CN104559994A - Biological gel breaker - Google Patents
Biological gel breaker Download PDFInfo
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- CN104559994A CN104559994A CN201310479346.XA CN201310479346A CN104559994A CN 104559994 A CN104559994 A CN 104559994A CN 201310479346 A CN201310479346 A CN 201310479346A CN 104559994 A CN104559994 A CN 104559994A
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- 108090000790 Enzymes Proteins 0.000 claims abstract description 44
- 102000004190 Enzymes Human genes 0.000 claims abstract description 44
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000000499 gel Substances 0.000 claims description 54
- 229940088598 enzyme Drugs 0.000 claims description 39
- 241000894006 Bacteria Species 0.000 claims description 35
- 239000003292 glue Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 230000006698 induction Effects 0.000 claims description 19
- 230000001939 inductive effect Effects 0.000 claims description 19
- 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 18
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 18
- 238000012986 modification Methods 0.000 claims description 16
- 230000004048 modification Effects 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 239000013612 plasmid Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 108010059892 Cellulase Proteins 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229940106157 cellulase Drugs 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- 238000005457 optimization Methods 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 5
- 230000015556 catabolic process Effects 0.000 claims description 5
- 238000006731 degradation reaction Methods 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 5
- 238000012797 qualification Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 239000013599 cloning vector Substances 0.000 claims description 4
- 238000013461 design Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 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 2
- 108020004705 Codon Proteins 0.000 claims description 2
- 102000012410 DNA Ligases Human genes 0.000 claims description 2
- 108010061982 DNA Ligases Proteins 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 2
- 229960005091 chloramphenicol Drugs 0.000 claims description 2
- 230000004087 circulation Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 150000002460 imidazoles Chemical class 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- 239000011347 resin Substances 0.000 claims description 2
- 229920005989 resin Polymers 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 7
- 229920002678 cellulose Polymers 0.000 abstract description 2
- 239000001913 cellulose Substances 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 230000003647 oxidation Effects 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 8
- 239000004160 Ammonium persulphate Substances 0.000 description 7
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 7
- 235000019395 ammonium persulphate Nutrition 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 229920001285 xanthan gum Polymers 0.000 description 7
- 239000000230 xanthan gum Substances 0.000 description 7
- 235000010493 xanthan gum Nutrition 0.000 description 7
- 229940082509 xanthan gum Drugs 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 5
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 229930182470 glycoside Natural products 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 239000004159 Potassium persulphate Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 238000012667 polymer degradation Methods 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 235000019394 potassium persulphate Nutrition 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000003375 selectivity assay Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
- C09K8/60—Compositions for stimulating production by acting on the underground formation
- C09K8/62—Compositions for forming crevices or fractures
- C09K8/66—Compositions based on water or polar solvents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2208/00—Aspects relating to compositions of drilling or well treatment fluids
- C09K2208/24—Bacteria or enzyme containing gel breakers
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Materials Engineering (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a biological gel breaker. The biological gel breaker is prepared from 60-75 parts by mass of mixing natural enzyme and 25-40 parts by mass of molecular modifying enzyme, wherein the natural enzyme is cellulose. The proportion of the natural enzyme and the molecular modifying enzyme can be regulated according to the practical fracturing needs, so that the modified biological gel breaker has characteristics of stable performance under a normal temperature condition, applicable temperature range of 20-130 DEG C, controllable reaction, long time of duration, specificity, no additional damages and less possibility to consume. The gel breaker is added in the later fracturing stage, good in gel breaking effect, complete in flowing back and free of residues.
Description
Technical field
The present invention relates to oilfield stimulation agent technical field, be specifically related to a kind of biological gel breaker agent.
