CN102174454A - Escherichia coli engineering bacterium for expressing recombinant sucrose phosphorylase - Google Patents
Escherichia coli engineering bacterium for expressing recombinant sucrose phosphorylase Download PDFInfo
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- CN102174454A CN102174454A CN201110009399.6A CN201110009399A CN102174454A CN 102174454 A CN102174454 A CN 102174454A CN 201110009399 A CN201110009399 A CN 201110009399A CN 102174454 A CN102174454 A CN 102174454A
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Abstract
The invention belongs to the field of biotechnology, and discloses an escherichia coli engineering bacterium for expressing recombinant sucrose phosphorylase, which is escherichia coli introduced with sucrose phosphorylase SPase gene derived from Leuconostoc mesenteroides ATCC 12291. The invention also discloses a construction method and application of the escherichia coli engineering bacteria for expressing the recombinant sucrose phosphorylase. The escherichia coli engineering bacteria for expressing the recombinant sucrose phosphorylase have the characteristics of convenience in operation, stable expression, high efficiency, low cost, easiness in purification and the like in large-scale production of the SPase.
Description
Technical field
The present invention designs biological technical field, specifically, relates to a kind of colibacillus engineering that efficiently expresses reorganization SPase gene.
Background technology
(SPase), a kind of glucanotransferase is the specific enzymes that glucoside bond is shifted in catalysis to sucrose phosphorylase, belongs to glycosyl hydrolase 13 families for EC 2.4.1.7, Sucrose Phosphorylase.The reaction that the main catalysis of this enzyme is two types: the one, the glucosyl group in the Cori's eater Cori is transferred to acceptor, as being acceptor, under this enzyme catalysis, can generate sucrose with D-fructose; The 2nd, the glucosyl group in the sucrose is transferred to acceptor, acceptor comprises the material of inorganic phosphate, water, phenolic hydroxy group, alcoholic extract hydroxyl group and carboxyl, as being acceptor with phosphoric acid, can generate Cori's eater Cori and D-fructose.
Use this catalysis characteristics, sucrose phosphorylase can be an acceptor with fructose, wood sugar, semi-lactosi and rhamnosyl, the oligose of the synthetic corresponding many glucose groups of catalysis, as 2-α-D-glucosyl-D-fructose, 1-α-D-glucosyl-D-wood sugar, 2-α-D-glucosyl-L-semi-lactosi, 2-alpha-D-glucose base-rhamnosyl etc.; Modify and transform the compound that contains alcoholic extract hydroxyl group, phenolic hydroxyl group and carboxyl,, and should reaction can synthesize the fabulous alpha-arbutin of whitening effect, tool industrial value as the synthetic arbutin of catalysis quinhydrones; The derivative of synthetic some Unstable Substance of catalysis improves its stability.
Sucrose phosphorylase mainly is distributed in the microorganisms such as bacterium, a small amount of distribution is arranged in the vegetable cell, industrially mainly obtain by biological fermentation, the production bacterial strain of this enzyme mainly contains Leuconostoc mesenteroides at present, Streptococcus mutans, Pseudomonas saccharophila, Bifidobacterium longum, Bifidobacterium adolescentis, and some genetic engineering bacteriums.
Hang down problems such as reaching the cost costliness in view of the natural SPase that directly extracts from wild bacterium exists complex process, extraction yield, searching is cheap, heterologous gene expression system is one of effective way that breaks through the traditional preparation process method efficiently.The intestinal bacteria growth fast, growth cycle is short, culture condition is simple, security is good, cheap, the scientific research personnel has mainly cloned 3 foreign genes at present, be respectively Leuconostoc mesenteroides 1149sp, Bifidobacterium adolescentis SucP, Bifidobacteriumlongum splP gene.
Report Leuconostoc mesenteroides ATCC 12291 such as Vandamme E.J. produce the highest bacterial strain (Biotechnology and Bioengneering, 1987,29 (1): 8-15) of SPase activity.The present invention for coming source sequence, selects for use intestinal bacteria as the host with the SPase gene of Leuconostoc mesenteroidesATCC 12291, has made up the active recombination bacillus coli of this high SPase.Intestinal bacteria genetic background is clear, breeding is fast, cost is low, expression amount is high, easy handling, is the first-selected system of expressing foreign protein.
Chinese patent CN101818166A discloses the method for clone's Yak blood superoxide dismutase, also adopting pET-22b (+) is carrier, utilize the advantage of the strong promoter T7 promotor of pET series to efficiently express SOD albumen, the present invention is utilizing pET-22b (+) to efficiently express the proteic while of SPase for expression vector, optimize the proteic expression condition of SPase, made the output of product be improved greatly.
Summary of the invention
Technical problem to be solved by this invention provides a kind of colibacillus engineering that efficiently expresses the recombinant sucrose Starch phosphorylase.
The technical problem that the present invention also will solve provides the construction process of above-mentioned colibacillus engineering.
