CN103555629A - Bacterial strain for producing medium-low-temperature beta-mannase as well as application thereof - Google Patents
Bacterial strain for producing medium-low-temperature beta-mannase as well as application thereof Download PDFInfo
- Publication number
- CN103555629A CN103555629A CN201310539350.0A CN201310539350A CN103555629A CN 103555629 A CN103555629 A CN 103555629A CN 201310539350 A CN201310539350 A CN 201310539350A CN 103555629 A CN103555629 A CN 103555629A
- Authority
- CN
- China
- Prior art keywords
- mannase
- beta
- guar gum
- obtains
- opus
- 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.)
- Granted
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 44
- 108090000790 Enzymes Proteins 0.000 claims abstract description 44
- 229920002907 Guar gum Polymers 0.000 claims abstract description 32
- 239000000665 guar gum Substances 0.000 claims abstract description 32
- 229960002154 guar gum Drugs 0.000 claims abstract description 32
- 235000010417 guar gum Nutrition 0.000 claims abstract description 32
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 60
- 239000003292 glue Substances 0.000 claims description 56
- 239000002609 medium Substances 0.000 claims description 21
- 238000000926 separation method Methods 0.000 claims description 14
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 230000003020 moisturizing effect Effects 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 8
- 239000000287 crude extract Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 229920002684 Sepharose Polymers 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 2
- 238000003379 elimination reaction Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 abstract description 40
- 230000000694 effects Effects 0.000 abstract description 13
- 239000000126 substance Substances 0.000 abstract description 9
- 239000003513 alkali Substances 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 5
- 238000009629 microbiological culture Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 150000003839 salts Chemical class 0.000 abstract description 2
- 238000012136 culture method Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 24
- 239000002585 base Substances 0.000 description 23
- 238000000034 method Methods 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 239000003921 oil Substances 0.000 description 12
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 9
- 229920013818 hydroxypropyl guar gum Polymers 0.000 description 9
- 239000004160 Ammonium persulphate Substances 0.000 description 8
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 8
- 235000019395 ammonium persulphate Nutrition 0.000 description 8
- 230000006378 damage Effects 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 7
- 229910021538 borax Inorganic materials 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- JRKICGRDRMAZLK-UHFFFAOYSA-L persulfate group Chemical group S(=O)(=O)([O-])OOS(=O)(=O)[O-] JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 7
- 239000004328 sodium tetraborate Substances 0.000 description 7
- 235000010339 sodium tetraborate Nutrition 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 208000014674 injury Diseases 0.000 description 5
- 239000011435 rock Substances 0.000 description 5
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 4
- -1 Carboxymethyl hydroxypropyl Chemical group 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 244000303965 Cyamopsis psoralioides Species 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 102000004533 Endonucleases Human genes 0.000 description 3
- 229920002752 Konjac Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 235000015110 jellies Nutrition 0.000 description 3
- 239000008274 jelly Substances 0.000 description 3
- 235000010485 konjac Nutrition 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000007639 printing Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000004753 textile Substances 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 244000241257 Cucumis melo Species 0.000 description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 239000006035 Tryptophane Substances 0.000 description 2
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 239000010779 crude oil Substances 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 150000002475 indoles Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000007669 thermal treatment Methods 0.000 description 2
- 229960004799 tryptophan Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical group O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 description 1
- 244000247812 Amorphophallus rivieri Species 0.000 description 1
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 1
- 102100032487 Beta-mannosidase Human genes 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 206010010214 Compression fracture Diseases 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 229920002581 Glucomannan Polymers 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004159 Potassium persulphate Substances 0.000 description 1
- 244000275012 Sesbania cannabina Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 244000250129 Trigonella foenum graecum Species 0.000 description 1
- 235000001484 Trigonella foenum graecum Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 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
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229940095054 ammoniac Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010055059 beta-Mannosidase Proteins 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000008398 formation water Substances 0.000 description 1
- 229940046240 glucomannan Drugs 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000252 konjac Substances 0.000 description 1
- 239000006101 laboratory sample Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 238000012667 polymer degradation Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 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
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 235000001019 trigonella foenum-graecum Nutrition 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses bacillus subtillis OPUS-001 for producing medium-low-temperature beta-mannase. Preservation number of the bacterial strain in China General Microbiological Culture Collection Center is CGMCC No.7361. The bacterial strain disclosed by the invention is simple in culture method, raw materials are easily available, enzyme yield of the bacterial strain is strong; produced alkali beta-mannase enzyme preparation is high in activity, alkaline-resistant and salt-resistant, has efficient gel breaking capability for oil field guar gum or modified guar gum fracturing fluid under a medium-low region temperature condition, is free of incompatibility with most of chemical assistants in the fracturing fluid, and can satisfy needs of oil field fracturing projects.
Description
Technical field
The invention belongs to microbial technology field, relate to bacterial strain and the application in the broken glue of middle low temperature well waterfrac treatment of fermentation, enzyme separating-purifying and this bacterial strain thereof that 'beta '-mannase is produced in a strain, and utilize this bacterial strain to prepare the method for 'beta '-mannase.Described 'beta '-mannase has middle low temperature, the alkali-proof feature had a liking for.
Background technology
Waterfrac treatment is an important technique measure of well production increment, intensified injection of wells.When ground high-pressure pump group by high-viscous liquid to substantially exceed in the discharge capacity Injection Well of formation absorption ability, after having suppressed the pressure that surpasses Around A Borehole terrestrial stress and tensile strength of rock near shaft bottom, in stratum, form crack.Along with injecting Zhong， crack, crack, extends forward gradually the liquid with propping agent.In stratum, formed like this sand packed fracture with sufficient length and one fixed width and height.Because man-made fracture has very high diafiltration ability, make oil gas can unobstructed inflow well in, play the effect of increasing yield and injection.
Aspect the selection of water-based fracturing liquid system viscosifying agent, the natural plants such as sesbania gum, konjak gum, fenugreek gum were once used in oil field, but at present, main that use is guar gum or derivatives thereof (hydroxypropylguar gum HPG, Carboxymethyl hydroxypropyl guar CMHPG, etc.).This class vegetable jelly is except konjak gum main component is glucomannan, and other all belong to polygalactomannan class, through linking agent (in low hot-well borax, middle high temperature organic boron), is cross-linked to form high-intensity frozen glue, has very strong prop-carrying capacity.After working fluid injects and to stitch together with propping agent, in order not make the oil reservoir water conservancy diversion crack of transformation stop up, must reduce viscosity colloid is broken after glue and returns and flow back to ground, to protect, be made the high-permeability in crack.
