CN102732538B - Novel high-temperature-resisting lipase gene and coding product thereof - Google Patents
Novel high-temperature-resisting lipase gene and coding product thereof Download PDFInfo
- Publication number
- CN102732538B CN102732538B CN201210127232.4A CN201210127232A CN102732538B CN 102732538 B CN102732538 B CN 102732538B CN 201210127232 A CN201210127232 A CN 201210127232A CN 102732538 B CN102732538 B CN 102732538B
- Authority
- CN
- China
- Prior art keywords
- lip34
- lipase
- recombinant
- product
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090001060 Lipase Proteins 0.000 title claims abstract description 66
- 239000013612 plasmid Substances 0.000 claims abstract description 41
- 102000004882 Lipase Human genes 0.000 claims abstract description 34
- 239000004367 Lipase Substances 0.000 claims abstract description 33
- 235000019421 lipase Nutrition 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 21
- 241000588724 Escherichia coli Species 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000013604 expression vector Substances 0.000 claims abstract description 9
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 230000006698 induction Effects 0.000 claims description 17
- 230000014509 gene expression Effects 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 230000029087 digestion Effects 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 230000005611 electricity Effects 0.000 claims description 8
- 230000009970 fire resistant effect Effects 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 238000001556 precipitation Methods 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 5
- 108091008146 restriction endonucleases Proteins 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 238000001042 affinity chromatography Methods 0.000 claims description 4
- 210000001072 colon Anatomy 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 239000012160 loading buffer Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- 230000009465 prokaryotic expression Effects 0.000 abstract description 3
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 239000000499 gel Substances 0.000 description 7
- 235000019626 lipase activity Nutrition 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 240000002044 Rhizophora apiculata Species 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000012882 sequential analysis Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 description 1
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 125000005908 glyceryl ester group Chemical group 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- -1 oxygen anion Chemical class 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a novel high-temperature-resisting lipase gene which is named as lip34 and which has a nucleotide sequence shown as SEQ ID NO.1. Also, the invention discloses a preparation method of the novel high-temperature-resisting lipase gene, a recombinant plasmid pET32a-lip34 containing the novel high-temperature-resisting lipase gene, and an expression vector thereof. The invention further discloses a high-temperature-resisting recombinant lipase and a preparation method thereof. The novel high-temperature-resisting lipase gene lip34 is excessively expressed in an escherichia coli prokaryotic expression system, and the production period of lipase is substantially shortened. The recombinant lipase obtained with the method has relatively high catalytic activity and thermal stability, and has good industrialized production and application prospects.
Description
Technical field
The present invention relates to a kind of novel lipase gene and coded product thereof, especially a kind of novel lipase gene and coded product thereof from mangrove forest soil microorganism.
Background technology
Lipase is a kind of enzyme being extensively present in animals and plants and microorganism, in lipid metabolism, plays an important role.On water-oil interface, the ester linkage hydrolyzing of lipase-catalyzed triacylglycerol, discharges and contains still less glyceryl ester or glycerine and the lipid acid of ester bond.In addition, also has plurality of enzymes activity, as the fractionation of the hydrolysis of the multiple ester of catalysis, synthetic and racemic mixture.Lipase reaction mild condition, has good stereoselectivity, and can not cause environmental pollution, therefore, in many industrial circles such as food, leather, medicine, feed and washing composition, is all widely used.
Occurring in nature exist microorganism 99% can not cultivate, the method for therefore screening new biological catalyst from the microorganism being separated to by traditional culture technique be have very much circumscribed.Training technology is exempted from utilization, directly from environmental microorganism, extracts genomic dna, is cloned in different bacteria carriers, and build in " metagenome library ", more therefrom screen, be the screening method having a extensive future.
Summary of the invention
One of object of the present invention is to solve the deficiencies in the prior art part and gene that a kind of lipase of can encode high catalytic efficiency, high stability and high resistance to cold and diseases is provided.
For achieving the above object, the technical scheme that the present invention takes is: a kind of novel fire resistant lipase gene, and described lipase gene called after lip34, the nucleotide sequence of described lipase gene is as shown in SEQ ID NO1.
High temperature resistant fatty enzyme gene described above is prepared gained by the following method: 4 positive colony of random choose from the grand genomic library of mangrove forest soil, order-checking, then use software to analyze institute's calling sequence, can obtain described high temperature resistant fatty enzyme gene.