The technical problem that the present invention will solve at last provides the application of above-mentioned colibacillus engineering in the preparation sucrose phosphorylase.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of colibacillus engineering of express recombinant sucrose phosphorylase, it is the intestinal bacteria that imported the sucrose phosphorylase SPase gene that derives from Leuconostoc mesenteroides Leuconostoc mesenteroides ATCC 12291.
Wherein, the genotype of described engineering bacteria is F
-OmpT gal dcm lon hsd SB (rB
-MB
-), described engineering bacteria contains expression vector pET-Spase, and wherein, described expression vector pET-Spase is that multiple clone site has been inserted sucrose phosphorylase SPase gene in plasmid pET-22b (+).
The construction process of the colibacillus engineering of above-mentioned express recombinant sucrose phosphorylase as shown in Figure 1, it comprises the steps:
(1) extracts Leuconostoc mesenteroides Leuconostoc mesenteroides ATCC 12291 total DNA;
(2) design primer, with PCR method clone SPase gene, primer adds that at 5 ' end the GCTCTAGA sequence constitutes Xba I restriction enzyme site, adds that at 5 ' end CCCAAGCT constitutes Hind III restriction enzyme site;
Upstream primer 5 '-GCTCTAGACGCAATCGTTACAGTTATTACTAGC-3 ';
Downstream primer 5 '-CCCAAGCTTGTTCTGAGTCAAATTATCACTGCTC-3 ';
(3) the SPase gene after the above-mentioned reorganization is inserted among the plasmid pET-22b (+), gets recombinant vectors pET-Spase;
(4) change recombinant vectors pET-Spase over to intestinal bacteria then, carry out the recombinant clone screening, obtain colibacillus engineering Escherichia coli sp1515.
The present invention selects to use pET-22b (+) expression vector, and this is because the pET system is the most powerful system of clonal expression recombinant protein function in intestinal bacteria (E.coli) since the dawn of human civilization.Goal gene is cloned on the pET system carrier, transcribed by force by phage t7 and translation signals is controlled; The t7 rna polymerase that expression has host cell to provide is induced.T7 polysaccharase mechanism is very effective and have selectivity: when fully inducing, nearly all cell resource all is used to express target protein; Behind the abduction delivering only several hours, target protein can account for more than 50% of total protein of cell usually.PET-22b (+) holds the purifying site of having designed 6 Histidines at C in addition, and Histidine can provide coordination electronics and some metal ion (as Ni
2+) chelating, 6 successive Histidines can make the albumen active adsorption in containing Ni
2+Chromatographic stuffing on, thereby can utilize the incompatible purified fusion protein of metal chelating, thereby greatly simplify the subsequent purification work of recombinant protein, be fit to very much mass preparation.
The application of the colibacillus engineering of above-mentioned express recombinant sucrose phosphorylase in the preparation sucrose phosphorylase.
Above-mentioned application, specifically, be with colibacillus engineering behind inducing culture, cytoclasis, the crude enzyme liquid that obtains makes sucrose phosphorylase through the affinity chromatography separation and purification.
Wherein, described method for inducing and cultivating is as follows: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in the LB substratum by volume, when OD=0.6～0.8, add IPTG to final concentration be 0.1～1.1mmol/L, inducing culture 4～16h under 25～40 ℃, the condition of 200rpm, centrifugal acquisition thalline.Preferred inducing culture condition is: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in the LB substratum by volume, when OD=0.6～0.8, add IPTG to final concentration 0.1mmol/L, inducing culture 10h under 25 ℃ of temperature, 200rpm condition, centrifugal acquisition thalline, through determination of activity, the SPase enzyme work of reorganization bacterium can reach 16.6U/mg.
Utilize expression of recombinant e. coli foreign protein major part all to use IPTG as inductor, but expensive and because of it to the toxicity of humans and animals, limited the industrialized development of gene engineering product.Lactose does not have toxicity and cheap, has crucial meaning in the industrial applications of recombination engineering bacteria.Whey powder is when producing cheese or casein food grade, with enzyme or acid the casein coagulation in the milk is separated the remaining byproduct in back.Main component in the whey powder is a lactose, and its inductor aspect as recombination bacillus coli does not cause too many concern.
Therefore, described method for inducing and cultivating can also adopt following method: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in synthetic medium by volume, when OD=0.6～0.8, add whey powder and glycerine to final concentration and be respectively 5～20g/L and 5～15g/L, inducing culture 6～24h under 20～40 ℃, the condition of 200rpm, centrifugal acquisition thalline.Optimum condition is: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in synthetic medium by volume, when OD=0.6～0.8, add whey powder and glycerine to final concentration and be respectively 20g/L and 5g/L, inducing culture 24h under 25 ℃, the condition of 200rpm, centrifugal acquisition thalline, this condition hypothallus OD
600Can reach 16.5, enzyme work can reach 82.5U/ml.Wherein, described synthetic medium comprises following component: NaH
2PO
42H
2O 4.0g/L, K
2HPO
414.6g/L, NH
4Cl 0.5g/L, (NH
4)
2SO
42.5g/L, (NH
4)
2-H-citrate 1.0g/L, Na
2SO
42.0g/L, micro-2ml/L, MgSO
4120g/L, VitB1 0.34g/mL, glucose 1g/L and penbritin 0.1g/L; Wherein, described trace element contains following component: CaCl
26H
2O0.74g/L, ZnSO
47H
2O 0.18g/L, MnSO
4H
2O 0.1g/L, Na
2-EDTA20.1g/L, FeCl
36H
2O 16.7g/L, CuSO
40.1g/L, CoCl
20.104g/L solvent is a deionized water.
