to one plant
The bacillus licheniformis engineering bacteria of gamma-glutamic acid.
Background technique
Polyurethane-epoxy resin is a kind of anionic high molecular polymer with extensive use, with various features: easily
It is dissolved in water；Good water holding capacity；Adsorbing metal ions；Slow release and biodegradable.Because of the special life of Polyurethane-epoxy resin
Object property, in agricultural, medicine, food, the fields such as cosmetics have extensive use.
Pertinent literature report, the producing strains of Polyurethane-epoxy resin are mainly by Bacillus subtillis, bacillus amyloliquefaciens and ground
Clothing bacillus etc..In general, the synthesis of Polyurethane-epoxy resin is mainly by endogenous and two kinds of approach of external source.Exogenous route is main
It is to add glutamic acid in the medium, glutamic acid is transported to intracellular to form Polyurethane-epoxy resin；And endogenous pathway is then main
It is glucose through glycolytic pathway, TCA circulation forms glutamic acid by α-ketoglutaric acid.So far, people mainly by with
Lower construction of strategy recombinant bacterial strain has to improve Polyurethane-epoxy resin yield: 1, strengthening glutamic acid route of synthesis；2, by-product synthesis way
The interruption of diameter；3, strengthen Polyurethane-epoxy resin synthesis expression of enzymes and ATP supply etc..By the genetic engineering transformation in terms of these, all
It is able to achieve the raising of Polyurethane-epoxy resin yield.
The supply level of NADPH plays a significant role in the synthesis process of target product.And NADPH Regeneration Ways are main
There is pentose phosphate pathway, the three approach such as TCA circulation and transhydrogenase approach, wherein phosphopentose rises in NADPH regenerative process
Main function.Glucose-6-phosphate dehydrogenase be glucose 6-phosphate enter pentose phosphate pathway first enzyme and its
The rate-limiting enzyme of entire pentose phosphate pathway, thus Zwf plays a significant role in NADPH regeneration.In research before, Wang
Et al (2012) is in bacillus subtilis by being overexpressed Zwf for the output increased of riboflavin 31%.However, Zwf is expressed
Someone does not study relationship between horizontal and Polyurethane-epoxy resin fermentation yield.
Summary of the invention
In view of this, the purpose of the present invention is to provide can be with the bacillus licheniformis engineering bacteria of high yield Polyurethane-epoxy resin.
First aspect present invention provides a kind of bacillus licheniformis, and the bacillus licheniformis is preserved in force in the date
Chinese university China typical culture collection center, deposit number are CCTCC No:M2016439, are named as bacillus licheniformis
Bacillus licheniformis (WX-02)/pHY-zwf, preservation date: on August 30th, 2016.
Second aspect of the present invention provides the construction method of bacillus licheniformis WX-02/pHY-zwf, step include:
It is overexpressed on the basis of plasmid pHY300PLK, building is overexpressed plasmid pHY-zwf, recombinant plasmid transformed bacillus licheniformis WX-02
After obtain bacillus licheniformis WX-02/pHY-zwf；The bacillus licheniformis WX-02, the bacterium deposit number are CCTCC No:
M208065 is named as bacillus licheniformis Bacillus licheniformisWX-02, preservation date: on April 28th, 2008.
Third aspect present invention provides the method that bacillus licheniformis WX-02/pHY-zwf produces Polyurethane-epoxy resin, step
Suddenly include:
(1) seed culture: bacillus licheniformis WX-02/pHY-zwf is connected to progress seed culture in LB culture medium and is obtained
Fermenting microbe；
(2) fermented and cultured: fermenting microbe being connected in fermentation medium and carries out fermented and cultured, the fermentative medium formula
Are as follows: 30-100g/L glucose；5-12g/L NH4Cl；5-15g/L sodium citrate；10-50g/L sodium glutamate；0.1-1.5g/
LK2HPO4·3H2O；0.1-1.5g/LMgSO4·7H2O；0.1-1.5g/LZnSO4·7H2O；0.1-1.5g/LCaCl2；0.10g/
LMnSO4·H2O；pH 6.0-8.0；The fermentation culture conditions are as follows: fermentation temperature is 30-40 DEG C, and liquid amount is container volume
10-30%, shaking speed 200-260r/min, fermentation period 34-38h.