Oil field main force gel breaker is persulphate at present, as ammonium persulphate, and Potassium Persulphate etc.As chemical substance, the speed temperature influence of the transformation period of persulphate and release free oxygen is very big; Take ammonium persulphate as example, and its transformation period in the time of 100 ℃ is 0.17h, and 80 ℃ is 2.1h, and 60 ℃ is 38.5h, reaches 1030h in the time of 40 ℃.This specific character determines that it exists uncontrollability in operation, that is: it is too fast that persulphate often breaks glue under hot environment, and when middle low temperature, cannot realize completely broken glue, and maybe must add activator can part onset.
In 40-55 ℃, under low temperature environment, increase the dosage of persulphate, although can realize the broken glue of of the fracturing fluid final aquation, can cause fracturing liquid viscosity too low simultaneously, thereby affect fracturing liquid, take grittiness energy, working security is poor; And level of residue is high, the row of returning lead low, fracturing effect is poor, causes oil gas extraction efficiency not high.If adopt the capsule breaker of modified packing, though can partly ensure of the fracturing fluid rheological property, persulphate is still difficult to the separation broken required enough free oxygens of glueization of fracturing fluid gel of sening as an envoy under the low temperature environment of down-hole; In order to meet the construction object of rapid break, need to append a large amount of capsule breakers, finally cause cost significantly to improve, increased the injury risk to stratum simultaneously.
And under the low temperature environment lower than 40 ℃, persulphate independent role cannot be realized thoroughly broken glue, must add activator material.And this Class Activation agent has intense stimulus smell conventionally, because itself being also chemical classes material, be there is to injury hidden danger in stratum; Also may exist simultaneously water-soluble poor, easily oxidized, see photolysis, poor stability, the shortcoming such as with high costs.
Meanwhile, the residual polymer molecular weight after the broken glue of persulphate is conventionally between 20-30 ten thousand, although apparent viscosity declines, reality still can cause certain injury to stratum.Sulfur-bearing superoxide toxicity is large simultaneously, and corrodibility is strong, and environment is had to certain pollution.
In late period in last century, oilfield engineering teacher starts biological enzyme to be applied to the broken glue of waterfrac treatment.Its principle is: specific glycan lytic enzyme can with vegetable jelly (being mainly guar gum and derivative thereof) in main component polygalactomannan formed well below sugared bond activation can transition thing, activated molecule are increased greatly, thereby significantly promote sugared bond rupture speed.For the biological enzyme of broken glue, follow " key " principle of enzyme coordination catalysis kinetics, only act on the specific glycosidic link in guar gum and derivant structure thereof, thereby be small molecules amount oligose by polymer degradation, and can be further under the synergy of the prozyme of other catalysiss, oligose is converted into monose and the disaccharides of irreducibility.And biological enzyme does not originally produce consumption before and after guar gum and derivative degraded thereof, just participate in reaction process, after reaction, restore to the original state again, continue to bring into play katalysis.So under lower concentration dosage, a large amount of guar gums and derivative thereof thoroughly can be decomposed at short notice to this machine-processed place that is its high efficiency.
β-Isosorbide-5-Nitrae-D-mannase (β-Isosorbide-5-Nitrae-D-mannanmannanohydrolase, EC.3.2.l.78; Hereinafter to be referred as 'beta '-mannase) be that a class can be hydrolyzed and take the restriction endonuclease of the polysaccharide (comprising mannosans, polygalactomannan, konjac glucomanna etc.) that β-Isosorbide-5-Nitrae-D-MANNOSE is main chain, be to be employed at present to prove the broken glue biological enzyme of optimal pressure break.
'beta '-mannase is of many uses, is widely used at present the fields such as food, medicine, papermaking, textile printing and dyeing, fodder industry.Different from above-mentioned Application Areas is, particular surroundings because of hydrocarbon-bearing pool, the higher salinity of enzyme require tolerance for broken glue, simultaneously because Oil Field Measure is generally alkalescence with fracturing liquid, requires biological enzyme under meta-alkali condition, (pH8.0-11.0) to keep higher vigor.Also to guarantee that it has with sterilant, the good compatibility of the oilfield chemical auxiliaries such as class material of showing to live simultaneously.
At present, alkaline ' beta '-mannase has been widely used in oil-gas field fracturing technique abroad, is particularly suited for the broken glue (20-50 ℃) of pressure break of middle low temperature Oil/gas Well, but there is no 20 ℃ of application reports under following cold condition.At home, relevant low temperature 'beta '-mannase report is more, but mostly is the acidity producing by natural mutagenic strain or engineering bacteria, neutral mannase, and is to be used as fodder additives or washing composition and medicine mostly, is not suitable with the specific requirement of oil reservoir.Aspect pH adaptability, alkaline ' beta '-mannase also has patent report, but at present just in washing composition and food and feeds sector application, (Ma Yan and waiting discloses: CN1266096).
In addition, low yield and the expensive application scale of 'beta '-mannase in Reservoir Development that limited, one of most important approach that can change this situation is screening and cultivates and can produce high bacterium producing multi enzyme preparation high vigor and alkali-proof 'beta '-mannase.
Summary of the invention
The present invention aims to provide a kind of microorganism strains of using alkali resistance 'beta '-mannase by the broken glue of low temperature well waterfrac treatment in fermentative production, and cultivation and the fermentation process of this bacterial strain are provided, and separation purification method and the application of 'beta '-mannase active ingredient in tunning.
In the plant root soil sample of the subtilis of production 'beta '-mannase provided by the present invention (Bacillus subtilis) Hong Jiannao lakeside, Shenmu County, OPUS-001 Shi You Shaanxi Province, screening obtains.(be called for short: CGMCC at this bacterial strain Yi China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101) preservation, preserving number is CGMCC No.7361, and preservation date is on March 22nd, 2013.
