CN103055464B - A kind of method utilizing laccase degraded octyl phenol - Google Patents
A kind of method utilizing laccase degraded octyl phenol Download PDFInfo
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- CN103055464B CN103055464B CN201210539590.6A CN201210539590A CN103055464B CN 103055464 B CN103055464 B CN 103055464B CN 201210539590 A CN201210539590 A CN 201210539590A CN 103055464 B CN103055464 B CN 103055464B
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 82
- DUIOKRXOKLLURE-UHFFFAOYSA-N 2-octylphenol Chemical compound CCCCCCCCC1=CC=CC=C1O DUIOKRXOKLLURE-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 48
- 102000004190 Enzymes Human genes 0.000 claims abstract description 48
- 239000007788 liquid Substances 0.000 claims abstract description 36
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- 230000015556 catabolic process Effects 0.000 claims abstract description 24
- 238000006731 degradation reaction Methods 0.000 claims abstract description 24
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 241000222355 Trametes versicolor Species 0.000 claims description 33
- 241000894006 Bacteria Species 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000000376 reactant Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 239000007979 citrate buffer Substances 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
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- 238000004255 ion exchange chromatography Methods 0.000 claims description 6
- 229920002477 rna polymer Polymers 0.000 claims description 6
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 claims description 5
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- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- NGPGDYLVALNKEG-UHFFFAOYSA-N azanium;azane;2,3,4-trihydroxy-4-oxobutanoate Chemical compound [NH4+].[NH4+].[O-]C(=O)C(O)C(O)C([O-])=O NGPGDYLVALNKEG-UHFFFAOYSA-N 0.000 claims description 3
- 238000010804 cDNA synthesis Methods 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- QKUSRAKPUWQSJS-UHFFFAOYSA-N diazanium 3-ethyl-2H-1,3-benzothiazole-6-sulfonate Chemical class [NH4+].[NH4+].[O-]S(=O)(=O)C1=CC=C2N(CC)CSC2=C1.[O-]S(=O)(=O)C1=CC=C2N(CC)CSC2=C1 QKUSRAKPUWQSJS-UHFFFAOYSA-N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 230000005611 electricity Effects 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 235000015099 wheat brans Nutrition 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 7
- 230000035484 reaction time Effects 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract 1
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- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 101150014615 LCC2 gene Proteins 0.000 description 4
- 101150075807 lcc1 gene Proteins 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- -1 Alkyl phenol Chemical compound 0.000 description 3
- 229920002271 DEAE-Sepharose Polymers 0.000 description 3
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- 239000000758 substrate Substances 0.000 description 3
- 101100325906 Bacillus subtilis (strain 168) ganA gene Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- 101100111413 Thermoanaerobacter pseudethanolicus (strain ATCC 33223 / 39E) lacZ gene Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 101150086432 lacA gene Proteins 0.000 description 2
- 101150001540 lacB gene Proteins 0.000 description 2
- 101150062200 lacC gene Proteins 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- LJYRLGOJYKPILZ-UHFFFAOYSA-N murexide Chemical compound [NH4+].N1C(=O)NC(=O)C(N=C2C(NC(=O)NC2=O)=O)=C1[O-] LJYRLGOJYKPILZ-UHFFFAOYSA-N 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 101100510607 Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) lccA gene Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- 150000004982 aromatic amines Chemical class 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
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- 102000057593 human F8 Human genes 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of method utilizing laccase degraded octyl phenol, does is step: preparation LacA enzyme liquid, regulates octyl phenol concentration 25 ~ 200? mg/L, adds enzyme liquid concentration 0.5 ~ 3U/mL that laccase is LacA, control pH3.5 ~ 6 of reaction system, temperature is degraded under remaining on 25 ~ 50 DEG C of conditions.What the present invention adopted is biological enzyme technique, has removal efficiency high, convenient, the outstanding features such as safety non-pollution; The more different isodynamic enzyme of the present invention, to the selectivity of octyl phenol, chooses the laccase the strongest to octyl phenol selectivity, adds amboceptor material, can improve the reaction efficiency of laccase, reduces the reaction time, thus reduces the use amount of laccase, reduces operating cost.Can the present invention degrade octyl phenol efficiently, when octyl phenol concentration is 100? during mg/L, under suitable catalyst condition, react 12? h degradation rate can reach 97.2%.