The beneficial effects of the present invention are: the present invention is horizontal by the synthesis that genetic engineering transformation increases NADPH intracellular, α -one
Glutaric acid can consume a large amount of NADPH during forming glutamic acid, so as to improve the yield of γ-PGA.By constructing 6- phosphoric acid grape
Glucocorticoid dehydrogenase over-express vector pHY-zwf, and convert to the previously stored production γ-PGA bacillus licheniformis WX-02 in laboratory
In, it has rebuild bacillus licheniformis and has produced γ-PGA engineering bacteria WX-02/pHY-zwf.Bacillus licheniformis WX- of the invention
02/pHY-zwf can be up to 51.13g/L during liquid fermentation with high yield Polyurethane-epoxy resin, Polyurethane-epoxy resin yield,
It lays a good foundation for later scientific research and industrialized production, there is boundless application prospect.
Detailed description of the invention
Fig. 1 is Expression element Ago-Gel figure in zwf over-express vector；
Fig. 2 is zwf gene overexpression vector plasmid map；
Fig. 3 is that recombinant vector converts bacillus licheniformis WX-02 bacterium colony PCR proof diagram；
Fig. 4 is that bacillus licheniformis WX-02/pHY-zwf fermentation termination in Optimal Medium measures Polyurethane-epoxy resin production
Measure schematic diagram.
Specific embodiment
First aspect present invention provides a kind of bacillus licheniformis, and the bacillus licheniformis is preserved in force in the date
Chinese university China typical culture collection center, deposit number are CCTCC No:M2016439, are named as bacillus licheniformis
Bacillus licheniformis(WX-02)/pHY-zwf。
Further, the present invention provides above-mentioned bacillus licheniformis to produce the application in Polyurethane-epoxy resin.This hair
Bright second aspect provides the construction method of bacillus licheniformis WX-02/pHY-zwf, and step includes: to cross table
Up on the basis of plasmid pHY300PLK, building is overexpressed plasmid pHY-zwf, after recombinant plasmid transformed bacillus licheniformis WX-02
To bacillus licheniformis WX-02/pHY-zwf；The bacillus licheniformis WX-02, the bacterium deposit number are CCTCC No:
M208065 is named as bacillus licheniformis Bacillus licheniformisWX-02.
Preferably, the overexpression plasmid pHY-zwf, promoter are P43 promoter in Bacillus subtillis 168, zwf
For 6- phosphate-dextrose dehydrogenase gene in bacillus licheniformis WX-02, terminator is amyL in bacillus licheniformis WX-02
Terminator.
Third aspect present invention provides the method that bacillus licheniformis WX-02/pHY-zwf produces Polyurethane-epoxy resin, step
Suddenly include:
(1) seed culture: bacillus licheniformis WX-02/pHY-zwf is connected to progress seed culture in LB culture medium and is obtained
Fermenting microbe；
(2) fermented and cultured: fermenting microbe being connected in fermentation medium and carries out fermented and cultured, the fermentative medium formula
Are as follows: 30-100g/L glucose；5-12g/L NH4Cl；5-15g/L sodium citrate；10-50g/L sodium glutamate；0.1-1.5g/
LK2HPO4·3H2O；0.1-1.5g/LMgSO4·7H2O；0.1-1.5g/LZnSO4·7H2O；0.1-1.5g/LCaCl2；0.10g/
LMnSO4·H2O；pH 6.0-8.0；The fermentation culture conditions are as follows: fermentation temperature is 30-40 DEG C, and liquid amount is container volume
10-30%, shaking speed 200-260r/min, fermentation period 34-38h.