Described lipase gene lip34 obtains from the grand genomic library of mangrove forest by the method for random screening, lip34 is the complete lipase gene of a 825bp size, utilizing BLAST to compare online its nucleotide sequence and carry out homology searching analysis and show, there is not similarity in described high temperature resistant fatty enzyme gene lip34 and known.
Another object of the present invention is to provide a kind of recombinant plasmid pET32a-lip34 that contains novel fire resistant lipase gene described above, according to the primers of lipase gene lip34, taking extract plasmid pUC18-lip34 as template, carry out pcr amplification, PCR product is through gel electrophoresis and reclaim product 1, gel reclaims product 1 through restriction enzyme BamHI and XhoI double digestion and reclaims product 2, plasmid pET32a is connected with recovery product 2 after XhoI double digestion through BamHI, obtain and connect product, i.e. recombinant plasmid pET32a-lip34.
In addition, the present invention also provides a kind of expression vector that contains novel fire resistant lipase gene described above, adopt and build with the following method gained: adopt electricity to turn method, the recombinant plasmid pET32a-lip34 of above-mentioned gained is transformed in e. coli bl21 competent cell, bacterial suspension renewal cultivation dilution spread are cultivated on amicillin resistance culture plate, picking positive transformant, obtains the expression vector colon bacillus BL21-pET32a-lip34 of novel fire resistant lipase gene.Described colon bacillus BL21-pET32a-lip34 is now preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Classification And Nomenclature is: colon bacillus Escherichia coli, preservation date is on 01 16th, 2012, and deposit number is CGMCCNo.5736.
A further object of the present invention is to provide a kind of preparation method of high temperature resistant recombinant lipase, comprises the following steps:
(1) structure of recombinant plasmid pET32a-lip34: according to the primers of lipase gene lip34, taking extract plasmid pUC18-lip34 as template, carry out pcr amplification, PCR product is through gel electrophoresis and reclaim product 1, gel reclaims product 1 through restriction enzyme BamHI and XhoI double digestion and reclaims product 2, plasmid pET32a is connected with recovery product 2 after XhoI double digestion through BamHI, obtains and connects product, i.e. recombinant plasmid pET32a-lip34;
(2) structure of lipase gene lip34 expression vector: adopt electricity to turn method, recombinant plasmid pET32a-lip34 is transformed in e. coli bl21 competent cell, bacterial suspension renewal cultivation dilution spread are cultivated on amicillin resistance culture plate to picking positive transformant;
(3) abduction delivering of target protein Lip34: the intestinal bacteria of recombinant plasmid transformed are coated on the flat board containing amicillin resistance, be inverted overnight incubation for 37 DEG C, picking mono-clonal is inoculated in LB liquid nutrient medium, 37 DEG C of shaking culture are spent the night, the ratio of 1: 100 is by volume forwarded to fresh in amicillin resistance LB substratum, is cultured to OD in 37 DEG C, 200rpm
600=0.6～0.8; Add IPTG inducing culture, after cultivation, through affinity chromatography purifying, obtain recombinant lipase Lip34.
As the preparation method's of a kind of high temperature resistant recombinant lipase of the present invention preferred implementation, the LB liquid nutrient medium in described step (3) contains 100 μ g/mL acillins.
As the preparation method's of a kind of high temperature resistant recombinant lipase of the present invention preferred implementation, the concentration of the IPTG adding in described step (3) is 0.2mmol/L, and inducing temperature is 30 DEG C, and induction time is 5h.
As the preparation method's of a kind of high temperature resistant recombinant lipase of the present invention preferred implementation, described preparation method also comprises step (4): the detection of target protein Lip34: get the bacterium liquid after 1.5mL induction, the centrifugal 1min of 12000 × g, abandon supernatant, by the resuspended precipitation of Tris-HCl (pH 8.0) of 50 μ L50mmol/L, add equal-volume 4 × Loading buffer, in boiling water, boil 10min, the centrifugal 10min of 13000 × g; Get 10 μ L supernatant liquors and carry out SDS-PAGE analysis, resolving gel concentration is 10%, concentrated gum concentration 5%.
Wherein, Lip34 prediction relative molecular weight is about 31.6kDa.Due to the protein fusion expression of pET32a (+) vector encoded Trx and six histidine-tagged etc. and objects, therefore the relative molecular weight of Lip34 recombinant lipase is about 46kDa.