Description
Technical field
The present invention relates to bioengineering and environmental contaminants process field, be specifically related to a kind of method utilizing laccase degraded octyl phenol.
Background technology
The use of OPEO starts from the forties in 20th century, uses increase rapidly to the eighties, has been the commercial second largest non-ionic surface active agent in the whole world at present.Alkyl phenol be OPEO in the environment through biodegradable main intermediate metabolites, although its final catabolite is CO
2and H
20, but because of the existence of phenyl ring, this process is slowly.Therefore, octyl phenol a large amount of enrichments are in the environment caused.Octyl phenol by water body or biological accumulation, enters body through biological chain on the one hand, and the food on the other hand by polluting enters in body.Just once found that alkyl phenol had estrogen active as far back as researcher in 1938, since the nineties numerous research show octyl phenol and some aquatiles and mammal population deterioration and extinction, fertility declines and forfeiture, the male biological effect such as to feminize are relevant.Recently, people start in depth to study the care between this kind of material and reproductive endocrine disruption.Find that octyl phenol can disturbance endocrine affect sexual development level, thus cause Female sexual precocity and male sperm quantity to reduce and the illness such as Quality Down.So a kind of method finding octyl phenol of effectively degrading is extremely urgent.
The method of current process octyl phenol mainly contains: chemical oxidization method, extraction, physisorphtion, biological degradation method etc.And these methods all exist that efficiency is low, secondary pollution, high in cost of production aspect problem.Bioanalysis mainly utilizes the metabolism of microorganism, and therefore the processing time is longer, and degradation efficiency is lower; The shortcoming of physico-chemical process various degrees and secondary pollution.Enzymatic process utilizes the character of enzyme to act on pollutant efficiently, is the method for phenolic comp ' ds pollution in a kind of new processing environment.Because the method is effective, easy to operate, efficiency is high, day by day receives the concern (Agriculture of Anhui science, 2009,37(30) of people: 14858-14861).
Laccase is a kind of oxidoreducing enzyme of cupric, belongs to oxidasic Lan Tong family.Have Substratspezifitaet widely, sphere of action comprises phenols, Polyphenols, amine, aryl amine etc.The substrate conversion such as phenols, fragrance or fatty amine, by capturing substrate molecule single electron, are corresponding free radical by laccase, by electron transmission, at three cores bunch by oxygen reduction Cheng Shui.And when there are some Small molecular as redox mediator, laccase oxidability strengthens.Therefore this patent utilizes laccase degraded octyl phenol, to obtain a kind of method of octyl phenol of degrading efficiently, thus alleviates the pollution of octyl phenol in environment.
Summary of the invention
The technical problem solved: the object of the invention is to propose a kind of good degrading effect, the method for octyl phenol pollutant removed by the catalytic efficiency laccase that utilizes that is high, convenient, safety non-pollution.
Technical scheme: a kind of method utilizing laccase degraded octyl phenol, step is: regulate octyl phenol concentration at 25 ~ 200mg/L, add the enzyme liquid concentration 0.5 ~ 3U/mL of laccase, control pH3.5 ~ 6 of reaction system, temperature is degraded under remaining on 25 ~ 50 DEG C of conditions.