Wherein, described method of cell disruption is ultrasonication.The concrete operations mode is preferably: with centrifugal gained thalline phosphoric acid buffer washed twice, resuspended, utilize the ultrasonic disruption cell on ice, get supernatant liquor after centrifugal and be the SPase crude enzyme liquid, gained SPase crude enzyme liquid is removed some cell debriss or other impurity with antifouling nickel post with 0.45 μ m membrane filtration.
Wherein, described affinity chromatography method is carried out affinity chromatography for using His Sepharose HP gel column.Concrete operation method is preferably: the protein liquid that obtains after the ultrasonication is combined with His Sepharose HP gel column, go out unconjugated albumen with lavation buffer solution, use the elution buffer wash-out then, obtain target protein.
Described binding buffer fluid component is as follows: 20mmol/L phosphoric acid is received, 40mmol/L imidazoles, 0.5mmol/L NaCl, transfers pH to 7.5.
Described washing buffer fluid component is as follows: 20mmol/L sodium phosphate, 100mmol/L imidazoles, 0.5mmol/L NaCl, transfer pH to 7.5.
Described elution buffer fluid component is as follows: 20mmol/L sodium phosphate, 500mmol/L imidazoles, 0.5mmol/L NaCl, transfer pH to 7.5.
Purification process of the present invention by using lavation buffer solution washed cell fragment impurity, can make SPase recombinant protein purity reach more than 90%.In addition, normally used affinity chromatography has the specificity height, the purification efficiency height, the product purity advantages of higher, but cost an arm and a leg, cost is higher; The present invention adopts metal Ni
2+The chelating affinity chromatography, it utilizes 6 coordination electronics and Ni that Histidine provided on the fusion rotein of expressing
2+Chelating, 6 successive Histidine sequences can make recombinant protein be adsorbed in containing metal Ni well
2+Chromatographic stuffing on, thereby utilize metal chelate chromatography to come purified fusion protein, simplified the downstream purification work of recombinant protein greatly, purity reaches more than 95%, its purification efficiency height, cost is low, is fit to very much mass preparation.
Beneficial effect: the advantage of the inventive method is:
1. the present invention revises the SPase gene by PCR method, removes terminator codon taa, makes when not changing the SPase aminoacid sequence, can utilize the terminator of plasmid to finish translation with 6 His albumen going up pET-22b (+) C-end.
2. use pET-22b as expression vector, be subjected to phage t7 to transcribe and translate (can select) signal control by force, the t7 rna polymerase that has host cell to provide is provided induces.T7 rna polymerase mechanism is very effective and have selectivity, and behind the abduction delivering only several hours, target protein can account for more than 50% of total protein of cell usually, can realize efficiently expressing of SPase gene.
3. the present invention utilizes on the fusion rotein of expression 6 Histidines that the characteristics of coordination electronics and some metal ion-chelant can be provided, and adopts metal Ni
2+The chelating affinity chromatography is come purified fusion protein, has greatly simplified the subsequent purification work of recombinant protein, and purity is up to more than 95%, cuts the purification efficiency height, cost is low, is fit to mass preparation.
Description of drawings
Intestinal bacteria Escherichia coli sp1515, depositary institution: Chinese typical culture collection center (CCTCC); Address: Wuhan Wuhan University, postcode: 430072; Deposit number: CCTCC NO:M 2011010; Preservation date: on January 8th, 2011.
The building process figure of Fig. 1 recombinant plasmid pET-SPase.
Fig. 2 SPase gene PCR amplified production.Wherein, M represents λ-EcoT14 digest DNAmarker molecular weight marker, 1,2,3 expression PCR products.
The double digestion checking of Fig. 3 pET-SPase recombinant plasmid.Wherein, M represents λ-EcoT14 digest DNAmarker molecular weight marker, and 1 expression pET-22b (+), 2 expression pET-SPase are through Xba I single endonuclease digestion product, and 3 expression pET-SPase are through Xba I and Hind III double digestion product.
The SDS-PAGE electrophoretogram of Fig. 4 pET-SPase.Wherein, M represents the molecular weight of albumen standard, 1 expression E.coliBL21 (DE3) tropina, 2 expressions are without IPTG inductive Escherichia coli sp1515 tropina, and 3 represent the Escherichia coli sp1515 tropina behind the IPTG abduction deliverings.
The influence of Fig. 5 inducing temperature to expressing.
Fig. 6 different IP TG concentration is to the influence of thalli growth and expression amount.