Background technology
Tradition gel breaker mainly contains following several: 1, be oxidized gel breaker: conventional oxidation gel breaker has Potassium Persulphate, ammonium persulphate etc.Because the activity of oxygenant is relevant with temperature, general when formation temperature is lower than 49 DEG C, the speed of reaction is just very slow, needs to add activator.Oxidation gel breaker have a lot of defects as: be 1. at high temperature swift in response with fracturing liquid, fracturing liquid is degraded in advance and lose conveying propping agent ability, even cause pressing crack construction failure; 2. it belongs to no special reactant, and any reactant such as tubing, stratum matrix and the hydro carbons etc. that can and run into react, and generates and the incompatible pollutent in stratum, causes formation damage; 3. be oxidized gel breaker probably just to have run out before arrival object crack, thus do not reach brokenly the object of glue.2, capsule oxidation gel breaker: capsule oxidation gel breaker is that superoxide is contained in separately in synthesis shell.The core material of capsule oxidation gel breaker is gel breaker, can adopt that contact with water be that solubilized becomes highly active solid strong oxidizer.The advantage of capsule oxidation gel breaker resides in reduced the impact of gel breaker on fracturing liquid rheological property; Increase gel breaker consumption, improve the flow conductivity of supporting crack.3, conventional enzyme breaker: enzyme has high catalytic capability and well active bioprotein, and its Morphology and structure of self when catalyzed reaction does not change, therefore can another reaction of catalysis again.Conventional enzyme breaker is the mixture of hemicellulase, cellulase, amylase and polygalacturonase, and it cannot be degraded specific polymkeric substance, does not reach desirable broken glue effect.In addition, although conventional enzyme breaker is a kind of fracturing liquid rubber-breaking agent preferably at low temperatures, it requires lower pH value.General enzyme activity when pH=6 is maximum, and high temperature, high ph-values can make enzyme lose activity.4, specific enzymes gel breaker: be directed to this, novel specific biological enzyme breaks colloid and ties up to its further investigation such as use temperature scope, pH scope, mainly for glucoside key screening specific hydrolase enzyme (LSE) of polysaccharide polymer sugar, they only decompose specific glycoside bond in polysaccharide polymer structure, can be monose and the disaccharides of irreducibility by polymer degradation.These specificitys are broken glue enzyme and are mainly contained Mierocrystalline cellulose glycoside bond specific enzymes, starch glycoside bond specific enzymes, guanidine glue glycoside bond specific enzymes etc.
The broken glue technology Problems existing of tradition: 1, speed is slow, and reaction times and activity thereof depend on temperature, and temperature, lower than 50 DEG C, is reacted very slow; 2, not environmentally, it belongs to no special reactant, and any reactant such as tubing, stratum matrix and the hydro carbons etc. that can and run into react, and generates and the incompatible pollutent in stratum, causes formation damage; 3, the broken glue time length is short, and oxidation gel breaker has probably just run out before arrival object crack, does not thus reach brokenly the object of glue; 4, residue is many, and the broken glue of oxidation has randomness, causes guanidine glue chain can not be degradable, and the molecular weight of about 20% is greater than 2.0 × 10
6polymkeric substance is not degraded substantially.
Summary of the invention
For overcoming the technological deficiency of traditional gel breaker, the invention provides a kind of new bio gel breaker XYPJ-2 gel breaker, this gel breaker is the biotechnological formulation of stabilization, it is the mixture of natural enzyme and molecular modification enzyme, be made up of the polysaccharide of special hydrolysis xanthan gum XYSW-1, can according to the ratio of both actual needs adjustment of pressure break, after transformation, the Performance comparision of gel breaker under normal temperature condition is stablized.The colloid formed due to the recyclable fracturing fluid gelatinizer of matching used Biological clean is with it met profit part and is broken glue, so this gel breaker added in the pressure break later stage, broken glue is effective, and the row of returning is thorough, without residue.
In order to reach as above object, the present invention takes following technical scheme:
A kind of biological gel breaker, is mixed by natural enzyme and molecular modification enzyme, it is characterized in that: described natural enzyme 60-75 part, described molecular modification enzyme 25-40 part according to the mass fraction.
Further preferably, described natural enzyme 65-70 part, described molecular modification enzyme 30-35 part according to the mass fraction.
Further preferably, described natural enzyme 68 parts, described molecular modification enzyme 32 parts according to the mass fraction.
Further, described natural enzyme is cellulase.