Described laccase is LacA, LacA is obtained by following method: cultivate bolt bacterium (Trametesversicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, 200mmol/L-pH5.0 citrate buffer solution, 1mmol/L guaiacol, 0.1mLTween80,0.3mmol/LCu
2+, condition of culture is 28 DEG C and cultivates 5d; Centrifugal 10min at having cultivated 1L zymotic fluid and being placed in the refrigerated centrifuge 4 DEG C of 10000rpm is collected supernatant and is laccase crude enzyme liquid; Then in supernatant, add ammonium sulfate to final concentration is 80%wt, places 24h and be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, precipitates and dissolves dialysis with 20mMpH7.5Tris-HClbuffer; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMpH7.5Tris-HClbuffer, elution buffer: 20mMpH7.0Tris-HClbuffer, the concentration gradient of NaCl is 0-0.6mol/L, elution volume is 800mL, elution speed: 1.5mL/min, every 4mL collects a pipe and detects laccase activity, obtains laccase LacA.
Described laccase LacA is obtained by following method:
(1) cultivation of bolt bacterium (Trametesversicolor)
The culture medium of bolt bacterium is PDA culture medium, includes glucose 20g/L, potato juice 20%wt, inoculates fresh bolt bacterium mycelia, and at 28 DEG C, 180rpm cultivates 3-4 days collecting by filtration mycelium;
(2) extraction of bolt bacterium total serum IgE
After getting the mycelium PBS cushioning liquid cleaning of the bolt bacterium of collection, at pulverized under liquid nitrogen mycelium to Powdered, collect in 2mL centrifuge tube, shake after adding 1mLTrizol, supernatant is shifted to 2mL centrifuge tube after the centrifugal 10min of 12000g at 4 DEG C, 2-3min is hatched at adding latter 30 DEG C of 200 μ L chloroform concussion 15s mixing, at 4 DEG C, the centrifugal 10min of 12000g gets supernatant, add supernatant 0.8 times of volume isopropyl alcohol mixing latter 4 DEG C at the centrifugal 10min of 12000g, be precipitated with the centrifugal 5min of 7500g at latter 4 DEG C of 75%wt ethanol water cleaning after removing supernatant, after making its natural drying, add DEPC water-soluble solution RNA to precipitate, as template ribonucleic acid, preserve at-20 DEG C,
(3) synthesis of cDNA
With bolt bacterium total serum IgE for template, reverse transcription is utilized to synthesize cDNA first chain:
Following template ribonucleic acid/Primer reactant liquor is prepared in microcentrifugal tube:
50μMOligodT1μL，
10mMdNTPMixture1μL，
Total serum IgE 1 μ g,
DEPC-H
2O7μL；
1min is placed on ice after being incubated 5min at mixing latter 65 DEG C;
Following cDNA synthesis reaction solution is prepared in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScriptBuffer4μL，
40U/μLRNaseInhibiter0.5μL，
200U/μLPrimeScriptRTase1μL，
RNasefreeH
2O4.5μL;
At 50 DEG C, be incubated 1h after the mixing of above-mentioned reactant liquor, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtained is immediately for the synthesis of cDNA second chain;
(4) clone of LacA gene
Design of primers:
LacA primer, carrier is pPICZ α B:
P5:GGGGAATTCATGGGTCTGCAGCGATT；
P6:GGGTCTAGATCACTGGTTAGCCTCGCTCA；
Reaction condition is 94 DEG C, 5min; Time out, adds ExTaq polymerase, adds 40 μ L paraffin oil sealings; 30 circulations (94 DEG C, 30s; 58 DEG C, 30s; 72 DEG C, 90s); 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations; Obtain gene LacA, with EcoRI, XbaI enzyme cutting, simultaneously expression vector also with identical enzyme respectively enzyme cut, the carrier after gene LacA cuts with enzyme is connected and spends the night, and transformation of E. coli, obtains recombinant plasmid pPICZ alpha B-LacA;
(5) preparation of restructuring laccase LacA
After recombinant plasmid pPICZ alpha A-LacA linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, recombinant yeast is lined on YPD flat board and activate, cultivate 2 days, be inoculated in 10mLBMGY fluid nutrient medium after growing single bacterium colony for 28 DEG C, in 30 DEG C, 200r/min shaking table is cultivated 48h, OD600 and is reached 2 ~ 6,3000rpm centrifugal 5min collection thalline, abandon supernatant, wash thalline 1 ~ 2 time with sterile purified water; Thalline BMMY inducing culture is diluted to OD600=1, replaces cotton plug with 4 layers of gauze, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); At cultured culture medium being placed in the refrigerated centrifuge 4 DEG C of 10000rpm, centrifugal 10min collects supernatant and is laccase crude enzyme liquid; First ultrafiltration apparatus concentrates laccase crude enzyme liquid; Then in concentrate, add the ammonium sulfate that final concentration is 80%wt, at 4 DEG C, place 24h to be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min at 4 DEG C, precipitate and dissolve dialysis with 20mMpH7.0Tris-HClbuffer; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharoseTMCL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMpH7.0Tris-HClbuffer, elution buffer: containing 0.4,0.6, the 20mMpH7.0Tris-HClbuffer of 1.0M, elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, obtain restructuring laccase LacA.
