WO2019179303A1 - Procédé de préparation et application d'une enzyme composite de dégradation de pétrole présent dans des boues huileuses - Google Patents

Procédé de préparation et application d'une enzyme composite de dégradation de pétrole présent dans des boues huileuses Download PDF

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Publication number
WO2019179303A1
WO2019179303A1 PCT/CN2019/076792 CN2019076792W WO2019179303A1 WO 2019179303 A1 WO2019179303 A1 WO 2019179303A1 CN 2019076792 W CN2019076792 W CN 2019076792W WO 2019179303 A1 WO2019179303 A1 WO 2019179303A1
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Prior art keywords
sludge
petroleum
fdh
follows
preparation
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PCT/CN2019/076792
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English (en)
Chinese (zh)
Inventor
张强
季蕾
王加宁
傅晓文
李天元
郭书海
陈贯虹
李琪
邢颖娜
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山东省科学院生态研究所
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Priority to KR1020197022007A priority Critical patent/KR20190111947A/ko
Priority to AU2019208247A priority patent/AU2019208247B2/en
Publication of WO2019179303A1 publication Critical patent/WO2019179303A1/fr

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    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/02—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with a cytochrome as acceptor (1.2.2)
    • C12Y102/02001—Formate dehydrogenase (cytochrome) (1.2.2.1)