Preferably, the condition of the seed culture are as follows: cultivation temperature is 30-39 DEG C, and liquid amount is the 15- of container volume
25%, shaking speed 160-200r/min, incubation time 8-12h.
It can be with lichens brood cell's bar of high yield Polyurethane-epoxy resin to one plant provided by the invention below in conjunction with specific embodiment
Bacterium engineering bacteria is further described.The embodiments described below is exemplary, and for explaining only the invention, and cannot be understood
For limitation of the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
It tests material unless otherwise specified, is that market is commercially available.
Embodiment 1
Present embodiments provide bacillus licheniformis WX-02/pHY-zwf and its construction method.
The clone of Expression element in 1 recombinant vector
Using the total DNA of Bacillus subtillis 168 as template, according to the genome of the NCBI Bacillus subtillis 168 announced
Sequence amplifies the sequence SEQ ID NO:1 of Promoter P43 using the method for PCR, and primer is respectively P43-F SEQ ID NO:
2,P43-R SEQ ID NO:3；Using the total DNA of bacillus licheniformis WX-02 as template, according to the lichens brood cell announced on NCBI
Bacillus WX-02 genome sequence amplifies glucose-6-phosphate dehydrogenase gene zwf sequence SEQ ID using the method for PCR
NO:4, primer are zwf-F SEQ ID NO:5, zwf-R SEQ ID NO:6；Using the total DNA of bacillus licheniformis WX-02 as mould
Plate amplifies amylase gene using the method for PCR according to the bacillus licheniformis WX-02 genome sequence announced on NCBI
The sequence SEQ ID NO:7 of amyL terminator, primer are TamyL-F SEQ ID NO:8, TamyL-R SEQ ID NO:9.
Agarose gel electrophoresis detection is carried out to Expression element in zwf over-express vector, Ago-Gel figure is for example attached
Shown in Fig. 1, wherein swimming lane 1 be 5K DNA marker (from top to bottom successively are as follows: 5000bp, 3000bp, 2000bp, 1500bp,
1000bp, 750bp, 500bp, 250bp, 100bp), swimming lane 2 is the band of the Promoter P43 in Bacillus subtillis 168
(305bp)；Swimming lane 3 is the band of 6- phosphate-dextrose dehydrogenase gene zwf in source bacillus licheniformis WX-02
(1479bp)；Swimming lane 4 is the amylase gene amyL terminator band (501bp) in bacillus licheniformis WX-02.
2 using P43 as the building of the over-express vector of promoter overexpression zwf gene
Zwf gene overexpression carrier, the table are constructed as starting vector using the expression vector pHY300PLK that laboratory saves
Up to carrier pHY300PLK can market be commercially available.First by P43 promoter, glucose-6-phosphate dehydrogenase gene zwf and amylase
The termination sub-piece of gene amyL is connected to together by the method for SOE-PCR.By EcoRI/XbaI double digestion, after purification and recovery
Connect with the pHY300PLK empty plasmid enzyme for also passing through EcoRI/XbaI double digestion, it is 16 DEG C, time 8h that enzyme, which connects temperature, and enzyme connects
The subsequent Transformed E .coli DH5 α of product chooses transformant and carries out bacterium colony PCR verifying, PCR is verified correct transformant chooses bacterium and be connected to
In PA bottle containing 5mL LB culture medium (50ug/mL ammonia benzyl antibiotic), takes out plasmid and be sequenced.
The zwf gene overexpression vector plasmid map is as shown in Fig. 2, with expression plasmid pHY300PLK for original matter
The Promoter P43 for deriving from Bacillus subtillis 168 is added in grain on its basis, from 6- in bacillus licheniformis WX-02
Glucose phosphate dehydrogenase gene zwf and in bacillus licheniformis WX-02 amylase gene amyL terminator, building
Recombinant plasmid pHY-zwf.