The physio-biochemical characteristics of subtilis OPUS-001 of the present invention and genetics characteristics are as follows:
(1) thalline feature: the μ m of individual cells (0.7-0.8) * (2.0-3.0), uniform coloring.Without pod membrane, peritrichous, can move.The μ m of gemma (0.6-0.9) * (1.0-1.5), oval to column, be positioned at thalline central authorities or slightly inclined to one side, after sporulation, thalline does not expand.While growing in liquid medium within, the normal wrinkle mould that forms.
(2) colony characteristics: bacterium colony surface irregularity is opaque, dirty white or micro-yellow.
(3) physico-chemical property: gram-positive microorganism, aerophil.Available protein, multiple sugar and starch, decompose tryptophane and form indoles.
(4) genetics characteristics: 16S rDNA analyzes, shows that it belongs to the similar but distinguishing monoid of bacillus subtilis Pseudomonas (Bacillus subtilis) and name as OPUS-001, the concrete visible sequence table of 16S rDNA sequence of bacterial strain OPUS-001.
The present invention also provides and utilizes described subtilis OPUS-001 to prepare the method for 'beta '-mannase, comprises the steps:
(1) subtilis OPUS-001 bacterial classification is inoculated on guar gum solid medium to 30-37 ℃, cultivation 1-2 days;
(2) solid medium list bacterium colony is connected in seed culture medium, 30-37 ℃, concussion cultivation 1-2 days, become seed liquor;
(3) seed liquor is accessed in fermention medium, 30-37 ℃, cultivation 2-5 days, centrifugal, obtain fermented liquid;
(4) fermented liquid, through separation and purification, obtains 'beta '-mannase;
Wherein said guar gum solid medium is: agar powder 20g/L, and guar gum 2-4g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, moisturizing is to 1L, pH 7.0-8.0; Described seed culture medium is: guar gum 2-4g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO4.7H
2o 0.2g/L, Pidolidone 5.0g/L, KCl 0.5g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, pH 7.0-8.0; Described fermention medium is: guar gum 2-4g/L, yeast soaks powder 5g/L, NaNO
35g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, pH 7.0-8.0.
In aforesaid method, the concussion speed described in step (2) is 150-200rpm/min.
In aforesaid method, the seed liquor described in step (3) and the volume ratio of fermention medium are preferably 1:20-50.
In aforesaid method, the described separation and purification of step (4) comprises the steps:
1. fermented liquid is centrifugal, get supernatant liquor elimination residue and obtain crude extract;
2. the saturated ammonium sulphate adding in crude extract, centrifugal after placing precipitation, get supernatant liquor; Supernatant liquor adds saturated ammonium sulphate, centrifugal after placing precipitation, obtains precipitation; Precipitation is dissolved in PBS, dialyses and obtains dialysing enzyme liquid;
3. the dialysis enzyme liquid of step (2), through Sepharose G-75 column chromatography for separation, obtains 'beta '-mannase elution peak component, and ultra-filtration membrane is concentrated, obtains aqueous 'beta '-mannase;
Further, the aqueous 'beta '-mannase that 3. step is obtained, through lyophilize, obtains powdery 'beta '-mannase.
Further, the saturated ammonium sulphate concentration of step described in is 2. 45-65%; The volume ratio of crude extract and saturated ammonium sulphate is 1:10, and the volume ratio of supernatant liquor and saturated ammonium sulphate is 0.5:1.
The present invention also provides the application of subtilis (Bacillus subtilis) OPUS-001 in the broken glue of middle low temperature well waterfrac treatment.Further, the described subtilis OPUS-001 broken glue alkali resistance 'beta '-mannase of preparation low temperature well waterfrac treatment that is used for fermenting.Its application direction also includes, but not limited to polymkeric substance de-plugging in petroleum industry field, containing poly-waste water and treatment of mud, crude oil quality-improving; And in fields such as food, medicine, papermaking, textile printing and dyeing, fodder industries.Middle low temperature of the present invention refers to 70 ℃ of following temperature, refers in particular to 10-70 ℃, more preferably 20-50 ℃.
Beta-mannase endonuclease capable efficient degradation guar gum and derivative thereof that method of the present invention obtains, its pH tolerance range 4.0-11.0, optimal reaction pH is 6.8-9.2, temperature tolerance range 10-70 ℃, optimum temperuture 30-50 ℃; Within the scope of said temperature and pH, enzyme activity can keep more than 85%.
OPUS-001 tunning of the present invention, and purified fermented liquid vigor component can prepare in the broken glue alkali resistance 'beta '-mannase of low temperature well waterfrac treatment, its application direction also comprises, but be not limited to the polymkeric substance de-plugging in petroleum industry field, containing poly-waste water and treatment of mud, crude oil quality-improving; And in fields such as food, medicine, papermaking, textile printing and dyeing, fodder industries.
Beneficial effect of the present invention:
Subtilis OPUS-001 of the present invention is the bacterial strain that a plant height produces 'beta '-mannase, its pH tolerance range of the 'beta '-mannase that produces wide, especially under alkaline condition, can also keep higher enzyme activity; Its temperature tolerance range is wide, especially at low temperature 10-20 ℃, also can maintain the enzyme activity that has been enough to the thorough aquation of frozen glue, is the temperature limitation that prior art fails to reach.
Strain culturing method of the present invention is simple, culture medium raw material is easy to get, strain enzyme-producing performance is strong, in tunning, 'beta '-mannase content is high, its product yield is higher than 3.2g/L, the high and alkaline-resisting salt tolerant of the alkaline ' beta '-mannase preparation vigor that produces, in oil field is had to efficient broken glue ability with guar gum or modified guar fracturing liquid under low interval temperature condition, and without incompatibility, can meet oil field compression fracture engineering demand with most chemical assistants in fracturing liquid.
Accompanying drawing explanation
Fig. 1 is the Sepharose G-75 column chromatography elution profile of the dialysis enzyme liquid that obtains of embodiment 3, and wherein peak 2 is middle low temperature 'beta '-mannase elution peak of the present invention.
Fig. 2 is the SDS-PAGE electrophorogram of low temperature liquid 'beta '-mannase in the present invention of obtaining of embodiment 3.
Fig. 3 is 'beta '-mannase thermal adaptability detected result of the present invention.