Meanwhile, the present invention also provides a kind of high temperature resistant recombinant lipase Lip34 that adopts method described above to prepare.The lipase hydrolysis activity of described Lip34 is 0.34U/mg, and Lip34 optimum temperuture is 85 DEG C, and optimal pH is 11.0.
Described recombinant lipase Lip34 obtains through the method purifying of affinity chromatography after lip34 expresses in escherichia coli prokaryotic expression system, described Lip34 has lipase conserved sequence, is positioned at the oxygen anion hole of HG dipeptides and the pentapeptide GASSGG (GA-X-S-X-G) of 120-125 position of aminoacid sequence 82-83 position.
The invention provides a kind of novel high temperature resistant fatty enzyme gene, this is gene constructed in plasmid pET32a, then this recombinant plasmid is transferred in escherichia coli prokaryotic expression system and is expressed and purifying.Show through bioinformatic analysis, the product of this genetic expression has higher catalytic activity and thermostability, has larger suitability for industrialized production and application potential.
Brief description of the drawings
Fig. 1 is the collection of illustrative plates of the agarose gel electrophoresis of the structure whole process of the expression vector that contains lipase gene lip34 of the present invention;
In Fig. 1, a:lip34PCR amplified production gel reclaims M:1kb ladder1:lip34; B:lip34DNA enzyme cuts back to close M:1kb ladder 1:lip34; C:pET32a plasmid enzyme restriction reclaims M:1kb ladder1:pET32a; D: recombinant plasmid enzyme is cut M:1kb ladder1:pET32-lip34.
The detection of expression collection of illustrative plates that Fig. 2 is lipase gene lip34;
In Fig. 2, M is Marker, and 1 is positive control, and 2 is negative contrast, 3 Lip34 that are abduction delivering.
Fig. 3 is the impact of inducing temperature on recombinant lipase Lip34 expression level;
In Fig. 3, M is Marker; 1-4 is the expression (temperature of 1-4 is respectively 18 DEG C, 25 DEG C, 30 DEG C, 37 DEG C) of Lip34 after differing temps induction.
Fig. 4 is the impact of IPTG concentration on recombinant lipase Lip34 expression level;
In Fig. 4, M is Marker, and 1 is contrast, and 2-8 is that (in 2-8, IPTG concentration is respectively 0mmol/L for the expression of Lip34 after different IP TG concentration induction, 0.05mmol/L, 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L).
Fig. 5 is the impact of induction time on recombinant lipase Lip34 expression level;
In Fig. 5, M is Marker, and 1 is contrast, and 2-9 is respectively induction 0h, 2h, 3h, 4h, 5h, 6h, 7h, the expression of Lip34 after 8h.
Fig. 6 is that the SDS-PAGE of the each component of recombinant lipase Lip34 purge process analyzes;
In Fig. 6,1 is the flowing liquid after chromatography, and 2 is a damping fluid of washing, and 3 is washed twice damping fluid, and 4 is an elutriant, and 5 is secondary elutriant, and 6 is three elutriants, and 7 is four elutriants, and 8 is precipitation.
Fig. 7 is the impact of temperature on recombinant lipase Lip34 activity.
Fig. 8 is the impact of pH on recombinant lipase Lip34 activity.
Embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with the drawings and specific embodiments, the invention will be further described.
Embodiment 1
1, the acquisition of novel lipase gene lip34
Adopt random clone sequencing analysis screening method, choose at random the order-checking of 4 positive colony, order-checking is completed by Shanghai Ying Jun biotech company, then uses software to analyze institute's calling sequence.Only there is the complete lipase gene of a 825bp size of No. 34 plasmid existence, called after lip34.
2, as follows according to the primers of lipase gene lip34:
lip34-F：5’-CGCGGATCCATGAGACAATTGAC-3’
lip34-R：5’-CCGCTCGAGTTAATTGTTAGTGTCCC-3’
3, pcr amplification
Reaction system is as follows:
4, PCR program is as follows:
5, the gel of lip34 gene amplification product reclaims
Carry out glue recovery according to step in E.Z.N.A gel extraction kit specification sheets.