The influence of Fig. 7 IPTG induction time to expressing.
The influence of two groups of different whey powder abduction deliverings of Fig. 8.
Two groups of different whey powders of Fig. 9 are induced thalli growth trend.
The SDS-PAGE electrophoretogram of Figure 10 SPase protein purification.Wherein, M represents the molecular weight of albumen standard, the Escherichia coli sp1515 tropina behind the 1 expression abduction delivering, the SPase albumen of 2 expressions behind the Ni-NTA column purification.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail.
All methods are ordinary method if no special instructions among the embodiment.Used plasmid pET-22b (+), intestinal bacteria bacterium DH5 α, BL21 (DE3) buy the company in Novagen; The primer is synthetic to be finished by the prompt base in the Invitrogen English Weihe River (Shanghai) trade Co., Ltd; Restriction enzyme Xba I, Hind III, λ-EcoT14I digest DNA Marker, Solution I ligase enzyme, molecular weight of albumen standard are all purchased the company in TaKaRa; Glue reclaims test kit in a small amount, plasmid extraction kit is purchased the company in Biomiga in a small amount; Bacterial genomes DNA extraction test kit is purchased in sky root biotech firm.
The structure of embodiment 1 Escherichia coli sp1515 genetic engineering bacterium.
One. design of primers
According to the SPase gene order (GenBank Accession NO.D90314.1) of Leuconostoc mesenteroides ATCC12291, use a pair of primer of Primerpremier 5 design amplification SPase genes, i.e. upstream primer P1 and downstream primer P2:
Upstream primer P1:5 '-GC
TCTAGACGCAATCGTTACAGTTATTACTAGC-3 ';
Downstream primer P2:5 '-CCC
AAGCTTGTTCTGAGTCAAATTATCACTGCTC-3 '.
5 ' end at P1, P2 is introduced Xba I, Hind III restriction enzyme site (underscore marks) respectively, and adds the protection base.
Two .PCR amplification Leuconostoc mesenteroides SPase fragment
(1) Leuconostoc mesenteroides extracting genome DNA
A) glycerine is frozen guarantee the Leuconostoc mesenteroides Leuconostoc mesenteroides ATCC that deposits 12291 line activation after, inoculate single bacterium colony in 30 ℃ of incubated overnight of 5ml MRS substratum;
B) the centrifugal collection thalline of bacterium liquid according to the operation of bacterial genomes DNA extraction test kit specification sheets, extracts the Leuconostoc mesenteroides genomic dna ,-20 ℃ of preservations.
The Leuconostoc mesenteroides genome of said extracted is through UV spectrophotometer measuring, and the OD260nm/280nm value illustrates that the DNA integrity is good between 0.7～0.9, and the purity height, can do the PCR experiment.
(2) pcr amplification
As template, utilize primer P1, P2 to carry out pcr amplification with the Leuconostoc mesenteroides genomic dna.The PCR reaction system is as shown in table 1, and the PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s, 54 ℃ of annealing 45s, 72 ℃ are extended 90s, totally 35 circulations.Amplified production detects with 1% agarose gel electrophoresis.
Table 1 PCR reaction system
As shown in Figure 2, pcr amplification product through 1% agarose gel electrophoresis at the visible specific band in about 1600bp place, consistent with the re-set target clip size.
Three. the clone of Leuconostoc mesenteroides SPase
The PCR product is connected to pET-22b (+) carrier and is transformed into the bacillus coli DH 5 alpha competent cell clone, screen positive recombinant plasmid with bacterium liquid PCR, plasmid double digestion method, positive recombinant plasmid is checked order, the recombinant plasmid called after pET-Spase plasmid that sequencing result is correct, specifically undertaken by following operation steps:
(1) double digestion reaction
PCR product and plasmid pET-22b (+) are carried out Xba I/Hind III double digestion respectively, and the ligation system is as shown in table 2.
According to shown in the table 2, configuration double digestion system is spent the night in 37 ℃ of water-baths, and 1% agarose gel electrophoresis detects and reclaim the purpose band.
Table 2 double digestion reaction system
(2) ligation
The PCR product inserts pET-22b (+) plasmid after Xba I and Hind III digestion Xba I and Hind III site connect into recombinant plasmid.
According to shown in the table 3, the configuration linked system is connected the PCR product behind the double digestion about 3 hours for 16 ℃ with Solution I ligase enzyme with linearizing pET-22b (+) carrier.
Table 3 ligation system
(3) transform and identify
Change recombinant plasmid over to the bacillus coli DH 5 alpha competence, extract recombinant plasmid and screen positive recombinant plasmid through bacterium liquid PCR and Xba I and Hind III enzyme blanking method, positive recombinant plasmid is checked order the recombinant plasmid called after pET-SPase plasmid that sequencing result is correct.Concrete operations are as follows:
Transform
According to " method on the molecular cloning handbook is made E.coli DH5 α competence and with recombinant plasmid CaCl
2Method is transformed into DH5 α competence
Bacterium liquid PCR identifies recon
With sterilization toothpick picking list bacterium colony, be inoculated in the 5ml LB liquid nutrient medium that contains penbritin, 37 ℃ of shaken overnight are cultivated.Get 1 μ l bacterium liquid and make template, press table 1 preparation PCR reaction solution, reaction conditions is identical with the PCR reaction conditions.With the bacillus coli DH 5 alpha of unconverted as negative control.With 1% agarose gel electrophoresis detected result.