The building of 3 bacillus licheniformis high yield Polyurethane-epoxy resin engineered strain WX-02/pHY-zwf will construct
Good over-express vector pHY-zwf converts bacillus licheniformis WX-02.Bacillus licheniformis WX-02 sense is done first
By state, then the activated spawn on plate chooses bacterium in the PA bottle containing 5mL LB, 37 DEG C are incubated overnight, and then connects with 5%
Kind amount is forwarded in growth medium, and 37 DEG C, 200rpm is cultivated to OD600Bacterium is collected to 0.85 or so, 5500rpm centrifugation 6min
Thallus is resuspended with washing culture medium in body, and 5500rpm is centrifuged 6min, 1mL washing culture medium is added afterwards in triplicate, thallus is resuspended,
Packing is into sterilized 1.5mLEP pipe, -80 DEG C of preservations.
15min is pre-chilled in electric revolving cup after drying on ice, then by the bacillus licheniformis WX-02 competence of 100ul
Cell and 10ul recombinant vector are added in electric revolving cup after mixing, and after 3-5min is pre-chilled on ice, are shocked by electricity under the conditions of 2.4kV, when electric shock
Between 4.8-5.2ms, be immediately added and 800ul recovery media and be transferred in 1.5mLEP pipe.37 DEG C, 100rpm cultivates 3h
Apply LB plate (Fourth Ring 20ug/mL antibiotic) afterwards.Bacterium is chosen after growing transformant to carry out bacterium colony PCR verifying and take out plasmid verifying,
Strain is saved after verifying is correct to get bacillus licheniformis WX-02/pHY-zwf.
It is as shown in Fig. 3 that recombinant vector converts bacillus licheniformis WX-02 bacterium colony PCR proof diagram, wherein swimming lane 1 is to adopt
With bacterium colony PCR verify band (2585bp), channel 2 be 5K marker (from top to bottom successively are as follows: 5000bp, 3000bp,
2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp).
Embodiment 2
The method that bacillus licheniformis WX-02/pHY-zwf produces Polyurethane-epoxy resin is present embodiments provided, lichens bud is passed through
Born of the same parents bacillus WX-02/pHY-zwf carries out fermenting experiment under the conditions of different fermentations, the method for obtaining high yield Polyurethane-epoxy resin.
The Polyurethane-epoxy resin that the fermenting experiment obtains is all made of following measuring method and quantitative approach:
The dehydrated alcohol of 2 times of volumes is added in 2.0mL fermentation liquid, concussion mixing is centrifuged 10min in 8000r/min.It loses
Supernatant is abandoned, centrifugation 2.0mL deionized water dissolving, concussion is uniformly.It takes 1.0mL solution in colorimetric cylinder, it is dense that 1mL is added
Hydrochloric acid hydrolyzes for 24 hours at 100 DEG C after sealing.After hydrolysis, filtered with 1mol/L NaOH constant volume 10mL, then through 0.22 μm of micropore
Film filtering.HPLC measurement uses Lichrospher C18 chromatographic column (25cm × 4.6mm).Detection wavelength 210nm.Mobile phase is
100mmol/L KH containing 5.0% methanol2PO4(with phosphoric acid tune pH value to 2.5).Flow velocity 1.0mL/min.Sample volume is 1.0 μ L.
Using Polyurethane-epoxy resin standard items as control, quantitative analysis is carried out to zymotic fluid product.
One, fermenting experiment of the bacillus licheniformis WX-02/pHY-zwf in ME fermentation medium
Picking WX-02 and WX-02/pHY-zwf bacterium colony are inoculated in respectively in 5mL LB culture medium, and 37 DEG C, 240r/min vibration
Swing overnight incubation.It is transferred in the fresh LB culture medium of 50mL with 2% inoculum concentration again, when OD600 is 1.0, with 1%
Inoculum concentration is inoculated into preprepared ME fermentation medium, and 37 DEG C, 240r/min, fermentation time 36h.
The LB culture medium: 10g/L peptone；5g/L yeast extract powder；10g/L sodium chloride；pH 7.0-7.2；Solid training
Feeding base adds agar 18g/L.