Fig. 4 is 'beta '-mannase pH adaptive detection result of the present invention.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.In addition, in following embodiment, if no special instructions, the experimental technique using is ordinary method, and material therefor, reagent etc. all can be bought from biological or chemical reagent company.
The substratum that following embodiment adopts and consist of as follows:
Guar gum solid medium: agar powder 20g/L, guar gum 2.5g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, moisturizing is to 1L;
Seed culture medium: guar gum 2.5g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, Pidolidone 5.0g/L, KC1 0.5g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, pH7.0-8.0;
Fermention medium: guar gum 2-4g/L, yeast soaks powder 5g/L, NaNO
35g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, 7.0-8.0.
Separation and the evaluation of embodiment 1 bacterial strain OPUS-001
1. the separation of bacterial strain OPUS-001
(1) sample collecting: from Hong Jiannao lakeside, Shenmu County, Shaanxi Province plant root soil;
(2) thermal treatment: pedotheque is made to suspension with aseptic deionized water, and thermal treatment is 10 minutes under 80 ℃ of conditions, kills thalline;
(3) enrichment culture: to adding nutrition source through in heat treated suspension, cultivate after 2 days under 37 ℃ of conditions, can obtain the spore-producing bacterium through enrichment;
(4) separation and purification: the microbial culture medium through enrichment culture is carried out to gradient dilution, and choosing extension rate is 10
-5, 10
-6with 10
-7three gradients on solid medium, carry out plate streaking, 37 ℃ of overnight incubation.Picking list bacterium colony is transferred in seed culture medium, and 37 ℃, concussion are cultivated 1 day, become seed liquor;
(5) seed liquor is accessed in fermention medium, 37 ℃, cultivate 3 days, choose the single strain that can utilize to greatest extent guar gum growth, called after bacterial strain OPUS-001.
2. the evaluation of bacterial strain OPUS-001
(1) thalline feature: the μ m of individual cells (0.7-0.8) * (2.0-3.0), uniform coloring.Without pod membrane, peritrichous, can move.The μ m of gemma (0.6-0.9) * (1.0-1.5), oval to column, be positioned at thalline central authorities or slightly inclined to one side, after sporulation, thalline does not expand.While growing in liquid medium within, the normal wrinkle mould that forms.
(2) colony characteristics: bacterium colony surface irregularity is opaque, dirty white or micro-yellow.
(3) physico-chemical property: gram-positive microorganism, aerophil.Available protein, multiple sugar and starch, decompose tryptophane and form indoles.
(4) 16S rDNA analyzes, and shows that it belongs to the similar but distinguishing monoid of bacillus subtilis Pseudomonas (Bacillus subtilis) and names as OPUS-001, is classified in subtilis (Bacillus subtilis).The center preservation of this bacterial strain Yi China Committee for Culture Collection of Microorganisms's common micro-organisms, its preserving number is CGMCC No.7361, preservation date: on March 22nd, 2013.
The fermentation of embodiment 2OPUS-001 bacterial strain
OPUS-001 bacterial classification is inoculated on guar gum solid medium, at 37 ℃, cultivates 2 days; Solid medium list bacterium colony is connected in the seed culture medium that fills 1/5-1/4 volume ratio, at 37 ℃, with 150rpm concussion, cultivates 1 day, become seed liquor; Seed liquor is added in fermention medium by 1/50 volume ratio, cultivate 3 days at 37 ℃, obtain fermented liquid.
The separation and purification of embodiment 3 'beta '-mannases
Carry out with the following method the 'beta '-mannase in separation and purification OPUS-001 bacterial strain fermentation liquor:
(1) by the fermented liquid of embodiment 1 at 3000rpm, 4 ℃ of centrifugal 15min, get supernatant, filter to obtain crude extract;
(2) in crude extract, add the saturated ammonium sulphate of the 45-65% of 10 times of amounts, solution is placed on magnetic stirring apparatus to 4 ℃ and spends the night; 20,000rpm, 4 ℃ of centrifugal 30min, retain supernatant liquor; Supernatant liquor adds saturated ammonium sulphate again to 0.5:1 (v/v), and recentrifuge is precipitated; Precipitation is dissolved in to PBS, with the dialysis of PBS solution, removes sulfate of ammoniac, the enzyme liquid of must dialysing;
(3) Sepharose G-75 column chromatography for separation for the dialysis enzyme liquid of step (2), after loading, with 0.1M NaCl aqueous solution wash-out, collect elutriant, every cut 1.5mL, collect altogether 30 cuts, through 280nm, detect, collect the elution peak component (as peak in Fig. 1 2) of 'beta '-mannase, with after PBS solution dialysis desalination, method with ultrafiltration and concentration, at 4 ℃, the centrifugal 10min of 15000rpm, obtains aqueous 'beta '-mannase;
(4) the liquid 'beta '-mannase that step (3) obtains, lyophilize, obtains freeze-drying 'beta '-mannase pulvis.
Fig. 2 is the SDS-PAGE electrophorogram of the liquid 'beta '-mannase that obtains of step (3), and molecular size range is about 38KD.