2,2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), I-hydroxybenzotriazole (HOBT), murexide (VA) or the guaiacol of final concentration 0.1mM is also added with in above-mentioned degraded system.
Described octyl phenol concentration is at 100mg/L.
Described enzyme liquid addition is for being less than 2.0U/mL.
Described degradation time is 12 ~ 24h.
Beneficial effect:
What the present invention adopted is biological enzyme technique, has removal efficiency high, convenient, the outstanding features such as safety non-pollution;
The more different isodynamic enzyme of the present invention, to the selectivity of octyl phenol, chooses the laccase the strongest to octyl phenol selectivity, adds amboceptor material, can improve the reaction efficiency of laccase, reduces the reaction time, thus reduces the use amount of laccase, reduces operating cost.
The present invention can degrade octyl phenol efficiently, and when octyl phenol concentration is 100mg/L, reaction 12h degradation rate can reach 97.2%.
Accompanying drawing explanation
Fig. 1 represents that in the present invention, five kinds of Isozymes of Laccases are to the degradation efficiency of octyl phenol;
Fig. 2 represents the efficiency of LacA enzymatic degradation octyl phenol in the present invention and the average degradation rate situation of change with octyl phenol concentration;
Fig. 3 represents the situation of change of efficiency with laccase addition of LacA enzymatic degradation octyl phenol in the present invention;
Fig. 4 represents the situation of change of efficiency with pH value of LacA enzymatic degradation octyl phenol in the present invention;
Fig. 5 represents the efficiency variation with temperature situation of LacA enzymatic degradation octyl phenol in the present invention;
Fig. 6 represents the impact of mediator material of the present invention on the efficiency of LacA enzymatic degradation octyl phenol.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in more detail, and listed embodiment is only the present invention is described and does not limit the present invention.
The preparation of embodiment 1 laccase
1.1 Coriolus Versicolors (Coriolusversicolor) are recombinated the preparation of laccase Lcc1 and Lcc2
1.1.1 the cultivation of Coriolus Versicolor (Coriolusversicolor)
Coriolus Versicolor (Coriolusversicolor, this microorganism can obtain from occurring in nature screening, also can buy and obtain (such as can purchased from Chinese industrial Microbiological Culture Collection administrative center) from commercial channels) culture medium be PDA: glucose 20g/L, potato juice 20%wt, inoculate fresh Coriolus Versicolor mycelia, at 28 DEG C, 180rpm cultivates 3-4 days collecting by filtration mycelium.