The ME fermentation medium: 20g/L glucose；20g/L sodium glutamate；12g/L sodium citrate；7g/L NH4Cl；
0.5g/LK2HPO4·3H2O；0.5g/LMgSO4·7H2O；0.5g/LZnSO4·7H2O；0.15g/LCaCl2；0.10g/
LMnSO4·H2O；pH 7.2.
In ME culture medium, the Polyurethane-epoxy resin maximum output of original strain WX-02 and recombinant bacterium WX-02/pHY-zwf
22.2g/L and 28.8g/L are respectively reached, engineering bacteria improves 27.1% than original strain.Two,
The fermentation of bacillus licheniformis WX-02/pHY-zwf Polyurethane-epoxy resin under different concentration of glucose
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
In the Optimal Medium for being 20-100g/L to preprepared concentration of glucose, 37 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 20-100g/L glucose；20g/L sodium glutamate；12g/L sodium citrate；7g/L
NH4Cl；0.5g/LK2HPO4·3H2O；0.5g/LMgSO4·7H2O；0.5g/LZnSO4·7H2O；0.15g/LCaCl2；0.10g/
LMnSO4·H2O；pH 7.2.
The Polyurethane-epoxy resin maximum output of bacillus licheniformis WX-02/pHY-zwf reaches 38.6g/L, and engineering bacteria is than former
Beginning bacterial strain improves 39.2%.
Three, bacillus licheniformis WX-02/pHY-zwf is in different NH4The fermentation of Polyurethane-epoxy resin under Cl concentration
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
To preprepared NH4Cl concentration is 37 DEG C, 240r/min, fermentation time 36h in the Optimal Medium of 5-15g/L.
The Optimal Medium: 20g/L glucose；20g/L sodium glutamate；12g/L sodium citrate；5-15g/L NH4Cl；
0.5g/LK2HPO4·3H2O；0.5g/LMgSO4·7H2O；0.5g/LZnSO4·7H2O；0.15g/LCaCl2；0.10g/
LMnSO4·H2O；pH 7.2.
The Polyurethane-epoxy resin maximum output of bacillus licheniformis WX-02/pHY-zwf reaches 36.2g/L, and engineering bacteria is than former
Beginning bacterial strain improves 38.6%.
Four, the fermentation of bacillus licheniformis WX-02/pHY-zwf Polyurethane-epoxy resin under different sodium citrate concentrations
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
Into the Optimal Medium of preprepared sodium citrate concentration 5-15g/L, 37 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 20g/L glucose；20g/L sodium glutamate；5-15g/L sodium citrate；7g/L NH4Cl；
0.5g/LK2HPO4·3H2O；0.5g/LMgSO4·7H2O；0.5g/LZnSO4·7H2O；0.15g/LCaCl2；0.10g/
LMnSO4·H2O；pH 7.2.
The Polyurethane-epoxy resin maximum output of bacillus licheniformis WX-02/pHY-zwf reaches 34.5g/L, and engineering bacteria is than former
Beginning bacterial strain improves 36.4%.
Five, the fermentation of bacillus licheniformis WX-02/pHY-zwf Polyurethane-epoxy resin under different concentration of sodium glutamate
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
Into the Optimal Medium of preprepared concentration of sodium glutamate 10-50g/L, 37 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 20g/L glucose；10-50g/L sodium glutamate；12g/L sodium citrate；7g/L NH4Cl；
0.5g/LK2HPO4·3H2O；0.5g/LMgSO4·7H2O；0.5g/LZnSO4·7H2O；0.15g/LCaCl2；0.10g/
LMnSO4·H2O；pH 7.2.
The Polyurethane-epoxy resin maximum output of bacillus licheniformis WX-02/pHY-zwf reaches 44.8g/L, and engineering bacteria is than former
Beginning bacterial strain improves 43.6%.