6, the double digestion of lip34 gene PCR product and product reclaim, and enzyme is cut product and detected through 1% agarose gel electrophoresis, and reaction system is as follows:
7, the extraction of plasmid pET32a
The E.coli mono-clonal that carries corresponding plasmid is inoculated into 5mL containing in 100 μ g/mL acillin LB nutrient solutions, 37 DEG C, 200rpm, shaking culture is spent the night.Extract plasmid according to step in OMEGA plasmid extraction kit specification sheets.
8, plasmid enzyme restriction and dephosphorylation
After plasmid extraction, carry out double digestion with BamHI and XhoI, then use 50 DEG C of processing of calf intestinal alkaline phosphatase.Reaction system is as follows:
9, ligation
In 0.2ml centrifuge tube, add following composition, after mixing 16 DEG C of connections of spending the night, connect product be stored in-20 DEG C for subsequent use or immediately for transform.
10, transform
(1) the competent preparation of electric Transformed E .coli BL21
1. inoculate the mono-bacterium colony of E.coli BL21 in LB liquid nutrient medium, 37 DEG C, the activation of 200rpm incubated overnight;
2. within 1: 5 by volume, be seeded to fresh 2% and spread cultivation in liquid, 18 DEG C, 200rpm shaking culture 3-4h to OD
600be 0.6 left and right, the centrifugal 10min of 5000 × g at 4 DEG C, abandons supernatant, collecting cell;
3. add the isopyknic precooling ddH of former substratum
2o re-suspended cell, the centrifugal 10min of 5000 × g at 4 DEG C, abandons supernatant, repeats once;
4. add the resuspended precipitation of 15% glycerine solution of equal-volume precooling, be distributed into the every pipe of 100 μ L and be sub-packed in 1.5mL centrifuge tube;
5. liquid nitrogen flash freezer, is stored in-80 DEG C of Ultralow Temperature Freezers.
(2) electricity of E.coli BL21 turns
1. take out from-80 DEG C of refrigerators the E.coli BL21 competent cell of preserving, be placed on ice and slowly thaw; Take out aseptic electric revolving cup and be placed on precooling on ice;
2. add about 500ng plasmid also softly to mix in competent cell, be placed in 1min on ice;
3. Eppendorf electroporation voltage is adjusted to 2500V;
4. the competent cell that has mixed plasmid DNA is transferred in electric revolving cup, softly rocks and make mixture drop down onto electric revolving cup bottom.Electric revolving cup is positioned in electroporation, and and electrode contact;
5. pulse electric shock is carried out in energising;
6. after having shocked by electricity, take out immediately the SOC liquid nutrient medium that electricity transforms cup and adds 800 μ L precoolings, the competent cell after promptly SOC substratum and electricity being turned mixes, and bacterial suspension is placed in to the centrifuge tube of 1.5mL, renewal cultivation 1h in 37 DEG C of shaking tables;
7. by the bacterium liquid gradient dilution coating selectivity flat board after renewal cultivation, be inverted in 37 DEG C of cultivations, cultivating 12-16h can picking positive transformant.
(3) screening of positive colony
Random picking transformant carries out bacterium colony PCR, employing universal primer M13 (+) (5 '-TGTAAAACGACGGCCAGT-3) and M13 (-) (5 '-CAGGAAACAGCTATGACC-3 ') carry out pcr amplification, whether meet the requirements to detect recombinant plasmid Insert Fragment by the effect of pcr amplification.Reaction system is as follows:
PCR program is as follows:
11, the screening of recombinant plasmid and enzyme are cut qualification
Random picking transformant is inoculated in 5mL containing in the LB liquid nutrient medium of amicillin resistance, 37 DEG C, 200rpm are cultivated 6-8h, extract plasmid DNA, by suitable restriction enzymes double zyme cutting for plasmid DNA, enzyme is cut system with step 6, positive colony is cut after qualification through further enzyme, called after pET32a-lip34.
The agarose gel electrophoretogram of the structure whole process of lipase gene lip34 expression vector is shown in accompanying drawing 1.
12, DNA sequencing, sequential analysis and sequence number
DNA sequencing is completed by Shanghai Ying Jun biotech company.The DNA sequence dna of mensuration is carried out in GenBank to similarity searching on NCBI and also carry out sequential analysis with DNAMAN 5.1.