1.1.2 the extraction of Coriolus Versicolor (Coriolusversicolor) total serum IgE
After getting the mycelium PBS cushioning liquid cleaning once of the Coriolus Versicolor (Coriolusversicolor) of collection, at pulverized under liquid nitrogen mycelium to Powdered, collect in 2mL centrifuge tube, add concuss after 1mLTrizol, supernatant is shifted to 2mL centrifuge tube after the centrifugal 10min of 12000g at 4 DEG C, 2-3min is hatched at adding latter 30 DEG C of 200 μ L chloroform concussion 15s mixing, at 4 DEG C, the centrifugal 10min of 12000g gets supernatant, add supernatant 0.8 times of volume isopropyl alcohol mixing latter 4 DEG C at the centrifugal 10min of 12000g, remove after supernatant with 75%wt ethanol water (DEPC process, eliminate mRNA enzyme) clean 2 times after at 4 DEG C the centrifugal 5min of 7500g be precipitated, after making its natural drying, add DEPC water-soluble solution RNA to precipitate, as template ribonucleic acid, preserve at-20 DEG C.
1.1.3 the synthesis of Coriolus Versicolor (Coriolusversicolor) cDNA
With Coriolus Versicolor (Coriolusversicolor) total serum IgE for template, reverse transcription is utilized to synthesize cDNA first chain (following reverse transcription agents useful for same all comes from kit " PrimeScriptTM1stStrandcDNASynthesisKit ", purchased from Takara company).
Following template ribonucleic acid/Primer reactant liquor is prepared in microcentrifugal tube:
50μMOligodT1μL，
10mMdNTPMixture1μL，
Total serum IgE 1 μ g,
DEPC-H
2O7μL；
1min is placed on ice after being incubated 5min at mixing latter 65 DEG C
Following cDNA synthesis reaction solution is prepared in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScriptBuffer4μL，
40U/μLRNaseInhibiter0.5μL，
200U/μLPrimeScriptRTase1μL
RNasefreeH
2O4.5μL;
At 50 DEG C, be incubated 1h after the mixing of above-mentioned reactant liquor, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtained is immediately for the synthesis of cDNA second chain.
1.1.4 the clone of Coriolus Versicolor (Coriolusversicolor) gene
Primer is designed respectively according to Coriolus Versicolor (Coriolusversicolor) laccase gene sequence (lcc1:HM137002 and lcc2:D84235) that NCBI announces:
P1:GC
gGAATTCgCTATCGGGCCTGTGACC, underscore represents EcoRI site;
P2:GGG
tCTAGAtTAGAGGTCGGATGAGTC, underscore represents XbaI site;
P3:CCC
aTCGATaGCCATTGGGCCCGTC, underscore represents ClaI site;
P4:CCC
tCTAGAtCAGAGGTCGGACGAG, underscore represents XbaI site.
The clone of gene lcc1 utilizes primer P1 and P2, and the clone of gene lcc2 utilizes primer P3 and P4.Reaction condition is 94 DEG C, 5min; Time out, adds ExTaq polymerase, adds 40 μ L paraffin oil sealings; 35 circulations (94 DEG C, 50s; 55 DEG C, 90s; 72 DEG C, 80s); 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Obtain gene lcc1 and lcc2, use EcoRI respectively, XbaI and ClaI and XbaI enzyme cutting, simultaneously expression vector pPICZ α A also with identical enzyme respectively enzyme cut, gene lcc1, carrier pPICZ α A after lcc2 cuts with enzyme is connected respectively and spends the night, and transformation of E. coli, obtains recombinant plasmid pPICZ alpha A-lcc1 and pPICZ α A-lcc2.
1.1.5 to recombinate the preparation of laccase
After recombinant plasmid pPICZ alpha A-Lcc1 linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, is lined by recombinant yeast on YPD flat board and activates, cultivate 2 days, be inoculated in 10mLBMGY fluid nutrient medium, in 30 DEG C after growing single bacterium colony for 28 DEG C, 200r/min shaking table cultivates 48h, OD
600reach the centrifugal 5min of 2 ~ 6,3000rpm and collect thalline, abandon supernatant, wash thalline 1 ~ 2 time with sterile purified water.Thalline BMMY inducing culture is diluted to OD
600=1, replace cotton plug with 4 layers of gauze, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V).