The mensuration of embodiment 4 beta-mannase enzyme activities
1. required reagent is surveyed in enzyme biopsy: 1) 0.38% melon sol solution, with acetic acid-sodium acetate buffer solution preparation of pH4.8-5.0; 2) 1% seminose standardized solution; 3) DNS reagent, gets Seignette salt 182g, is dissolved in 500mL distilled water and heats, in hot solution, add successively 3,5-dinitrosalicylic acid 6.3g, NaOH 21g, phenol 5g, S-WAT 5g, be stirred to moltenly, coolingly with distilled water, be settled to 1000mL afterwards, store in brown reagent bottle, under room temperature, place and use for 15 days later, use validity period one and a half months; 4) enzyme standard model, after getting liquid 'beta '-mannase after 2mL purifying and adding distilled water to be uniformly dissolved, is settled to 100mL; Freeze-drying freeze-drying 'beta '-mannase pulvis, with distilled water stepwise dilution, is controlled OD
280value is between 0.5-1.0;
2. draw respectively 1% seminose standardized solution 1.0,2.0,3.0,4.0,5.0,6.0mL, in 50mL volumetric flask, makes every mL respectively containing seminose 200,400,600,800 with distilled water, the reference liquid of 1000,1200 μ g.Respectively get different concns reference liquid 0.5mL in test tube, add acetic acid-sodium acetate buffer solution of 2mL pH4.8-5.0, add 2.5mL DNS reagent and boil 5 minutes.After cold, add water 5mL, shake up, 520nm place measures optical density(OD), with 0.5mL water, replaces reference liquid to make blank.The optical density value of gained of take is X-coordinate, with 1/2nd value of corresponding standard sweet dew sugar concentration (that is: 0.100,0.200,0.300; 0.400,0.500,0.600; this is the mg number of seminose in every pipe) be ordinate zou, drawing standard curve, and calculate regression equation;
3. in test tube A and B, add substrate solution 2mL, 50 ℃ of preheatings add 0.5mL enzyme liquid in A pipe after 5 minutes, 50 ℃ are accurately reacted 10 minutes, take out and respectively add 2.5mL DNS reagent, in B pipe, add 0.5mL enzyme liquid, boil immediately 5 minutes, after cold, respectively add water 5mL, shake up, 520nm place measures optical density(OD) (B pipe is blank), and finds corresponding reducing sugar content and be converted to unit of enzyme from typical curve;
4. enzyme is lived and is defined: under specified temp and pH (50 ℃, pH4.8-5.0), in 1min, decompose beta-mannase in melon glue and produce the required enzyme amount of reduction group that is equivalent to 1 μ mol seminose, be defined as 1 Ge Meihuo unit (real is international unit IU), with μ mol/min, represent;
5. enzyme activity calculates
By formula (1), calculate enzyme activity:
In formula,
X-enzyme activity (μ mol seminose/min.mL)
The relative seminose mg numerical value of OD value of K-find from mark liquid curve
The enzyme amount (0.5 * 1/n) of W-actual participation reaction
N-enzyme liquid dilution general times
180-be the molecular weight of seminose
10-reaction times (min)
10
3—1mmol=10
3×1μmol
The absolute difference of the twice independent measurement result obtaining under repeated condition should not surpass 3% of mean value.
6. the freeze-drying 'beta '-mannase pulvis vigor that after measured, prepared by freeze-drying is 5.5-6.5 * 10
5iU/g; The liquid 'beta '-mannase that purifying obtains finally adjusts to 1.0 * 10 by actual measured value
6iU/L left and right, for follow-up broken glue Performance Detection.
Embodiment 5 'beta '-mannases break glue Performance Detection
Fracturing fluid system formula (the low hot-well of North China branch office of China Petrochemical Industry adopts) comprising:
Base fluid formula: 0.28% hydroxypropylguar gum (HPG, one-level)+1%KCl+0.3%CX307(breakdown of emulsion cleanup additive)+0.5%AS-6(anti-clayswelling agent), use 0.05%Na
2cO
3(pH adjusting agent) adjusts system pH is 7.8 left and right; The per-cent of wherein said component refers to the final concentration of each component in base fluid.
Crosslinked fluid: 1% borax soln, crosslinked than being 100:4.
(1) preparation of base fluid: take 2.8g hydroxypropylguar gum pulvis, slowly join in 1000mL water while stirring, stir 15 minutes to after being uniformly dissolved, normal temperature swelling 4 hours, by final concentration requirement, add successively other each components in base fluid formula, with 0.05%Na
2cO
3adjust pH value to 7.5-8.0;
(2) the liquid 'beta '-mannase (starting point concentration 1.0 * 10 purifying of the present invention being obtained
6iU/L) with 100 times of tap water dilutions, be work enzyme liquid, work enzyme liquid should be now with the current.
Each laboratory sample is got base fluid prepared by 100mL step (1), in each sample, add respectively 0.10-0.30mL work enzyme liquid make base fluid in enzyme final concentration be that 10-30mg/L(is by starting point concentration), using and do not add the base fluid of enzyme liquid as blank, after stirring, add respectively 4mL1% linking agent borax solution (the crosslinked 100:4 that compares, v/v), rapid stirring 60 seconds, makes base fluid be cross-linked to the state of hanging.Each sample frozen glue is put into 200mL volumetric flask, be placed in 50 ℃ of waters bath with thermostatic control, observe Gel Breakdown state in 1-3h, measure breaking glue solution viscosity.Breaking glue solution viscosity Yong Pinshi viscometer determining, gets mean value three times.
After measured, the 'beta '-mannase liquid of 10-30mg/L can be in 45-90 minute by described fracturing fluid system reduced viscosity to 5mPa.s, and do not add the blank group of enzyme liquid still to keep high viscosity.
The thermal adaptability of embodiment 6 'beta '-mannases detects
According to the method for the step of embodiment 4 (1), configure respectively 0.35% guar gum base fluid and hydroxypropylguar gum base fluid, in base fluid, other components and content thereof are all identical with component and content thereof described in embodiment 4 base fluid formulas.Respectively get 100mL guar gum base fluid, establish experimental group 1, experimental group 2 and control group, every group of 13 samples.Experimental group 1 adds liquid 'beta '-mannase liquid, and final concentration is 10mg/L; The liquid 'beta '-mannase liquid that experimental group 2 adds, final concentration is 30mg/L; Control group does not add 'beta '-mannase liquid.Each sample, after stirring, adds 6mL borax linking agent (1% borax solution, the crosslinked 100:6 that compares, v/v) stir and be cross-linked to the state of hanging, put into constant temperature water bath, every group of sample is respectively 10,15,20,25,30,35,40,45,50,55, in 60,65,70 ℃, break glue experiment, observe the broken gluey condition after 2 hours, measure breaking glue solution viscosity, result is as Fig. 3.
The result of Fig. 3 shows, 'beta '-mannase of the present invention all can effectively reduce fracturing fluid gel viscosity under 10-70 ℃ of condition.Under 30mg/L consumption, frozen glue viscosity all can approach or be down to below 5mPa.s in 2 hours, its optimum temperuture is 30-50 ℃, more particularly in 10-20 ℃ of low-temperature range, also can maintain the enzyme activity that has been enough to the thorough aquation of frozen glue, break through the tolerable temperature limit of original 'beta '-mannase relevant report herein; 'beta '-mannase of the present invention, under 10mg/L consumption, also showing preferably broken glue ability is 35 ℃ in its optimum temperuture.Control group all keeps high fracturing fluid gel viscosity, and frozen glue is for hanging viscosity state.