13, the abduction delivering of target protein Lip34 and detection
The intestinal bacteria of recombinant plasmid transformed are coated on the flat board containing amicillin resistance, be inverted overnight incubation for 37 DEG C.Picking mono-clonal is inoculated in LB liquid nutrient medium (containing 100 μ g/mL acillins), and 37 DEG C of shaking culture are spent the night; The ratio of 1: 100 is by volume forwarded to fresh in amicillin resistance LB substratum, is cultured to OD in 37 DEG C, 200rpm
600=0.6～0.8; Add IPTG to final concentration 1.0mmol/L, inducing culture 4h under 37 DEG C of 200rpm conditions.Get the bacterium liquid after 1.5mL induction, the centrifugal 1min of 12000 × g, abandons supernatant; By the resuspended precipitation of 50 μ LTris-HCl (50mmol/L, pH 8.0), add equal-volume 4 × Loadingbuffer, in boiling water, boil 10min, the centrifugal 10min of 13000 × g; Get 10 μ L supernatant liquors and carry out SDS-PAGE analysis, resolving gel concentration is 10%, and concentrated gum concentration 5%, is shown in accompanying drawing 2.
14, the optimization of Lip34 abduction delivering condition
(1) determining of best inducing temperature
When induction, add IPTG to final concentration 1.0mmol/L, respectively at 18 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, lower induction 8h, with the differential expression (Fig. 3) of SDS-PAGE testing goal albumen.
(2) determining of best IPTG concentration
When induction, add IPTG to be respectively 0.05mmol/L, 0.1mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L to final concentration, in 30 DEG C of induction 8h, with the differential expression (Fig. 4) of SDS-PAGE testing goal albumen.
(3) determining of best induction time
When induction, add IPTG to final concentration 1.0mmol/L, in 30 DEG C of inductions, sample respectively at 0h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, with the differential expression (Fig. 5) of SDS-PAGE testing goal albumen.
Embodiment 2
1, the purifying of recombinant lipase Lip34
Use the method for affinity chromatography to carry out purifying.Get the bacterium liquid after 600mL induction, in 4 DEG C, the centrifugal 10min of 5000 × g, collect thalline; Within 20: 1, (V: V) ratio adds lysis buffer; Ultrasonication 40 times under 300W power (each/3s, interval time 3s); Through 4 DEG C, the centrifugal 30min of 10000 × g, get supernatant again; Supernatant is added in miniature gravitational stratification post, collects flowing liquid; Wash post twice with lavation buffer solution, each 4mL, collects flowing liquid; Wash post 4 times with elution buffer, each 0.5mL, collects respectively all flowing liquids; Each component of collecting is got respectively 10 μ L and is done SDS-PAGE analysis (Fig. 6).
2, lipase activity determination
The tuurbidimetry that the mensuration of lipase activity utilizes test kit to provide: return to zero as blank using 50mmol/L Tris-HCl; Get substrate buffer solution 4mL and add 0.9%NaCl 50 μ L, measure its absorbancy under 420nm wavelength, be denoted as As, this As value is equivalent to the absorbance of the concentration of standard pipe (454 μ mol/L); To the Lip34 enzyme liquid that adds 5 μ L purifying in 1mL triacylglycerol substrate, put upside down after mixing and measure immediately A420, be denoted as A1, under the thermograde of 37 DEG C, react 20min, measure A420, be denoted as A2, activity unit's basis formula of every milliliter of lipase enzyme liquid: (A1-A2)/As × 4.54, then convert and obtain the lipase activity of unit mass according to the lipase concentration of measuring.
3, the optimization of the condition of lipase activity
(1) determining of Lip34 optimum temperature
The thermograde of 5 DEG C, 15 DEG C, 25 DEG C, 35 DEG C, 45 DEG C, 55 DEG C, 65 DEG C, 75 DEG C, 85 DEG C, 95 DEG C is set, and the measuring method of lipase activity, with 2, uses Microsoft Excel to carry out data processing (Fig. 7).
(2) determining of the suitableeest action pH of Lip34
PH gradient (2,3,4,5,6,7,8,9,10,11,12) is set, and the measuring method of lipase activity, with 2, uses Microsoft Excel to carry out data processing (Fig. 8).
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (9)
1. a novel fire resistant lipase gene, is characterized in that, described lipase gene called after lip34, and the nucleotide sequence of described lipase gene is as shown in SEQ ID NO 1.