At the culture medium cultivating (being cultured to the 13rd day for best) is placed in the refrigerated centrifuge 4 DEG C of 10000rpm, centrifugal 10min collects supernatant and is laccase crude enzyme liquid.First ultrafiltration apparatus concentrates laccase crude enzyme liquid; Then in concentrate, add the ammonium sulfate that final concentration is 80%wt, at 4 DEG C, place 24h to be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min, precipitation 20mMTris-HClbuffer(pH7.0 at 4 DEG C) dissolve dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMTris-HClbuffer(pH7.0), elution buffer: containing 0.4,0.6, the 20mMTris-HClbuffer(pH7.0 of 1.0M), elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, obtain restructuring laccase Lcc1.
By above-mentioned same method, obtain restructuring laccase Lcc2.
1.2 prepare laccase LacA and LacB and LacC by the direct cultivation and fermentation of original bacteria bolt bacterium (Trametesversicolor)
Cultivate bolt bacterium (Trametesversicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, citrate buffer solution (200mmol/L, pH5.0), 1mmol/L guaiacol, 0.1mLTween80,0.3mmol/LCu2+, condition of culture is 28 DEG C and cultivates 5d.Centrifugal 10min at having cultivated zymotic fluid (1L) and being placed in the refrigerated centrifuge 4 DEG C of 10000rpm is collected supernatant and is laccase crude enzyme liquid.Then in supernatant, add the ammonium sulfate that final concentration is 80%wt, at 4 DEG C, place 24h to be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min, precipitation 20mMTris-HClbuffer(pH7.5 at 4 DEG C) dissolve dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMTris-HClbuffer(pH7.5), elution buffer: 20mMTris-HClbuffer(pH7.0), the concentration gradient of NaCl is 0-0.6mol/L, and elution volume is 800mL, elution speed: 1.5mL/min, every 4mL collects a pipe and detects laccase activity, three protein peak figure detected by Protein Detection instrument, collect three kinds of laccases respectively, and called after laccase LacA and LacB and LacC.
The preparation of 1.3 bolt bacterium Trametesversicolor restructuring laccase LacA, LacB and LacC
1.3.1 the cultivation of bolt bacterium Trametesversicolor
This microorganism of bolt bacterium Trametesversicolor can be screened from occurring in nature, mutagenic obtained, also can buy and obtain (such as can purchased from Chinese industrial Microbiological Culture Collection administrative center), the same 1.1.1 of cultural method from commercial channels.
1.3.2 the extraction of bolt bacterium Trametesversicolor total serum IgE
The same 1.1.2 of method.
1.3.3 the synthesis of bolt bacterium TrametesversicolorcDNA
The same 1.1.3 of method.
1.3.4 the clone of bolt bacterium Trametesversicolor gene
According to the bolt bacterium Trametesversicolor laccase gene sequence (lccA:AB212732 that NCBI announces; LccB:AB212722; LccC:AB212734) primer is designed respectively:
LacA primer: (carrier pPICZ α B)
P5:GGG
gAATTCaTGGGTCTGCAGCGATT; Underscore represents EcoRI site
P6:GGG
tCTAGAtCACTGGTTAGCCTCGCTCA; Underscore represents XbaI site
LacB primer: (carrier pPICZ α C)
P7:GGG
GAATTCATGGGCAGGGTCTCATCTCTCTG；
P8:GGG
TCTAGATTAGAGGTCGGATGAGTCAAGAGCG；
LacC primer: (carrier pPICZ α B)
P9:GGG
GAATTCATGGGCAAGTTTCACTCTTTTGTGAA；
P10:GGG
TCTAGATCAGAGGTCGGACGAGTCCAAA；
Reaction condition is 94 DEG C, 5min; Time out, adds ExTaq polymerase, adds 40 μ L paraffin oil sealings; 30 circulations (94 DEG C, 30s; 58 DEG C, 30s; 72 DEG C, 90s); 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations.Obtain gene lacA, lacB and lacC, all use EcoRI, XbaI enzyme cutting, simultaneously expression vector also with identical enzyme respectively enzyme cut, carrier after gene lacA, lacB cut with enzyme with lacC is connected respectively and spends the night, transformation of E. coli, obtains recombinant plasmid pPICZ alpha B-LacA, pPICZ α C-LacB and pPICZ α B-LacC.