Further, the preparation of described molecular modification enzyme comprises the steps:
1) amplification of design of primers and goal gene
Gene order according to broken glue preparation OA designs and synthesizes primer, and the broken glue preparation OA recombinant plasmid after codon optimized is that template carries out pcr amplification, and PCR reaction system is 25 μ l, and reaction parameter is: 95 DEG C of denaturation 2min; 95 DEG C of 15s, 50 DEG C of 30s, 72 DEG C of 3min, totally 30 circulations; Last 72 DEG C extend 10min, 16 DEG C of preservations; Get 9 μ l reaction product to identify for 1% agarose gel electrophoresis;
2) structure of broken glue preparation OA cloning vector
PCR primer after reclaiming glue respectively and pET28a carrier carry out EcoRI and SalI double digestion, and spending the night in 16 DEG C with T4DNA ligase enzyme after glue reclaims connects; Connection product conversion is entered E.coli Top10 competent cell, be coated on the LB flat board containing kantlex; Extract plasmid and carry out EcoRI and SalI double digestion, with 1% agarose gel electrophoresis qualification, will the positive colony called after pET28a-OA obtained be screened, and its bacterium liquid is sent to biotech company's order-checking;
3) target protein is induced to express
Empty plasmid pET28a and cloning vector pET28a-OA chemical method are proceeded in competent cell E.coli BL21star (DE3) respectively, 37 DEG C of incubated overnight on the LB solid medium of chloramphenicol resistance are being received containing card, select single bacterium colony next day to be inoculated in 5ml respectively and to contain in the LB substratum of kantlex, during in 37 DEG C of shaking culture to OD600=0.4 ~ 0.6, the bacterium liquid that taking-up 1ml does not induce is as contrast before induction, the IPTG that final concentration is 1mmol/L is added in residue culture, continue inducing culture, SDS-PAGE analysis is carried out after taking out after 6h that inoculum is centrifugal and removing supernatant,
4) analysis of expression of recombinant proteins form
The bacterium that will spend the night is inoculated in 20ml LB liquid nutrient medium by 1:100, be placed in 37 DEG C when being cultured to bacteria concentration OD600=0.4 ~ 0.6, adding IPTG to final concentration is 1.0mmol/L, inducing culture 6h, 10000rpm, centrifugal 10min collects thalline, add the ultrasonic degradation liquid (pH8.0 of 1/10 volume, 50mM Tris-HCl, 50mM NaCl) ultrasonic disruption 10min in ice bath, centrifugally carries out SDS-PAGE analysis to upper cleer and peaceful precipitation respectively afterwards;
5) optimization of abduction delivering time
Induce on the unified basis for 1.0mmol/L of final concentration at IPTG, change induction time, the bacterium liquid of enlarged culturing is inoculated in LB liquid nutrient medium by 1:100, when OD600=0.4 ~ 0.6, get the centrifugal supernatant that goes of 1mL bacterium liquid when inducing 0h, 2h, 4h, 6h, 8h, 10h respectively and carry out SDS-PAGE electrophoresis, determine that the suitableeest induction time is 6-7h;
6) optimization of IPTG induced concentration
The bacterium that will spend the night is inoculated in LB liquid nutrient medium by 1:100, when its OD600=0.4 ~ 0.6, by final concentration be 0.2,0.4,0.6,0.8,1.0,1.2mmol/L adds IPTG, 37 DEG C of induction 6h, get the centrifugal supernatant that goes of bacterium liquid 1mL and carry out SDS-PAGE electrophoresis, determine that the best induced concentration of IPTG is 0.8-0.9mmol/L;
7) optimization of inducing temperature
The bacterium that will spend the night is inoculated in LB liquid nutrient medium by 1:100, be placed in 25 DEG C, 28 DEG C, 37 DEG C cultivations respectively, when being OD600=0.4 ~ 0.6 to bacteria concentration, adding IPTG respectively to final concentration is 1.0mmol/L, inducing culture 6h, get the centrifugal supernatant that goes of bacterium liquid and carry out SDS-PAGE electrophoresis, determine that best inducing temperature is 37 DEG C;
8) purifying of recombinant protein
The bacterium that will spend the night is inoculated in 150mL substratum by 1:100, and 37 DEG C are cultured to bacteria concentration OD600 value when being 0.4 ~ 0.6, and adding IPTG to final concentration is 1.0mmol/L, after inducing culture 10h, 10000rpm, centrifugal 15min precipitate thalline, and the thalline collected is positioned over-20 DEG C spend the night after for subsequent use.The ultrasonic degradation liquid of 1/10 volume is added, broken 5 times of ice-bath ultrasonic ripple after collecting induction thalline; Collected by centrifugation supernatant, be splined on the 5ml Talon resin post that balanced through level pad (1 × PBS), after slow flow velocity loading, unconjugated foreign protein is washed away with 6 times of column volume level pads, finally with the level pad wash-out target protein containing 150mM imidazoles, and the target protein solution of wash-out is carried out SDS-PAGE analysis after dialyzate (pH8.0,20mMTris-HCl, 50mM NaCl) 4 DEG C of dialyzed overnights.