Definitions

  • the invention relates to a preparation method and application of a sludge petroleum degradation composite enzyme, and belongs to the technical field of soil treatment.
  • PAHs polycyclic aromatic hydrocarbons
  • Sludge treatment is the most difficult. Sludge is produced in the upstream exploration and development of oil, oil and gas gathering, sewage treatment, tank bottom cleaning and downstream petroleum refining process. Due to process equipment and human operation, a large amount of oil, mud and water containing petroleum hydrocarbons will be produced. The oily sludge produced by the mixture leaking into the environment. The oily sludge in the oilfield is a large and wide-ranging pollution source with high oil content and high heavy oil composition.
  • the oil-degrading enzyme preparation is obtained by separation and drying;
  • the petroleum-degrading microorganism is Acinetobacter aceti, the species preservation number: CGMCC No. 3915, and the depository: China Microbial Culture Collection Management Committee Microbiology center.
  • the invention degrades the oil-contaminated soil by fixing the enzyme system of the microorganism having the function of degrading petroleum through the adsorbent, and the degradation efficiency is remarkably improved, the microbial degradation rate is increased by 30 to 50 times, and the stability is improved by the crude enzyme solution. 15 to 20 times.
  • the invention aims at the deficiencies of the prior art and provides a preparation method and application of a sludge petroleum degradation composite enzyme.
  • a method for preparing a sludge oil degradation composite enzyme the steps are as follows:
  • Acinetobacter calcoaceticus cells are disrupted, centrifuged, and the supernatant is taken to obtain a petroleum degrading enzyme solution 21 # ;
  • the strain storage number of Acinetobacter calcoaceticus is CGMCC No. 3915.
  • This bacterium is a known strain and does not involve the preservation of strains.
  • the Acinetobacter calcoaceticus cells are cultured as follows:
  • Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) is transferred to LB medium at a mass ratio of 1% to 2%, and seed cultured for 14 to 16 hours at 28 to 32 ° C and 150 to 180 rpm. Seed liquid
  • step b Transfer the seed liquid prepared in step a to the LB medium at a mass ratio of 4% to 5%, and expand the culture for 14 to 16 hours at 28 to 32 ° C and 150 to 180 rpm to prepare the bacterial liquid. ;
  • the bacterial liquid prepared in the step b is centrifuged to collect the precipitate, and the Acinetobacter calcoaceticus cell body is obtained.
  • the cells in the step (1) are broken, and the steps are as follows:
  • Acinetobacter calcoaceticus cells are mixed uniformly with a pH 7.5 phosphate buffer at a mass to volume ratio of 1:15, in units of g/ml; under 320W ultrasonic conditions, intermittent ultrasound is used.
  • the treatment method was carried out for cell disruption for 17 min, each ultrasonic disruption time was 2 s, and the intermittent time was 2 s.
  • the centrifugation condition is: centrifugation at 5000 r/min for 2 min.
  • the formate dehydrogenase is a formate dehydrogenase CbFDH, and the amino acid sequence is shown in SEQ ID NO.
  • the preparation steps of the formate dehydrogenase CbFDH are as follows:
  • step (ii) transferring the genetically engineered strain E. coli BL21-fdh constructed in step (i) to a seed medium at a seeding rate of 1% to 2% by mass, at 28 to 32 ° C, 150 to 180 rpm.
  • the seed is cultured for 10 to 12 hours to prepare an Escherichia coli seed liquid;
  • step (iii) transferring the Escherichia coli seed liquid of step (ii) to a fermentation medium at a seeding rate of 4% to 5% by mass, and culturing for 16 to 18 hours at 28 to 32 ° C and 150 to 180 rpm to collect genetic engineering.
  • the strain Escherichia coli BL21-fdh was disrupted by cells, centrifuged, and the supernatant was collected to prepare formate dehydrogenase CbFDH.
  • the genetic engineering strain E. coli BL21-fdh is constructed as follows:
  • the formate dehydrogenase gene fdh derived from Candida boidinii was amplified and ligated into the E. coli expression vector pET28a(+) to construct a recombinant expression vector pET28a(+)-fdh carrying the fdh gene.
  • the host strain Escherichia coli BL21 (DE3) was picked and transformed, and the recombinant Escherichia coli BL21-fdh expressing formate dehydrogenase was obtained by screening.
  • the seed medium component is as follows:
  • Peptone 10 g / L peptone 10 g / L
  • yeast extract 5 g / L yeast extract 5 g / L
  • NaCl 10 g / L ampicillin (ampicillin) 100 ⁇ g / mL.
  • the fermentation medium components are as follows:
  • Peptone 10g / L yeast extract 5g / L, Na 2 HPO 4 ⁇ 12H 2 O 9g / L, KH 2 PO 4 6.8g / L, (NH 4 ) 2 SO 4 3.3g / L, glucose 0.5g / L , lactose 2g / L, MgSO 4 ⁇ 7H 2 O 0.5g / L, CaCl 2 0.02g / L, glycerol 0.5% by volume.
  • step (iii) the cell is broken, and the steps are as follows:
  • the genetically engineered strain Escherichia coli BL21-fdh was mixed uniformly with a pH 7.5 phosphate buffer at a mass to volume ratio of 1:15 to 25 g/ml; under 195 W ultrasonic conditions, The cells were disrupted by intermittent ultrasonic treatment for 6 min, each ultrasonic disruption time was 3 s, and the intermittent time was 5 s.
  • the centrifugation condition is: centrifugation at 3000 r/min for 2 min.
  • the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase is 1:4.
  • the mass ratio of protein to sodium formate is 1:5.
  • the sodium formate concentration in the sludge oil degradation complex enzyme treatment solution is 167 mmol/L.
  • the sludge and the sludge petroleum degradation composite enzyme treatment liquid are mixed at a mass to volume ratio of 1:20.
  • the present invention firstly mixes the petroleum-degrading enzyme system derived from Acinetobacter calcoaceticus with formate dehydrogenase, and finds that it can be used for treating petroleum degradation and repair in high-concentration petroleum-contaminated sludge.
  • the adsorbent such as diatomaceous earth
  • the oil in the sludge can be reduced in a short time, has an efficient oil pollution treatment capability, and has low production cost and has broad application prospects;