2. one kind contains the recombinant plasmid pET32a-lip34 of novel fire resistant lipase gene as claimed in claim 1, according to the primers of lipase gene lip34, taking extract plasmid pUC18-lip34 as template, carry out pcr amplification, PCR product is through gel electrophoresis and reclaim product 1, gel reclaims product 1 through restriction enzyme BamHI and XhoI double digestion and reclaims product 2, plasmid pET32a is connected with recovery product 2 after XhoI double digestion through BamHI, obtain and connect product, i.e. recombinant plasmid pET32a-lip34.
3. an expression vector that contains novel fire resistant lipase gene as claimed in claim 1, it is characterized in that, adopt and build with the following method gained: adopt electricity to turn method, the recombinant plasmid pET32a-lip34 of claim 2 gained is transformed in e. coli bl21 competent cell, bacterial suspension renewal cultivation dilution spread are cultivated on amicillin resistance culture plate, picking positive transformant, obtains the expression vector colon bacillus BL21-pET32a-lip34 of high temperature resistant fatty enzyme gene.
4. a preparation method for high temperature resistant recombinant lipase, is characterized in that, comprises the following steps:
(1) structure of recombinant plasmid pET32a-lip34: according to the primers of lipase gene lip34, taking extract plasmid pUC18-lip34 as template, carry out pcr amplification, PCR product is through gel electrophoresis and reclaim product 1, gel reclaims product 1 through restriction enzyme BamHI and XhoI double digestion and reclaims product 2, plasmid pET32a is connected with recovery product 2 after XhoI double digestion through BamHI, obtains and connects product, i.e. recombinant plasmid pET32a-lip34;
(2) structure of lipase gene lip34 expression vector: adopt electricity to turn method, recombinant plasmid pET32a-lip34 is transformed in e. coli bl21 competent cell, bacterial suspension renewal cultivation dilution spread are cultivated on amicillin resistance culture plate to picking positive transformant;
(3) abduction delivering of target protein Lip34: the intestinal bacteria of recombinant plasmid transformed are coated on the flat board containing amicillin resistance, be inverted overnight incubation for 37 DEG C, picking mono-clonal is inoculated in LB liquid nutrient medium, 37 DEG C of shaking culture are spent the night, the ratio of 1:100 is forwarded to freshly in amicillin resistance LB substratum by volume, is cultured to OD in 37 DEG C, 200rpm
600=0.6～0.8; Add IPTG inducing culture; After cultivation, through affinity chromatography purifying, obtain recombinant lipase Lip34.
5. the preparation method of high temperature resistant recombinant lipase as claimed in claim 4, is characterized in that, the LB liquid nutrient medium in described step (3) contains 100 μ g/mL penbritins.
6. the preparation method of high temperature resistant recombinant lipase as claimed in claim 4, is characterized in that, the concentration of the IPTG adding in described step (3) is 0.2mmol/L, and inducing temperature is 30 DEG C, and induction time is 5h.
7. the preparation method of high temperature resistant recombinant lipase as claimed in claim 4, it is characterized in that, described preparation method also comprises step (4): the detection of target protein Lip34: get the bacterium liquid after 1.5mL induction, the centrifugal 1min of 12000 × g, abandon supernatant, the resuspended precipitation of Tris-HCl that is 8.0 with the pH of 50 μ L50mmol/L, adds equal-volume 4 × Loadingbuffer, in boiling water, boil 10min, the centrifugal 10min of 13000 × g; Get 10 μ L supernatant liquors and carry out SDS-PAGE analysis, resolving gel concentration is 10%, concentrated gum concentration 5%.
8. a high temperature resistant recombinant lipase Lip34 who is obtained by high temperature resistant fatty enzyme gene expression described in claim 1.