Six, the fermentation picking of bacillus licheniformis WX-02/pHY-zwf Polyurethane-epoxy resin in Optimal Medium
Bacterium colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.Again with
1% inoculum concentration is transferred in the fresh LB culture medium of 50mL, when OD600 is 1.0, is inoculated into 1% inoculum concentration pre-
First in ready Optimal Medium, 37 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 30-100g/L glucose；5-12g/L NH4Cl；5-15g/L sodium citrate；10-50g/L
Sodium glutamate；0.1-1.5g/LK2HPO4·3H2O；0.1-1.5g/LMgSO4·7H2O；0.1-1.5g/LZnSO4·7H2O；
0.1-1.5g/LCaCl2；0.10g/LMnSO4·H2O；pH 6.0-8.0.
The Polyurethane-epoxy resin maximum output of bacillus licheniformis WX-02/pHY-zwf reaches 51.13g/L, sees attached drawing 4, work
Journey bacterium improves 49% than original strain.
Embodiment 3
It present embodiments provides bacillus licheniformis WX-02/pHY-zwf and produces Polyurethane-epoxy resin in Optimal Medium
Method, specific steps include:
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
Into preprepared Optimal Medium, 40 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 100g/L glucose；12g/L NH4Cl；5g/L sodium citrate；10g/L sodium glutamate；
1.5g/LK2HPO4·3H2O；1.5g/LMgSO4·7H2O；1.5g/LZnSO4·7H2O；1.5g/LCaCl2；0.15g/
LMnSO4·H2O；pH 8.0.
Gained fermentation results and embodiment 2 are almost the same, and Polyurethane-epoxy resin yield has compared to original strain obviously to be mentioned
It is high.
Embodiment 4
It present embodiments provides bacillus licheniformis WX-02/pHY-zwf and produces Polyurethane-epoxy resin in Optimal Medium
Method, specific steps include:
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
Into preprepared Optimal Medium, 30 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 50g/L glucose；8g/L NH4Cl；10g/L sodium citrate；10g/L sodium glutamate；
0.8g/LK2HPO4·3H2O；0.8g/LMgSO4·7H2O；0.8g/LZnSO4·7H2O；0.8g/LCaCl2；0.15g/
LMnSO4·H2O；pH 7.0.
Gained fermentation results and embodiment 2 are almost the same, and Polyurethane-epoxy resin yield has compared to original strain obviously to be mentioned
It is high.
Embodiment 5
It present embodiments provides bacillus licheniformis WX-02/pHY-zwf and produces Polyurethane-epoxy resin in Optimal Medium
Method, specific steps include:
Picking colony WX-02/pHY-zwf is inoculated in 5mL LB culture medium, and 37 DEG C, 240r/min shaken cultivation is stayed overnight.
It is transferred in the fresh LB culture medium of 50mL with 1% inoculum concentration, when OD600 is 1.0, is inoculated with 1% inoculum concentration again
Into preprepared Optimal Medium, 34 DEG C, 240r/min, fermentation time 36h.
The Optimal Medium: 30g/L glucose；10g/L NH4Cl；5g/L sodium citrate；50g/L sodium glutamate；
0.8g/LK2HPO4·3H2O；0.8g/LMgSO4·7H2O；0.8g/LZnSO4·7H2O；0.8g/LCaCl2；0.15g/
LMnSO4·H2O；pH 6.0.