  • formate dehydrogenase is formate dehydrogenase CbFDH (amino acid sequence is SEQ ID NO. 1)
  • the oil-degrading enzyme system will further improve the repair efficiency of high-concentration petroleum-contaminated sludge, and the inventor is surprised. It was found that when the recombinant bacteria were cultured in a specific medium to obtain the formate dehydrogenase CbFDH, the stability of the formate dehydrogenase CbFDH was significantly improved.
  • Acinetobacter calcoaceticus was purchased from the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the strain collection number was CGMCC No.3915;
  • Candida boidinii is purchased from the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the strain collection number is CGMCC 2.2378;
  • Plasmid pET28a(+) was purchased from Shandong Vaughan Biotechnology Co., Ltd.;
  • E. coli BL21 (DE3) is a commercially available product.
  • Acinetobacter calcoaceticus cells were cultured as follows:
  • Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) was transferred to LB medium at a mass ratio of 2%, and the seeds were cultured at 30 ° C, 160 rpm for 15 h to prepare a seed liquid;
  • the seed liquid prepared in the step a is transferred to the LB medium at a mass ratio of 4% to 5%, and the culture liquid is expanded at 30 ° C and 160 rpm for 15 hours to prepare a bacterial liquid;
  • the bacterial liquid prepared in the step b is centrifuged to collect the precipitate, and the Acinetobacter calcoaceticus cell body is obtained.
  • a formate dehydrogenase gene fdh derived from Candida boidinii is obtained by amplification, the gene sequence is shown in SEQ ID NO. 2, and the upstream amplification primer sequence is shown in SEQ ID NO.
  • the increased formate dehydrogenase gene fdh was ligated into the E. coli expression vector pET28a(+), and the recombinant expression vector pET28a(+)-fdh carrying the fdh gene was constructed, and the host strain Escherichia coli BL21(DE3) was transformed and the transformant was picked. , the recombinant Escherichia coli BL21-fdh expressing formic acid dehydrogenase was screened; for specific step conditions, refer to the instruction manual of the E. coli expression vector pET28a (+);
  • the seed medium components are as follows:
  • Peptone 10 g / L peptone 10 g / L
  • yeast extract 5 g / L yeast extract 5 g / L
  • NaCl 10 g / L ampicillin (ampicillin) 100 ⁇ g / mL.
  • step (iii) Transfer the E. coli seed solution of step (ii) to the fermentation medium at a 4% by mass inoculum, and incubate at 30 ° C, 160 rpm for 16 h, and collect the genetically engineered strain E. coli BL21-fdh.
  • the cells were disrupted by centrifugation at 3000 r/min for 2 min, and the supernatant was collected to obtain a formate dehydrogenase CbFDH; the amino acid sequence was determined as shown in SEQ ID NO.
  • the fermentation medium components are as follows:
  • Peptone 10g / L yeast extract 5g / L, Na 2 HPO 4 ⁇ 12H 2 O 9g / L, KH 2 PO 4 6.8g / L, (NH 4 ) 2 SO 4 3.3g / L, glucose 0.5g / L , lactose 2g / L, MgSO 4 ⁇ 7H 2 O 0.5g / L, CaCl 2 0.02g / L, glycerol 0.5% by volume.
  • the genetically engineered strain E. coli BL21-fdh was mixed with phosphate buffer at pH 7.5 in a ratio of mass ratio of 1:20, unit g/ml; under ultrasonic conditions of 195 W, intermittent ultrasonic treatment was used. Cell disruption was carried out for 6 min, each ultrasonic disruption time was 3 s, and the intermittent time was 5 s.
  • a method for preparing a sludge oil degradation composite enzyme the steps are as follows:
  • Example 1 The Acinetobacter calcoaceticus cells prepared in Example 1 were disrupted by centrifugation at 5000 r/min for 2 min, and the supernatant was taken to obtain a petroleum degrading enzyme solution 21 # ;
  • Acinetobacter calcoaceticus cells in a ratio of mass ratio of 1:20 to phosphate buffer pH 7.5, unit g/ml; under ultrasonic conditions of 320 W, use intermittent ultrasonic treatment to perform cells Broken for 17min, each ultrasonic breaking time 2s, intermittent time 2s;
  • IPTG solution was added to a final concentration of 0.5 mmol / L for induction.
  • the method for preparing the sludge petroleum degradation composite enzyme according to the third embodiment is different in that the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase protein is 1:3.
  • the preparation method of the sludge petroleum degradation composite enzyme as described in Example 3 is that the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase protein is 1:5.
  • the preparation method of the sludge petroleum degradation composite enzyme as described in Example 3 is that the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase protein is 2:3.
  • the preparation method of the sludge petroleum degradation composite enzyme as described in Example 3 is that the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase protein is 1:1.
  • the preparation method of the sludge petroleum degradation composite enzyme as described in Example 3 is that the mass ratio of the petroleum degrading enzyme solution 21 # to the formate dehydrogenase protein is 2:1.
  • the enzyme preparation was prepared as described in Example 3 of Chinese Patent Document CN103484447A (Application No. 201310456751.X).
  • the experimental group 1-8 was reacted with CK group at 30 ° C and 150 rpm for 12 h, and the reaction was terminated by adding dichloromethane.
  • the oil degradation rate of the sludge was determined by gravimetric method. The specific steps are as follows:
  • Degradation rate (%) (control group oil weight - enzymatic hydrolysis test group oil weight) ⁇ 100% / control group oil weight
  • Experimental group number Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Experimental group 5 Experimental group 6 Experimental group 7 Experimental group 8 CK group Petroleum degradation rate (%) 35.1 35.6 32.0 39.7 29.9 26.6 10.3 10.7 -
  • Experimental group number Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Experimental group 5 Experimental group 6 Experimental group 7 Experimental group 8 CK group Petroleum degradation rate (%) 35.3 - 31.7 42.1 28.6 25.9 8.3 6.5 -