9. high temperature resistant recombinant lipase Lip34 as claimed in claim 8, is characterized in that, the lipase hydrolysis activity of described Lip34 is 0.34U/mg, and Lip34 optimum temperuture is 85 DEG C, and optimal pH is 11.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210127232.4A CN102732538B (en) | 2012-04-26 | 2012-04-26 | Novel high-temperature-resisting lipase gene and coding product thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210127232.4A CN102732538B (en) | 2012-04-26 | 2012-04-26 | Novel high-temperature-resisting lipase gene and coding product thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102732538A CN102732538A (en) | 2012-10-17 |
| CN102732538B true CN102732538B (en) | 2014-08-06 |
Family
ID=46988838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210127232.4A Expired - Fee Related CN102732538B (en) | 2012-04-26 | 2012-04-26 | Novel high-temperature-resisting lipase gene and coding product thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102732538B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103865941B (en) * | 2012-12-11 | 2018-02-16 | 丰益(上海)生物技术研发中心有限公司 | A kind of lipase mould from branch spore, its coding gene sequence and application thereof |
| CN103361374A (en) * | 2013-07-23 | 2013-10-23 | 扬州日兴生物科技股份有限公司 | Chitosanase gene derived from soil metagenome library and obtaining method and application of chitosanase gene |
| CN103834626B (en) * | 2013-12-31 | 2017-01-11 | 广东药学院 | High temperature-resistant lipase and coded product thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500868A (en) * | 2002-11-13 | 2004-06-02 | �й���ѧԺ�����о��� | High temperature lipase, coding gene order and uses thereof |
-
2012
- 2012-04-26 CN CN201210127232.4A patent/CN102732538B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500868A (en) * | 2002-11-13 | 2004-06-02 | �й���ѧԺ�����о��� | High temperature lipase, coding gene order and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| 张煜星等.铜绿假单胞菌脂肪酶Lipase基因的原核表达.《安徽农业科学》.2008,(第35期), |
| 耐高温酸性脂肪酶菌株NJY-1-3的选育及发酵条件的研究;闫丽娟等;《食品科技》;20100331;第35卷(第3期);全文 * |
| 铜绿假单胞菌脂肪酶Lipase基因的原核表达;张煜星等;《安徽农业科学》;20081231(第35期);全文 * |
| 闫丽娟等.耐高温酸性脂肪酶菌株NJY-1-3的选育及发酵条件的研究.《食品科技》.2010,第35卷(第3期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102732538A (en) | 2012-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103361326B (en) | Partial glyceride lipase mutant with improved thermal resistance, mutant plasmid, recombination strain and preparation method | |
| CN109576244B (en) | Novel lipase, preparation and application thereof | |
| CN107828806A (en) | A kind of β alpha-glucosidase genes of new resistance to glucose and its application | |
| CN102732538B (en) | Novel high-temperature-resisting lipase gene and coding product thereof | |
| CN109182298A (en) | A kind of recombinant lipase mutant, engineering bacteria and application | |
| CN101748069A (en) | recombinant blue-green algae | |
| CN106916837A (en) | Hyperosmosis glycerine protein kinase gene RkHog1 and its recombinant expression carrier | |
| CN104312931B (en) | Torulaspora delbrueckii and application thereof | |
| CN104726477A (en) | Lipase coding gene and engineering strain thereof | |
| CN109929822A (en) | A kind of Aspergillus oryzae lipase mutant and its application | |
| CN108103036A (en) | A kind of novel laccase enzyme and its gene, engineering bacteria, preparation and application | |
| CN107365755A (en) | A kind of novel lipase and its gene, engineering bacteria and preparation method | |
| CN107384893A (en) | A kind of thermostable lipase and its preparation and application | |
| CN101402947B (en) | Novel gene of esterase and recombinant expression system | |
| CN110358751A (en) | A kind of recombinant lipase mutant, encoding gene, recombination engineering and application | |
| CN101165172B (en) | Recombination methyl nourishment bacillus and application thereof | |
| CN105349557A (en) | Malic enzyme gene RKME2 and recombinant expression vector thereof | |
| CN101824401B (en) | Glucanase and coding nucleic acid and expression thereof | |
| CN108570459A (en) | A kind of method of high-efficiency fermenting production recombinant bacteria laccase | |
| CN107739733A (en) | aspartate transaminase and preparation method thereof | |
| CN109055344A (en) | A kind of algin catenase and its application with hot recovery characteristics | |
| CN109929853A (en) | The application of the heat shock protein gene in Thermophilic Bacteria source | |
| CN100395332C (en) | L-sorbosone dehydrogenase, gene encoding same and use thereof | |
| CN103710364A (en) | Laccase gene Lac7 and expression protein and application thereof | |
| CN109234251A (en) | Protein and the nucleic acid molecules for encoding the protein are preparing the application in phosphohydrolase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140806 Termination date: 20150426 |
|
| EXPY | Termination of patent right or utility model |