Gained fermentation results and embodiment 2 are almost the same, and Polyurethane-epoxy resin yield has compared to original strain obviously to be mentioned
It is high.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Hubei University
The bacillus licheniformis engineering bacteria of<120>one plant heights production Polyurethane-epoxy resin
<130> 2016
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 305
<212> DNA
<213>P43 promoter in Bacillus subtillis 168
<400> 1
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt taccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300
atgaa 305
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tgataggtgg tatgttttcg 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
ttcatgtgta cattcctc 18
<210> 4
<211> 1479
<212> DNA
<213>glucose-6-phosphate dehydrogenase gene zwf in bacillus licheniformis WX-02
<400> 4
ttgaaaaaag atcaaatgga accaaaagca gttatcgtaa tatttggcgc aacaggggat 60
ttggcaaaac gaaaactata tccttctatc cacaggctct acgaaaacgg acaaatcggg 120
aatgaattcg cggttgtcgg agtcggcaga cgcccttgga caaatgaaga ctttcgcagt 180
actgtacagc aatcggtttc gaagtttccg ttaaacgaaa aggatgtaga cgagtttaca 240
tctcacttct actatcatcc ttttgatgtt acaaattccg gctcataccg ggaattgaac 300
gaacttttag agaagctgga aagtacatat gatattccga ataaccggat gttttactta 360
gcgatggctc ctgaattttt cggaacgatt gcaaagttcc taaaatcaga gggtgtcact 420
tcaacaacag gctggtcaag gctcgtgatc gagaagcctt tcggacatga tctgccgagc 480
gcaaaatcct tgaatcagga aattcgtgaa gcctttacag aagatcaaat ctatcggatt 540
gaccattatc taggaaagca aatggtgcag aacatcgaag tcatccgctt tgccaacgcg 600
atttttgagc cgctttggac aaacagatac atctcgaata ttcaaatcac gtcaagtgaa 660
gatttgggtg tggaagacag agcgagatat tatgaaaaat ccggtgcgct tcgagacatg 720
gttcaaaacc acattcttca aatggtggcg ctgcttgcga tggaaccgcc gatcaagctg 780
aacacggaag agatccgcag cgaaaaagtg aaggtgctca gagcccttcg tccgattcga 840
aaagacgaag tcgatcagtt ttttgtgcgc ggccagtatg acgcgggtgt cgttgacgaa 900
aaacaggttc cggcttatcg cgatgagcaa aatgtagcaa aagagtcgaa tacggaaacg 960
ttcgttgccg gcaaactgct gatcgacaac ttcagatggg ccggtgtgcc gttctacatc 1020
cggacaggga aacggatgca gaaaaaatca acgcagattg tcgtccaatt taaagacatt 1080
ccgatgaatc tttattacgg aaacggcaac acgatgcatc ctaacctgct ggtgatccat 1140
attcaacctg atgaaggcat cacccttcat ttaaacgcaa ggaagcttgg aggcggcact 1200
tttgcccagc cgatcaagct cgactactgc cataactgcg gggacggcat caacacgcct 1260
gaagcttatg aaaagctgat tttggactgt ttgcacggag acgcgacaaa ctttgcccac 1320
tgggatgaag ttgcgctttc ctggagtttt gttgatgcga tttctcaaaa ctgggaggaa 1380
aacaaaaccc tttcccctaa ctatgaggcg ggctctatgg gaccgaaagc ttctgatgac 1440
ctgctcgcaa aagacggctt tcattggtgg ccgctttaa 1479
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<400> 5
ttgaaaaaag atcaaatgga 20
<210> 6
<211> 19
<212> DNA
<213>artificial sequence
<400> 6
ttaaagcggc caccaatga 19
<210> 7
<211> 501
<212> DNA
<213>in bacillus licheniformis WX-02 amylase gene amyL terminator
<400> 7
aagagcagag aggacggatt tcctgaagga aatccgtttt tttattttgc ccgtcttata 60
aatttctttg attacatttt ataattaatt ttaacaaagt gtcatcagcc ctcaggaagg 120
acttgctgac agtttgaatc gcataggtaa ggcggggatg aaatggcaac gttatctgat 180
gtagcaaaga aagcaaatgt gtcgaaaatg acggtatcgc gggtgatcaa tcatcctgag 240
actgtgacgg atgaattgaa aaagcttgtt cattccgcaa tgaaggagct caattatata 300
ccgaactatg cagcaagagc gctcgttcaa aacagaacac aggtcgtcaa gctgctcata 360
ctggaagaaa tggatacaac agaaccttat tatatgaatc tgttaacggg aatcagccgc 420
gagctggacc gtcatcatta tgctttgcag cttgtcacaa ggaaatctct caatatcggc 480
cagtgcgacg gcattattgc g 501
<210> 8
<211> 20
<212> DNA
<213>artificial sequence
<400> 8
aagagcagag aggacggatt 20
<210> 9
<211> 21
<212> DNA
<213>artificial sequence
<400> 9
cgcaataatg ccgtcgcact g 21
Claims (6)
1. a kind of bacillus licheniformis, it is characterised in that: the deposit number of the bacillus licheniformis is CCTCC No:
M2016439 is named as bacillus licheniformis Bacillus licheniformis (WX-02)/pHY-zwf.