Double digestion is identified recon
With sterilization toothpick picking list bacterium colony, be inoculated in the 5ml LB liquid nutrient medium that contains penbritin, 37 ℃ of shaken overnight are cultivated.Extract recombinant plasmid according to a small amount of plasmid extraction kit specification sheets, press table 2 preparation double digestion reaction solution, the enzyme tangent condition is also identical.Detect with 1% agarose gel electrophoresis.
As shown in Figure 2, bacterium liquid pcr amplification product is slightly larger than 1500bp approximately, conform to the expection clip size, as shown in Figure 3, the pET-SPase recombinant plasmid of screening is cut to 1515bp and two fragments of 5340bp by Xba I and Hind III enzyme, conform to the expection clip size, show the positive pET-SPase recombinant plasmid of recombinant plasmid of screening.
The plasmid order-checking
Deliver to the order-checking of Invitrogen company through the pET-SPase recombinant plasmid of bacterium liquid PCR, double digestion evaluation and screening, the result is shown in SEQ ID No.1.Sequence is submitted to ncbi database and GenBank Accession NO.D90314.1 carries out the BLAST comparative analysis, the result shows that above-mentioned clone's Leuconostoc mesenteroides SPase sequence is correct.
Four. make up Escherichia coli sp1515 genetic engineering bacterium
Recombinant plasmid pET-SPase is transformed into e. coli bl21 (DE3), step in concrete operations step and the present embodiment
Three. the conversion operation among the clone of Leuconostoc mesenteroides SPase is identical.
Filter out called after Escherichia coli sp1515 behind the positive colony.
One .IPTG induces Escherichia coli sp1515 genetic engineering bacterium to express
(1) the single bacterium colony of picking reorganization bacterium overnight incubation in the LB substratum is done contrast with bacteria samples and the E.coli BL21 (DE3) that contains empty carrier;
(2) next day, with experimental group and control group by volume 3% inoculum size be seeded in the fresh 50mL LB substratum, 37 ℃, the 200r/min shaking culture;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, add inductor IPTG to final concentration be 0.5mmol/L, induced 6 hours for 30 ℃;
(4) centrifugal (4 ℃, 8000r/min 15min) collects thalline to bacterium liquid.
Two. expression product SDS-PAGE electrophoresis detection
(1) expression product of above-mentioned centrifugal collection is washed twice with damping fluid (10mmol/L potassiumphosphate buffer pH 6.5) after, get 1ml and carry out ultrasonication (200W～400W, ultrasonic 3s, intermittently 5s, 2min altogether), 4 ℃, the centrifugal 15min of 10000r/min.
(2) draw 10 μ l samples, detect (concentrating gum concentration 4.5%, resolving gel concentration 10%), the record result data with conventional SDS-PAGE method.
Getting Escherichia coli sp1515 genetic engineering bacterium inductive protein supernatant liquor and carry out the SDS-PAGE electrophoresis detection, is control group with E.coli BL21 (DE3) with without inductive reorganization bacterium tropina.As shown in Figure 4, control group has more a protein band than experimental group at 55kD, conforms to reorganization SPase protein molecular weight.Show that thus this differential protein may be the SPase albumen of expressing in the experimental group in recombination bacillus coli, but the activity of expression product needs also further to detect.
Three. the enzyme activity determination of expression product
Carry out enzyme activity determination to expressing product albumen liquid, use without inductive bacterium liquid eggs to compare in vain.
(1) SPase determination of activity: get supernatant 20 μ L, join (1.5mL phosphoric acid buffer pH 6.5,1.5mL sucrose, 30 μ L EDTA, 100 μ L NADP in the 3.35mL reaction system
+, 100 μ L glucose 1,6-bisphosphate, 50 μ L MgCl
2, 20 μ L α-Pu Taotang transphosphorylases, 10 μ L glucose-6-phosphate dehydrogenases), 25 ℃ of water-bath balances of reaction system 5min measures the variation of NADPH light absorption value immediately at the 340nm place.
(2) method of calculation: enzyme activity (U/ml)=(OD
s-OD
0) * 3.32 (ml) ÷ (6.2 * 0.02 (ml))=Δ OD * 26.8.OD
0: the reaction initial absorbance; OD
s: absorbancy behind the 1min; 6.2:NADPH optical extinction coefficient (cm at 340nm
2/ μ mol).
(3) determining the protein quantity: protein content determination adopts the Bradford method, is standard substance with bovine serum albumin BSA.