'beta '-mannase of the present invention all can effectively reduce fracturing fluid gel viscosity under 10-70 ℃ of condition, and its suitable concentration scope is 10-30mg/L.'beta '-mannase of the present invention, under differing temps environment, by its addition of suitable adjusting or action time, can be realized thoroughly broken glue of gelled fracturing fluid, guarantees Oil Field Measure transformation requirement.
Hydroxypropylguar gum base fluid is that experimental group is carried out above-mentioned broken glue experiment, experimental result shows that 'beta '-mannase of the present invention all can effectively reduce fracturing fluid gel viscosity under 10-70 ℃ of condition, in the concentration range of 10-30mg/L, frozen glue viscosity all can approach or be down to below 5mPa.s in 2 hours.
Further experimental result shows, 'beta '-mannase of the present invention, and the fracturing liquid to the guar gum that contains 2.0-4.5g/L or hydroxypropylguar gum base fluid, all can effectively reduce frozen glue viscosity.
The pH adaptive detection of embodiment 7 'beta '-mannases
According to the method for the step of embodiment 4 (1), configure respectively 0.28% guar gum base fluid and hydroxypropylguar gum, in base fluid, other components and content thereof are all identical with component and content thereof described in embodiment 4 base fluid formulas.Respectively get 100mL guar gum base fluid, establish experimental group 1, experimental group 2 and control group, every group of 6 samples.Every group of sample is adjusted to respectively after different pH6.0,7.0,8.0,9.0,10.0,11.0, and experimental group 1 adds liquid 'beta '-mannase liquid, and final concentration is 10mg/L; The liquid 'beta '-mannase liquid that experimental group 2 adds, final concentration is 30mg/L; Control group does not add 'beta '-mannase liquid.Each sample, after stirring, adds 4mL linking agent (1% borax solution, the crosslinked 100:4 that compares, v/v), stir and be cross-linked to the state of hanging, put into 50 ℃ of constant temperature water baths and break glue experiment, observe the broken gluey condition after 2 hours, measure breaking glue solution viscosity, result is as Fig. 4.
Fig. 4 result shows, 'beta '-mannase of the present invention all has broken glue ability within the scope of pH6.0-11.0; When pH7.0-9.0, the broken glue ability of enzyme is the strongest.By continuation, adjust the consumption of enzyme, in 30mg/L consumption, can further guarantee that each experimental group frozen glue viscosity all can approach or be down to below 5mPa.s in 2 hours.
'beta '-mannase of the present invention, at the general applicability of wider pH range, makes it to meet the formula requirement of all kinds of fracturing fluid systems in oil field, has reduced to greatest extent the vigor bringing because of incompatibility and has suppressed.
Hydroxypropylguar gum base fluid is that experimental group is carried out above-mentioned broken glue experiment, experimental result shows that 'beta '-mannase of the present invention all has preferably broken glue ability within the scope of pH6.0-11.0, in the concentration range of 10-30mg/L, when pH7.0-9.0, frozen glue viscosity all can approach or be down to below 5mPa.s in 2 hours.
Further experimental result shows, 'beta '-mannase of the present invention, and the fracturing liquid to the guar gum that contains 2.5-4.5g/L or hydroxypropylguar gum, all can effectively reduce frozen glue viscosity.
The breaking glue solution level of residue of embodiment 8 'beta '-mannases is measured
(1) according to shown in table 1, by the 'beta '-mannase of the present invention of different concns, act on respectively with 1% borax solution (the crosslinked 100:6 that compares, v/v) crosslinked 3.8% guar gum or carboxymethyl guar gum base fluid, 200mg/L APS in contrast, being placed in 50 ℃ of Constant Temp. Ovens heats 4 hours, make the completely broken glue of base fluid, breaking glue solution is mixed, accurately measure 50mL and be transferred to prior load weighted centrifuge tube, with after the centrifugal 20min of rotating speed of 1800rpm, be placed in Constant Temp. Oven and be dried to after constant weight, weigh, and calculate residue content.Result is as table 1.
Table 1 result shows, the 'beta '-mannase group (10 that adds different concns, 20,30,40,50mg/L), breaking glue solution level of residue is all less than 400mg/L, lower than the prescribed value (500mg/L) in gel breaker method for testing performance > > for industry standard SY/T6380-2008 < < pressure break; APS group is higher than 600mg/L;
(2) breaking glue solution of step (1) filters, from filtration situation, add the control group filtration velocity (12h can not filter) of ammonium persulphate (APS) well below the breaking glue solution filtration velocity (filtration velocity < 1h) that adds 'beta '-mannase of the present invention, illustrate that beta-mannase endonuclease capable of the present invention effectively reduces of the fracturing fluid viscous force; Observe residue state in filtrate, only add residue in the control group of APS and be state of aggregation, can not show a candle to the residue adding after 'beta '-mannase effect of the present invention disperses, also show the high molecular polymer that beta-mannase endonuclease capable of the present invention is thoroughly degraded in fracturing liquid, thereby be conducive to fracturing fluid residue, return row.
After the broken glue of table 1. 'beta '-mannase of the present invention and ammonium persulphate, level of residue is compared
The breaking glue solution core damage experiment of embodiment 9 'beta '-mannases
Select the artificial rock core of different rate of permeation to carry out the experiment of thing mould, under 50 ℃ of constant temperature, displacement simulation local water also calculates the initial rate of permeation K of rock core
0; With fracturing liquid (fracturing liquid described in embodiment 4, wherein base fluid pH is 8.0) saturated core, broken glue enzyme group adds the 'beta '-mannase of the present invention of final concentration 20mg/L, and control group adds the APS of final concentration 300mg/L, closure systems thermostatic effect 3h;
1. reaction is carried out reverse displacement until constant pressure calculates the rate of permeation K that respectively organizes rock core after the row of returning with simulated formation water after finishing
1;
2. core damage rate is calculated, injury rate (λ; %) numerical computational formulas: λ=(K
0-K
1)/K
0; Result is as table 2;
3. table 2 result shows, broken glue enzyme group is significantly less than APS group to the injury rate of rock core; Point out 'beta '-mannase of the present invention can effectively avoid vegetable jelly residue to be detained stratum, form filter cake and stop up hole.