2. bacillus licheniformis described in claim 1 is producing the application in Polyurethane-epoxy resin.
3. a kind of construction method of bacillus licheniformis as described in claim 1, step includes: to be overexpressed plasmid
On the basis of pHY300PLK, building is overexpressed plasmid pHY-zwf, obtains lichens after recombinant plasmid transformed bacillus licheniformis WX-02
Bacillus WX-02/pHY-zwf；The bacillus licheniformis WX-02, the bacterium deposit number are CCTCC No:M208065, life
Entitled bacillus licheniformis Bacillus licheniformisWX-02.
4. the construction method of bacillus licheniformis as claimed in claim 3, it is characterised in that: the overexpression plasmid pHY-
Zwf, promoter are P43 promoter in Bacillus subtillis 168, and zwf is 6- phosphoric acid-grape in bacillus licheniformis WX-02
Glucocorticoid dehydrogenase gene, terminator are amyL terminator in bacillus licheniformis WX-02.
5. the method that the fermentation of bacillus licheniformis described in claim 1 produces Polyurethane-epoxy resin, step include:
(1) seed culture: bacillus licheniformis WX-02/pHY-zwf is connected to progress seed culture in LB culture medium and is fermented
Strain；
(2) fermented and cultured: fermenting microbe being connected in fermentation medium and carries out fermented and cultured, the fermentative medium formula are as follows:
30-100g/L glucose；5-15g/L NH4Cl；5-15g/L sodium citrate；10-50g/L sodium glutamate；0.1-1.5g/
LK2HPO4·3H2O；0.1-1.5g/LMgSO4·7H2O；0.1-1.5g/LZnSO4·7H2O；0.1-1.5g/LCaCl2；0.10g/
LMnSO4·H2O；pH 6.0-8.0；The fermentation culture conditions are as follows: fermentation temperature is 30-40 DEG C, and liquid amount is container volume
10-30%, shaking speed 200-260r/min, fermentation period 30-38h.
6. the method that bacillus licheniformis fermentation as claimed in claim 5 produces Polyurethane-epoxy resin, it is characterised in that: the seed
The condition of culture are as follows: cultivation temperature is 30-39 DEG C, and liquid amount is the 15-25%, shaking speed 160-200r/ of container volume
Min, incubation time 8-12h.
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| CN107904197A (en) * | 2017-09-13 | 2018-04-13 | 湖北大学 | A kind of method and engineering bacteria for improving bacillus licheniformis fermenting and producing shikimic acid |
| CN109913515B (en) * | 2018-04-28 | 2022-10-21 | 湖北大学 | Method for increasing yield of poly gamma-glutamic acid by improving glycerol metabolism of bacillus |
| CN108486030B (en) * | 2018-05-03 | 2021-03-23 | 湖北大学 | Preparation method and application of bacillus for high-yield poly-gamma-glutamic acid through over-expression of phoP gene |
| CN108410789B (en) * | 2018-05-07 | 2021-09-10 | 湖北大学 | Microorganism for producing poly-gamma-glutamic acid and construction method and application thereof |
| CN110305917B (en) * | 2019-07-27 | 2022-10-14 | 湖北大学 | Application of rex gene of bacillus in improving yield of poly-gamma-glutamic acid |
| CN114276952B (en) * | 2021-12-06 | 2023-08-18 | 湖北工业大学 | Method for improving bacillus licheniformis spore formation efficiency |
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