Respectively to do not induce and in the expression crude enzyme liquid of IPTG inductive Escherichia coli sp1515 SPase carry out determination of activity.The result shows that inductive reorganization bacterium does not detect enzymic activity, and the SPase activity is 8.5U/mg in IPTG inductive Escherichia coli sp1515 genetic engineering bacterium.
By further research, optimized the expression condition of genetic engineering bacterium to inducing temperature, IPTG induced concentration and induction time.
One. the influence of different inducing temperatures
(1) the single bacterium colony of picking reorganization bacterium overnight incubation in the LB substratum;
(2) next day, with seed liquor by volume 3% inoculum size be seeded in the fresh 50mL LB substratum, 37 ℃, the 200r/min shaking culture;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, add IPTG to final concentration 0.1mmol, under 20 ℃, 25 ℃, 30 ℃, 35 ℃, induce 10h respectively.The result as shown in Figure 5.
Two. the influence of different IP TG concentration
(1) (2) are all identical with influence (1) (2) operation of the different inducing temperatures of present embodiment step 1;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, add IPTG respectively to final concentration 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.7mmol/L, 0.9mmol/L, 1.1mmol/L, induce 10h for 25 ℃.The result as shown in Figure 6.
Three. the influence of different induction times
(1) (2) are all identical with influence (1) (2) operation of the different inducing temperatures of present embodiment step 1;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, add IPTG to final concentration 0.1mmol/L, 25 ℃, induce 4h, 6h, 8h, 10h, 12h, 14h, 16h respectively.The result as shown in Figure 7.
Four. the test under the best inductive condition
The best inductive condition of each factor of coming out according to step 1 two three screenings is tested
(1) (2) are all identical with influence (1) (2) operation of the different inducing temperatures of present embodiment step 1;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, add IPTG to final concentration 0.1mmol/L, 25 ℃, induce 10h respectively.
(4) collect thalline, reach 16.6U/mg through its enzyme work of SPase determination of activity.
One. the mensuration of lactose-content in the whey powder
(1) configuration of standard substance and sample
Lactose: the standard model that is configured to 1g/L
Whey powder: with pure water whey powder is mixed with concentration 1g/L, centrifugal (8000r/min 15min) got supernatant liquor and is the whey powder lysate to remove objectionable impurities in 1 hour for 60 ℃ of water-baths.The used whey powder is a high protein whey powder, purchases in IPT interface commerce and trade (Shanghai) Co., Ltd..
(2) adopt high performance liquid phase to detect: chromatographic column is Bio-Rad Aminex HPX-87H (7.8mm * 300mm), UltiMate3000 workstation, ultraviolet and a differential detector.Sample is with 0.22 μ m membrane filtration, last sample 20 μ L.Mobile phase place 5mmol/L sulfuric acid, 0.6mL/min, 55 ℃.
(3) calculate by measuring, lactose-content is 71.33% in the whey powder.
Two. reagent is prepared
(1) synthetic medium component: NaH
2PO
42H
2O 4.0g/L, K
2HPO
414.6g/L, NH
4Cl 0.5g/L, (NH
4)
2SO
42.5g/L, (NH
4)
2-H-citrate 1.0g/L, Na
2SO
42.0g/L, micro-2ml/L, MgSO
4120g/L, VitB1 0.34g/mL, glucose 1g/L and penbritin 0.1g/L; Wherein, described trace element contains following component: CaCl
26H
2O0.74g/L, ZnSO
47H
2O 0.18g/L, MnSO
4H
2O 0.1g/L, Na
2-EDTA20.1g/L, FeCl
36H
2O 16.7g/L, CuSO
40.1g/L, CoCl
20.104) g/L, solvent is a water.
(2) whey powder treatment process: with pure water whey powder is mixed with concentration 100g/L (lactose-content), centrifugal (8000r/min 15min) got supernatant liquor and is the whey powder lysate to remove objectionable impurities in 1 hour for 60 ℃ of water-baths.After adding glycerine to concentration is 25g/L, and sterilization (110 ℃, 10min) stand-by.
Three. whey powder is induced the expression of SPase at Escherichia coli sp1515
(1) the single bacterium colony of picking reorganization bacterium overnight incubation in the LB substratum is done contrast with bacteria samples and the E.coli BL21 (DE3) that contains empty carrier;
(2) next day, with experimental group and control group by volume 3% inoculum size be seeded in the fresh 100mL synthetic medium, 37 ℃, the 200r/min shaking culture;
(3) treat that bacterium liquid grows to OD
600By 0.6～0.8 o'clock, divide two groups to induce, first group adds inductor whey powder to final concentration is 20g/L; Second group adds the inductor whey powder and glycerol mixture to final concentration is respectively 20g/L and 5g/L, induces 24 hours for 25 ℃;
(4) centrifugal (4 ℃, 8000r/min 15min) collects thalline to bacterium liquid.
Four. interpretation of result
Enzyme activity determination: method is with embodiment 2 step 4. the enzyme activity determination of expression product.