Results and analysis:
1, structure and the qualification of glue preparation OA recombinant expression vector is broken
Carry out pcr amplification with the primer pair goal gene designed, after 1% agarose gel electrophoresis detects, obtain the specificity target DNA fragment (figure mono-A) that a size is about 1473bp, consistent with expected results.
Be that template carries out pcr amplification with recombinant plasmid, obtain the fragment (figure bis-B) that size is about 1473bp, in the same size with goal gene.Simultaneously by recombinant plasmid through EcoRI and SalI double digestion, produce the fragment (figure mono-B) of 1473bp.Show goal gene correctly insertion vector, construction of recombinant plasmid success.
2, abduction delivering and the expression-form qualification of glue preparation OA gene is broken
SDS-PAGE electrophoresis result shows, the bacterium containing recombinant expression vector through IPTG induction great expression size can be about the new albumen of 53.4kD, and proceed to empty carrier pET28a bacterium, proceed to recombinant vectors pET28a-OA and without IPTG induction bacterium and do not express this albumen (figure bis-) containing the bacterium of any carrier.The molecular weight of new albumen is about 53.4kD, in the same size with desired value, and illustration purpose is protein induced expresses successfully.
3, different inducing temperature is on the impact of target protein expression amount
SDS-PAGE electrophoresis result display (figure tri-), the expression amount of target protein when 28 DEG C of inductions is higher than 23 DEG C, but expression amount is the highest 37 DEG C time.
4, the purifying of fusion rotein
The His-tag utilizing recombinant protein N end to contain carries out metal ion-chelant column chromatography, and the sample of purifying, through SDS-PAGE electrophoresis detection, is shown as single band (figure tetra-), the efficiently purifying that can be realized this albumen by cobalt post affinity chromatography is described.
The beneficial effect of biological gel breaker of the present invention compared with traditional gel breaker is as table 1.
Table 1 biological gel breaker of the present invention and traditional gel breaker contrast table
Accompanying drawing explanation
Figure 1A is broken the pcr amplification product of glue preparation OA gene; B is the qualification result of recombinant plasmid pET28a-OA
The SDS-PAGE of Fig. 2 target protein analyzes
The abduction delivering of albumen for the purpose of A
M is albumen Marker; No. 1 is restructuring contrast; No. 2 is restructuring induction; No. 3 is empty control plasmid; No. 4 is empty plasmid induction; No. 5 is the contrast of empty bacterium; No. 6 is the induction of empty bacterium.
The analysis of protein expression form for the purpose of B
The SDS-PAGE of target protein expression-form analyzes M: standard protein; 1: the thalline of not inducing; 2: the supernatant after inducible strain ultrasonic degradation; 3: the precipitation after inducible strain ultrasonication; 4: inducible strain whole cell albumen
Fig. 3 differing temps is on the impact of target protein expression amount
M: Protein Marker; 1: do not induce bacterium; 2:23 DEG C of induction; 3:28 DEG C of induction; 4:37 DEG C of induction.
The SDS-PAGE of Fig. 4 purifying protein analyzes
M: Protein Marker; 1: the target protein of purifying
Embodiment:
Below in conjunction with embodiment to further instruction of the present invention, in embodiment, be mass fraction, but the present invention is not limited thereto.
Embodiment 1
A kind of biological gel breaker, cellulase 68 parts, molecular modification enzyme 32 parts.
Embodiment 2
A kind of biological gel breaker, cellulase 65 parts, molecular modification enzyme 35 parts.
Embodiment 3
A kind of biological gel breaker, cellulase 70 parts, molecular modification enzyme 30 parts.