Table 2. 'beta '-mannase of the present invention and ammonium persulphate core damage rate comparison result
Claims (9)
1. subtilis (Bacillus subtilis) OPUS-001 who produces 'beta '-mannase, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCCNo.7361.
2. the application of subtilis OPUS-001 as claimed in claim 1 in the broken glue of middle low temperature well waterfrac treatment.
3. according to the application of claim 2, it is characterized in that the described subtilis OPUS-001 broken glue 'beta '-mannase of preparation low temperature well waterfrac treatment that is used for fermenting.
4. a production method for 'beta '-mannase, is characterized in that, comprises the steps:
(1) subtilis OPUS-001 bacterial classification is inoculated on guar gum solid medium to 30-37 ℃, cultivation 1-2 days;
(2) solid medium list bacterium colony is connected in seed culture medium, 30-37 ℃, concussion cultivation 1-2 days, become seed liquor;
(3) seed liquor is accessed in fermention medium, 30-37 ℃, cultivation 2-5 days, centrifugal, obtain fermented liquid;
(4) fermented liquid, through separation and purification, obtains 'beta '-mannase;
Wherein said guar gum solid medium is: agar powder 20g/L, and guar gum 2-4g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, moisturizing is to 1L, pH7.0-8.0; Described seed culture medium is: guar gum 2-4g/L, yeast soaks powder 5g/L, K
2hPO
45g/L, MgSO4.7H
2o 0.2g/L, Pidolidone 5.0g/L, KCl 0.5g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, pH7.0-8.0; Described fermention medium is: guar gum 2-4g/L, yeast soaks powder 5g/L, NaNO
35g/L, K
2hPO
45g/L, MgSO
47H
2o 0.2g/L, MnSO
45 * 10
-5g/L, FeSO
47H
2o 1.5 * 10
-6g/L, CuSO
45H
2o 1.6 * 10
-8g/L, moisturizing is to 1L, pH7.0-8.0.
5. production method according to claim 4, is characterized in that, described in step (2), shaking speed is 150-200rpm/min.
6. production method according to claim 4, is characterized in that, in step (3), the volume ratio of seed liquor and fermention medium is 1:20-50.
7. production method according to claim 4, is characterized in that, the described separation and purification of step (4) comprises the steps:
1. fermented liquid is centrifugal, get supernatant liquor elimination residue and obtain crude extract;
2. the saturated ammonium sulphate adding in crude extract, centrifugal after placing precipitation, get supernatant liquor; Supernatant liquor adds saturated ammonium sulphate, centrifugal after placing precipitation, obtains precipitation; Precipitation is dissolved in PBS, dialyses and obtains dialysing enzyme liquid;
3. the dialysis enzyme liquid of step (2), through Sepharose G-75 column chromatography for separation, obtains 'beta '-mannase elution peak component, and ultra-filtration membrane is concentrated, obtains aqueous 'beta '-mannase.
8. production method according to claim 7, is characterized in that, the aqueous 'beta '-mannase lyophilize 3. obtaining in step, obtains powdery 'beta '-mannase.
9. production method according to claim 7, is characterized in that, the saturated ammonium sulphate concentration of step described in is 2. 45-65%; The volume ratio of crude extract and saturated ammonium sulphate is 1:10, and the volume ratio of supernatant liquor and saturated ammonium sulphate is 0.5:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310539350.0A CN103555629B (en) | 2013-11-04 | 2013-11-04 | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310539350.0A CN103555629B (en) | 2013-11-04 | 2013-11-04 | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103555629A true CN103555629A (en) | 2014-02-05 |
| CN103555629B CN103555629B (en) | 2015-11-11 |
Family
ID=50009995
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310539350.0A Active CN103555629B (en) | 2013-11-04 | 2013-11-04 | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103555629B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152428A (en) * | 2014-08-12 | 2014-11-19 | 云南师范大学 | Mannase ManAGN25 with low temperature activity and gene thereof |
| CN105621630A (en) * | 2016-01-18 | 2016-06-01 | 黑龙江大学 | Beta-mannase for decoloration and application of beta-mannase |
| CN108504694A (en) * | 2018-03-28 | 2018-09-07 | 大连知微生物科技有限公司 | A kind of superhigh temperature biology base gel breaker, its environment-friendly preparation method thereof and application |
| CN111334493A (en) * | 2020-04-02 | 2020-06-26 | 南京工业大学 | Medium-low temperature endo β -mannase, and coding gene and application thereof |
| CN113528115A (en) * | 2021-08-06 | 2021-10-22 | 陕西森瑞石油技术开发有限公司 | Low-temperature guar gel breaker for oil-gas well fracturing, preparation method and application |
| CN114621891A (en) * | 2021-12-10 | 2022-06-14 | 华东理工大学 | Pseudomonas strain for producing beta-mannase and preparation method of low-temperature gel breaking enzyme |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999064619A2 (en) * | 1998-06-10 | 1999-12-16 | Novozymes A/S | Novel mannanases |
| CN1266096A (en) * | 1999-03-05 | 2000-09-13 | 中国科学院微生物研究所 | Method for producing alkaline 'beta'-mannase |
| CN101838620A (en) * | 2009-03-17 | 2010-09-22 | 大连百奥泰科技有限公司 | Bacillus subtilis and alkali-resisting and salt-resisting oil field fracturing enzyme and application thereof |
| CN101838528A (en) * | 2009-03-17 | 2010-09-22 | 大连百奥泰科技有限公司 | Environment-friendly and bio-available green fracturing fluid system |
| CN101880523A (en) * | 2010-07-02 | 2010-11-10 | 大连百奥泰科技有限公司 | Enzyme-microbe coupled fracturing fluid system and preparation method and applications thereof |
| CN102154247A (en) * | 2011-02-15 | 2011-08-17 | 华东理工大学 | Method for preparing beta-mannanase by using natural Bacillus subtilis |
-
2013
- 2013-11-04 CN CN201310539350.0A patent/CN103555629B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999064619A2 (en) * | 1998-06-10 | 1999-12-16 | Novozymes A/S | Novel mannanases |
| CN1266096A (en) * | 1999-03-05 | 2000-09-13 | 中国科学院微生物研究所 | Method for producing alkaline 'beta'-mannase |