Two groups of tests are lived inducing the sampling in 6,11,22,24 hours of beginning back to survey the SPase enzyme respectively, and growth tendency and enzyme are lived respectively the result shown in Fig. 8,9.Interpolation whey powder and glycerol mixture to final concentration are respectively 20g/L and 5g/L when inducing, and induce 24 hours for 25 ℃, and the work of SPase enzyme can reach 82.5U/mL under this condition.
The growth of cultivating the reorganization bacterium with synthetic medium is much better than to use the LB substratum, may be that synthetic medium provides the more somatomedin of horn of plenty, and better to the pH buffering effect, the growth of suitable reorganization bacterium; Only add the whey powder treatment solution when the growth of adding in addition 5g/L glycerine thalline again and the active all effects of SPase are better than, reason may be, thalline is the power consumption process to the active transport that is utilized as of lactose, and utilizing has delay.From the result, utilize synthetic medium, be better than inducing with IPTG among the embodiment 3 effect of recombinant bacterial strain with the expression effect of whey powder inductor.Therefore, whey powder can be used as carbon source and inductor simultaneously, compares expensive and virose IPTG, greatly reduces fermentation costs.
One. the preparation of nickel post
When using the nickel post for the first time, cross post with distilled water earlier, exist to wash until air repeatedly if any air and all discharge; Use 10ml (containing the 20mM imidazoles) binding buffer liquid and 10ml elution buffer (containing the 40mM imidazoles) to cross post respectively then;
Cross post with 10ml binding buffer liquid more at last.
Two. the crude enzyme liquid purifying
(1) preparation of crude enzyme liquid
Crude enzyme liquid prepares: after the expression product of centrifugal collection is washed twice with damping fluid (10mmol/L potassiumphosphate buffer pH 6.5), get 1ml and carry out ultrasonication (200W～400W, ultrasonic 3s, 5s intermittently, 2min altogether), 4 ℃, the centrifugal 15min of 10000r/min, get supernatant and get crude enzyme liquid, crude enzyme liquid can upper prop after with 0.45 μ m membrane filtration;
(2) purifying
His Sepharose HP gel column washs back 10ml binding buffer liquid (20mmol/L sodium phosphate with the 10ml distilled water, the 40mmol/L imidazoles, 0.5mol/L NaCl, pH=7.5) balance, above-mentioned ready crude enzyme liquid is crossed post, target protein is combined with gel column, there are not bonded albumen lavation buffer solution (20mmol/L sodium phosphate, 100mmol/L imidazoles, 0.5mol/LNaCl, pH=7.5) remove, use elution buffer (20mmol/L potassiumphosphate, 500mmol/L imidazoles, 0.5mol/L NaCl at last, pH=7.5) wash-out obtains target protein.
Three .SDS-PAGE identify albumen
Conventional SDS-PAGE method detects (concentrating gum concentration 4.5%, resolving gel concentration 10%), and the record result as shown in figure 10.
Claims (10)
1. the colibacillus engineering of an express recombinant sucrose phosphorylase is characterized in that it is the intestinal bacteria that imported the sucrose phosphorylase SPase gene that derives from Leuconostoc mesenteroides Leuconostoc mesenteroides ATCC 12291.
2. the colibacillus engineering of express recombinant sucrose phosphorylase according to claim 1, the genotype that it is characterized in that described engineering bacteria is F
-OmpT gal dcm lon hsd SB (rB
-MB
-), described engineering bacteria contains expression vector pET-Spase, and wherein, described expression vector pET-Spase is that multiple clone site has been inserted sucrose phosphorylase SPase gene in plasmid pET-22b (+).
3. according to the colibacillus engineering of the described express recombinant sucrose phosphorylase of claim 1, it is characterized in that described strain classification called after intestinal bacteria (Escherichia coli) sp1515, be preserved in Chinese typical culture collection center on January 8th, 2011, its preserving number is CCTCC NO:M 2011010.
4. the construction process of the colibacillus engineering of the described express recombinant sucrose phosphorylase of claim 1 is characterized in that it comprises the steps:
(1) extracts Leuconostoc mesenteroides Leuconostoc mesenteroides ATCC 12291 total DNA;
(2) design primer, with PCR method clone SPase gene, primer adds that at 5 ' end the GCTCTAGA sequence constitutes Xba I restriction enzyme site, adds that at 5 ' end CCCAAGCT constitutes Hind III restriction enzyme site;
Upstream primer 5 '-GCTCTAGACGCAATCGTTACAGTTATTACTAGC-3 ';
Downstream primer 5 '-CCCAAGCTTGTTCTGAGTCAAATTATCACTGCTC-3 ';
(3) the SPase gene after the above-mentioned reorganization is inserted among the plasmid pET-22b (+), gets recombinant vectors pET-Spase;
(4) change recombinant vectors pET-Spase over to intestinal bacteria then, carry out the recombinant clone screening, obtain colibacillus engineering.
5. the application of the colibacillus engineering of the described express recombinant sucrose phosphorylase of claim 1 in the preparation sucrose phosphorylase.