Embodiment 4
A kind of biological gel breaker, cellulase 60 parts, molecular modification enzyme 40 parts.
Embodiment 5
A kind of biological gel breaker, cellulase 75 parts, molecular modification enzyme 25 parts.
Embodiment 6
Gel breaker stoste described in Example 1-5, thin up is that 0.03%XYPJ-2 gel breaker is for subsequent use.The xanthan gum stoste of 24g is taken, the water of 776g at the use for laboratory precision electronic balance that is 0.01g.Xanthan gum stoste is slowly poured into water and Keep agitation, configures the xanthan gum of 3%, stir and make it fully dissolve, be divided into impartial two parts, numbering A, B.In A, B, add 0.03%XYPJ-2 gel breaker and 0.03% ammonium persulphate gel breaker respectively, then fully stir, make gel breaker dispersed, then put into the thermostat water bath of 40 DEG C, observe, measure and record the change of A, B viscosity in 120min.Experimental result is as table 2.
Table 20.03%XYPJ-2 gel breaker, 0.03% ammonium persulphate breaking glue solution viscosity change (40 DEG C) in time
As can be seen from Table 2: when temperature is 40 DEG C, it is thorough that XYPJ-2 gel breaker breaks glue than traditional gel breaker ammonium persulphate, solves the problem that low temperature on oil field breaks glue difficulty.
Embodiment 7
Gel breaker stoste described in Example 1-5, thin up is that 0.03%XYPJ-2 gel breaker is for subsequent use.The xanthan gum stoste of 24g is taken, the water of 776g at the use for laboratory precision electronic balance that is 0.01g.Xanthan gum stoste is slowly poured into water and Keep agitation, configures the xanthan gum of 3%, stir and make it fully dissolve, be divided into impartial two parts, numbering A, B.In A, B, add 0.03%XYPJ-2 gel breaker and 0.03% ammonium persulphate gel breaker respectively, then fully stir, make gel breaker dispersed, then put into the thermostat water bath of 60 DEG C, observe, measure and record the change of A, B viscosity in 120min.Experimental result is as table 2.
Table 30.03%XYPJ-2 gel breaker, 0.03% ammonium persulphate breaking glue solution viscosity change (60 DEG C) in time
As shown in Table 3: when temperature is 60 DEG C, although traditional gel breaker ammonium persulphate serves certain effect, broken glue degree is still obviously inferior to XYPJ-2 gel breaker.After XYPJ-2 gel breaker breaks glue, viscosity is low, without residue, and formation fanout free region, and not by the impact of construction environment, easy to use.
Claims (5)
1. a biological gel breaker, is mixed by natural enzyme and molecular modification enzyme, it is characterized in that: described natural enzyme 60-75 part, described molecular modification enzyme 25-40 part according to the mass fraction.
2. biological gel breaker according to claim 1, is characterized in that: described natural enzyme 65-70 part, described molecular modification enzyme 30-35 part according to the mass fraction.
3. biological gel breaker according to claim 1, is characterized in that: described natural enzyme 68 parts, described molecular modification enzyme 32 parts according to the mass fraction.
4. the biological gel breaker according to any one of claim 1-3, is characterized in that: described natural enzyme is cellulase.