| CN101838620A (en) * | 2009-03-17 | 2010-09-22 | 大连百奥泰科技有限公司 | Bacillus subtilis and alkali-resisting and salt-resisting oil field fracturing enzyme and application thereof |
| CN101838528A (en) * | 2009-03-17 | 2010-09-22 | 大连百奥泰科技有限公司 | Environment-friendly and bio-available green fracturing fluid system |
| CN101880523A (en) * | 2010-07-02 | 2010-11-10 | 大连百奥泰科技有限公司 | Enzyme-microbe coupled fracturing fluid system and preparation method and applications thereof |
| CN102154247A (en) * | 2011-02-15 | 2011-08-17 | 华东理工大学 | Method for preparing beta-mannanase by using natural Bacillus subtilis |
Non-Patent Citations (3)
| Title |
|---|
| 李希明 等: "生物破胶酶研究及应用", 《石油钻采工艺》, vol. 28, no. 2, 30 April 2006 (2006-04-30), pages 52 - 55 * |
| 柴萍萍: "枯草芽孢杆菌（Bacillus subtilis）WY45产β-甘露聚糖酶的纯化与性质研究", 《中国优秀硕士学位论文全文数据库·工程科技I辑》, 15 May 2005 (2005-05-15) * |
| 韦赟 等: "枯草芽孢杆菌产β-甘露聚糖酶固体发酵条件的优化*", 《微生物学通报》, 31 December 2006 (2006-12-31), pages 84 - 89 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152428A (en) * | 2014-08-12 | 2014-11-19 | 云南师范大学 | Mannase ManAGN25 with low temperature activity and gene thereof |
| CN105621630A (en) * | 2016-01-18 | 2016-06-01 | 黑龙江大学 | Beta-mannase for decoloration and application of beta-mannase |
| CN108504694A (en) * | 2018-03-28 | 2018-09-07 | 大连知微生物科技有限公司 | A kind of superhigh temperature biology base gel breaker, its environment-friendly preparation method thereof and application |
| CN111334493A (en) * | 2020-04-02 | 2020-06-26 | 南京工业大学 | Medium-low temperature endo β -mannase, and coding gene and application thereof |
| CN113528115A (en) * | 2021-08-06 | 2021-10-22 | 陕西森瑞石油技术开发有限公司 | Low-temperature guar gel breaker for oil-gas well fracturing, preparation method and application |
| CN114621891A (en) * | 2021-12-10 | 2022-06-14 | 华东理工大学 | Pseudomonas strain for producing beta-mannase and preparation method of low-temperature gel breaking enzyme |
| CN114621891B (en) * | 2021-12-10 | 2023-08-04 | 华东理工大学 | Pseudomonas strain for producing beta-mannase and preparation method of low-temperature gel breaking enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103555629B (en) | 2015-11-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103555629B (en) | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase | |
| Castellane et al. | Production of exopolysaccharide from rhizobia with potential biotechnological and bioremediation applications | |
| CN101619300B (en) | Sphingomonas sp. TP-5 , method and application thereof for producing welan gum | |
| CN104109646B (en) | Slime reducing agent suitable for heavy oil wells with different mineralization and application | |
| Ma et al. | Application of guar gum degrading bacteria in microbial remediation of guar-based fracturing fluid damage | |
| CN104373094B (en) | Low-permeability reservoir microbial oil recovery composite preparation and use method thereof | |
| CN101203615A (en) | Bacterial cellulose-containing formulations and method of producing effective bacterial cellulose-containing formulations | |
| EP0073599B1 (en) | Polyvalent metal ion chelating agents for xanthan solutions | |
| CA2104560C (en) | Injection of dextrins for subterranean microbial processes | |
| CN110170516B (en) | Heavy metal contaminated soil remediation agent | |
| CN101864284A (en) | Elm bark powder particles, preparation method for elm bark powder particles, elm bark powder vegetable gum and application of elm bark powder vegetable gum | |
| Aboulrous et al. | Application of natural polymers in engineering | |
| CN109679871A (en) | A kind of method of PAM-SA immobilized microorganism degradation oily waste water | |
| AU2017308633A1 (en) | Compositions comprising and methods of making bio-polymers | |
| US7824569B2 (en) | Soluble salt produced from a biopolymer and a process for producing the salt | |
| CN113999660B (en) | Plugging material, plugging agent and plugging method | |
| CN103232966B (en) | Middle and high temperature resisting exopoly water insoluble polysaccharide protoplast fusion engineering bacterium and application thereof | |
| Abdurrahman et al. | A short review of biopolymers for enhanced of oil recovery in mature fields | |
| Ha et al. | Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains | |
| Bajestani et al. | Biosynthesis and physicochemical characterization of a bacterial polysaccharide/polyamide blend, applied for microfluidics study in porous media | |
| CN103614127A (en) | Microorganism and lipopeptide combined low-temperature oil reservoir oil extraction and paraffin removal and inhibition technology | |
| CN108504694A (en) | A kind of superhigh temperature biology base gel breaker, its environment-friendly preparation method thereof and application | |
| CN104694450B (en) | The thermophilic ground bacillus cereus of generation emulsifying agent and its application in high solidifying oil exploitation | |
| CN104585242A (en) | Artificial wetland plant growth promoting agent | |
| CN103087959B (en) | Preparation for producing instant xanthan gum and use method of preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: FU NA Effective date: 20150819 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20150819 Address after: 116000 No. 78 industrious street, Zhongshan District, Liaoning, Dalian 2-101 Applicant after: Zhao Jing Applicant after: Fu Na Address before: 116000 No. 78 industrious street, Zhongshan District, Liaoning, Dalian 2-101 Applicant before: Zhao Jing |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhao Jing Inventor after: Fu Na Inventor before: Zhao Jing |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20170616 Address after: Dalian high tech Industrial Park, 116000 Liaoning province Lixian Street 32 floor of building B room 103 Patentee after: Dalian know Microorganism Technology Co., Ltd. Address before: 116000 No. 78 industrious street, Zhongshan District, Liaoning, Dalian 2-101 Co-patentee before: Fu Na Patentee before: Zhao Jing |
|
| TR01 | Transfer of patent right |