6. application according to claim 5 is characterized in that colibacillus engineering behind inducing culture, cytoclasis, and the crude enzyme liquid that obtains makes sucrose phosphorylase through the affinity chromatography separation and purification.
7. application according to claim 6, it is characterized in that described method for inducing and cultivating is as follows: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in the LB substratum by volume, when OD=0.6～0.8, add IPTG to final concentration be 0.1～1.1mmol/L, inducing culture 4～16h under 20～40 ℃, the condition of 200rpm, centrifugal acquisition thalline.
8. application according to claim 6, it is characterized in that described method for inducing and cultivating is as follows: with colibacillus engineering in 37 ℃ of following incubated overnight, 3～5% be inoculated in synthetic medium by volume, when OD=0.6～0.8, add whey powder and glycerine to final concentration and be respectively 5～20g/L and 5～15g/L, inducing culture 6～24h under 20～40 ℃, the condition of 200rpm, centrifugal acquisition thalline.
9. application according to claim 8 is characterized in that described synthetic medium comprises following component: NaH
2PO
42H
2O 4.0g/L, K
2HPO
414.6g/L, NH
4Cl 0.5g/L, (NH
4)
2SO
42.5g/L, (NH
4)
2-H-citrate 1.0g/L, Na
2SO
42.0g/L, micro-2ml/L, MgSO
4120g/L, VitB1 0.34g/mL, glucose 1g/L and penbritin 0.1g/L; Wherein, described trace element contains following component: CaCl
26H
2O 0.74g/L, ZnSO
47H
2O 0.18g/L, MnSO
4H
2O 0.1g/L, Na
2-EDTA 20.1g/L, FeCl
36H
2O 16.7g/L, CuSO
40.1g/L, CoCl
20.104g/L.
10. application according to claim 6 is characterized in that described affinity chromatography method is to use HisSepharose HP gel column to carry out affinity chromatography.
Priority Applications (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148455A (en) * | 2015-03-25 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of derivant and application thereof |
| CN106367458A (en) * | 2016-09-13 | 2017-02-01 | 华南农业大学 | Application of recombinant sucrose phospholylase in preparation of functional oligosaccharides |
| CN107236696A (en) * | 2017-07-31 | 2017-10-10 | 江南大学 | A kind of sucrose phosphorylase recombined bacillus subtilis in expression L. mesenteroides sources |
| CN109486782A (en) * | 2018-11-26 | 2019-03-19 | 江南大学 | A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency |
| CN110343654A (en) * | 2019-08-15 | 2019-10-18 | 江南大学 | A kind of genetic engineering bacterium producing sucrose phosphorylase |
| CN110452845A (en) * | 2019-08-15 | 2019-11-15 | 江南大学 | A kind of Escherichia coli producing sucrose phosphorylase |
-
2011
- 2011-01-17 CN CN201110009399.6A patent/CN102174454A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| <第六届全国化学工程与生物化工年会论文集> 20101231 隋姗姗等 乳糖诱导蔗糖磷酸化酶基因在重组大肠杆菌中的表达 全文 1-10 , * |
| 20061231 Jin-Ha Lee等 Molecular cloning of a gene encoding the sucrose phosphorylase from Leuconostoc mesenteroides B-1149 and the expression in Escherichia coli 612-620 1－10 第39卷, * |
| 20101231 侯顾伟等 蔗糖磷酸化酶制备及应用的研究进展 17-20 1-10 , 第6期 * |
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| CN106148455A (en) * | 2015-03-25 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of derivant and application thereof |
| CN106367458A (en) * | 2016-09-13 | 2017-02-01 | 华南农业大学 | Application of recombinant sucrose phospholylase in preparation of functional oligosaccharides |
| CN106367458B (en) * | 2016-09-13 | 2019-11-15 | 华南农业大学 | Recombinant sucrose phosphorylase is preparing the application in functional oligose |
| CN107236696A (en) * | 2017-07-31 | 2017-10-10 | 江南大学 | A kind of sucrose phosphorylase recombined bacillus subtilis in expression L. mesenteroides sources |
| CN107236696B (en) * | 2017-07-31 | 2019-11-26 | 江南大学 | A kind of sucrose phosphorylase recombined bacillus subtilis for expressing the source L.mesenteroides |
| CN109486782A (en) * | 2018-11-26 | 2019-03-19 | 江南大学 | A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency |
| CN109486782B (en) * | 2018-11-26 | 2020-06-09 | 江南大学 | Method for improving sucrose phosphorylase expression efficiency through molecular chaperone co-expression |
| CN110343654A (en) * | 2019-08-15 | 2019-10-18 | 江南大学 | A kind of genetic engineering bacterium producing sucrose phosphorylase |
| CN110452845A (en) * | 2019-08-15 | 2019-11-15 | 江南大学 | A kind of Escherichia coli producing sucrose phosphorylase |
| CN110452845B (en) * | 2019-08-15 | 2021-03-02 | 江南大学 | Escherichia coli for producing sucrose phosphorylase |
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