5. the biological gel breaker according to any one of claim 1-3, is characterized in that, the preparation of described molecular modification enzyme comprises the steps:
1) amplification of design of primers and goal gene
Gene order according to broken glue preparation OA designs and synthesizes primer, and the broken glue preparation OA recombinant plasmid after codon optimized is that template carries out pcr amplification, and PCR reaction system is 25 μ l, and reaction parameter is: 95 DEG C of denaturation 2min; 95 DEG C of 15s, 50 DEG C of 30s, 72 DEG C of 3min, totally 30 circulations; Last 72 DEG C extend 10min, 16 DEG C of preservations; Get 9 μ l reaction product to identify for 1% agarose gel electrophoresis;
2) structure of broken glue preparation OA cloning vector
PCR primer after reclaiming glue respectively and pET28a carrier carry out EcoRI and SalI double digestion, and spending the night in 16 DEG C with T4DNA ligase enzyme after glue reclaims connects; Connection product conversion is entered E.coli Top10 competent cell, be coated on the LB flat board containing kantlex; Extract plasmid and carry out EcoRI and SalI double digestion, with 1% agarose gel electrophoresis qualification, will the positive colony called after pET28a-OA obtained be screened, and its bacterium liquid is sent to biotech company's order-checking;
3) target protein is induced to express
Empty plasmid pET28a and cloning vector pET28a-OA chemical method are proceeded in competent cell E.coli BL21star (DE3) respectively, 37 DEG C of incubated overnight on the LB solid medium of chloramphenicol resistance are being received containing card, select single bacterium colony next day to be inoculated in 5ml respectively and to contain in the LB substratum of kantlex, during in 37 DEG C of shaking culture to OD600=0.4 ~ 0.6, the bacterium liquid that taking-up 1ml does not induce is as contrast before induction, the IPTG that final concentration is 1mmol/L is added in residue culture, continue inducing culture, SDS-PAGE analysis is carried out after taking out after 6h that inoculum is centrifugal and removing supernatant,
4) analysis of expression of recombinant proteins form
The bacterium that will spend the night is inoculated in 20ml LB liquid nutrient medium by 1:100, be placed in 37 DEG C when being cultured to bacteria concentration OD600=0.4 ~ 0.6, adding IPTG to final concentration is 1.0mmol/L, inducing culture 6h, 10000rpm, centrifugal 10min collects thalline, add the ultrasonic degradation liquid (pH8.0 of 1/10 volume, 50mM Tris-HCl, 50mM NaCl) ultrasonic disruption 10min in ice bath, centrifugally carries out SDS-PAGE analysis to upper cleer and peaceful precipitation respectively afterwards;
5) optimization of abduction delivering time
Induce on the unified basis for 1.0mmol/L of final concentration at IPTG, change induction time, the bacterium liquid of enlarged culturing is inoculated in LB liquid nutrient medium by 1:100, when OD600=0.4 ~ 0.6, get the centrifugal supernatant that goes of 1mL bacterium liquid when inducing 0h, 2h, 4h, 6h, 8h, 10h respectively and carry out SDS-PAGE electrophoresis, determine that the suitableeest induction time is 6-7h;
6) optimization of IPTG induced concentration
The bacterium that will spend the night is inoculated in LB liquid nutrient medium by 1:100, when its OD600=0.4 ~ 0.6, by final concentration be 0.2,0.4,0.6,0.8,1.0,1.2mmol/L adds IPTG, 37 DEG C of induction 6h, get the centrifugal supernatant that goes of bacterium liquid 1mL and carry out SDS-PAGE electrophoresis, determine that the best induced concentration of IPTG is 0.8-0.9mmol/L;
7) optimization of inducing temperature
The bacterium that will spend the night is inoculated in LB liquid nutrient medium by 1:100, be placed in 25 DEG C, 28 DEG C, 37 DEG C cultivations respectively, when being OD600=0.4 ~ 0.6 to bacteria concentration, adding IPTG respectively to final concentration is 1.0mmol/L, inducing culture 6h, get the centrifugal supernatant that goes of bacterium liquid and carry out SDS-PAGE electrophoresis, determine that best inducing temperature is 37 DEG C;
8) purifying of recombinant protein
The bacterium that will spend the night is inoculated in 150mL substratum by 1:100, and 37 DEG C are cultured to bacteria concentration OD600 value when being 0.4 ~ 0.6, and adding IPTG to final concentration is 1.0mmol/L, after inducing culture 10h, 10000rpm, centrifugal 15min precipitate thalline, and the thalline collected is positioned over-20 DEG C spend the night after for subsequent use.The ultrasonic degradation liquid of 1/10 volume is added, broken 5 times of ice-bath ultrasonic ripple after collecting induction thalline; Collected by centrifugation supernatant, be splined on the 5ml Talon resin post that balanced through level pad (1 × PBS), after slow flow velocity loading, unconjugated foreign protein is washed away with 6 times of column volume level pads, finally with the level pad wash-out target protein containing 150mM imidazoles, and the target protein solution of wash-out is carried out SDS-PAGE analysis after dialyzate (pH8.0,20mMTris-HCl, 50mM NaCl) 4 DEG C of dialyzed overnights.
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