Abstract

L'invention concerne un procédé de préparation d'une enzyme composite de dégradation de pétrole présent dans des boues huileuses, comprenant les étapes suivantes : (1) la réalisation d'une rupture de cellules sur un corps bactérien d'Acinetobacter calcoaceticus, la centrifugation et l'extraction d'un surnageant pour obtenir une solution d'enzyme de dégradation de pétrole 21#; et (2) le mélange de la solution d'enzyme de dégradation de pétrole 21# avec de la formiate déshydrogénase pour obtenir une enzyme composite de dégradation de pétrole présent dans des boues huileuses. L'invention concerne en outre une enzyme composite de dégradation de pétrole présent dans des boues huileuses, obtenue à l'aide du procédé de préparation selon l'invention et une application de celle-ci en récupération de boues huileuses contaminées par du pétrole.
PCT/CN2019/076792 2018-03-22 2019-03-04 Procédé de préparation et application d'une enzyme composite de dégradation de pétrole présent dans des boues huileuses WO2019179303A1 (fr)

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KR1020197022007A KR20190111947A (ko) 2018-03-22 2019-03-04 슬러지 석유 분해 복합 효소의 제조 방법 및 응용
AU2019208247A AU2019208247B2 (en) 2018-03-22 2019-03-04 Preparation method and application of sludge petroleum degrading complex enzyme

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CN201810237841.2 2018-03-22
CN201810237841.2A CN108486006B (zh) 2018-03-22 2018-03-22 一种油泥石油降解复合酶的制备方法与应用

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CN110564635B (zh) * 2019-03-01 2020-05-12 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) 一株高效降解石油的雷氏普罗维登斯菌l1与应用
CN110724650B (zh) * 2019-10-21 2021-11-02 天津大学 一种石油降解菌tdyn1t及其应用
CN111454935A (zh) * 2020-04-25 2020-07-28 北京博泰至淳生物科技有限公司 一种用于污水脱氮的固定化酶及其制备方法和应用
CN112795525A (zh) * 2021-03-25 2021-05-14 辽宁大学 一种乙酸钙不动杆菌培养基及其在降解双酚a的中的应用

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