1.3.5 the preparation of restructuring laccase LacA, LacB and LacC
The same 1.1.5 of method, obtains the pure enzyme of restructuring laccase LacA, LacB and LacC.
Embodiment 2 difference restructuring Isozymes of Laccase is to the selectivity of octyl phenol
Five kinds of laccases are added respectively to 2U/mL in the reaction system containing octyl phenol 50mg/L; Regulate pH to 4.5 with citrate buffer solution, control temperature is at 40 DEG C; To detect the residual quantity of octyl phenol after reacting 24h under the rotating speed of 100rpm in shaking bath.As shown in Figure 1, after 24h has been carried out in reaction, the degradation rate of five kinds of laccases Lcc1, Lcc2, LacA, LacB and LacC to octyl phenol is respectively 43.6%, 0%, 80.7%, 75.3%, 61.1% to concrete outcome.Show that laccase Lcc2 can not act on octyl phenol, and the degradation effect of laccase LacA to octyl phenol is best.1.9,1.1,1.3 times of Lcc1, LacB, LacC respectively.
The detection method of octyl phenol is as follows: first to the reaction system of 10mL, add isopyknic benzinum and carry out liquid-liquid extraction, each sample extraction three times, to ensure that the octyl phenol in reaction system extracts completely, standing 30min gets upper strata petroleum ether solution 2mL respectively after the complete layering of solution, and Nitrogen evaporator dries up rear 2mL methyl alcohol dissolution process sample.Carry out analysis to the octyl phenol residual quantity HPLC in above-mentioned reaction system to detect.Liquid chromatograph is Agilent1260, EclipseXDB-C18 chromatographic column (4.6 × 250mm, 5 μm), and mobile phase is V(acetonitrile): V(water (3%wt acetic acid))=80:20, flow velocity is 1mL/min, sample size 20 μ l, running time 15min.
The computing formula of octyl phenol degradation rate of the present invention is:
Degradation rate (%)=[(initial concentration-residual concentration)/initial concentration] × 100%.
The effect of embodiment 3 laccase LacA degraded octyl phenol pollutant under different octyl phenol concentration
Adding laccase LacA to concentration under the concentration of the octyl phenol of reaction system is respectively 25mg/L, 50mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L is 2U/mL, pH to 4.5 is regulated with citrate buffer solution, control temperature at 40 DEG C, to detect the residual quantity of octyl phenol after reacting 24h under the rotating speed of 100rpm in shaking bath.Result of the test is as shown in Figure 2: degradation rate reduces gradually along with the increase of octyl phenol concentration; When the concentration of octyl phenol is lower than 100mg/L, the average degradation rate linearly growth trend of octyl phenol, and when concentration continues to increase, the ascendant trend of the average degradation rate of octyl phenol is slow.Therefore, concentration of substrate should control within the specific limits (octyl phenol concentration controls at 100mg/L) to obtain better degradation effect.
Embodiment 4 studies the impact of laccase addition on laccase LacA degraded octyl phenol
Choosing octyl phenol concentration is 100mg/L, adopt the citric acid-sodium citrate buffer solution of pH4.5, adding laccase to concentration is respectively 0.5U/mL, 1.0U/mL, 1.5U/mL, 2.0U/mL, 2.5U/mL, 3.0U/mL, control temperature at 40 DEG C, to detect the residual quantity of octyl phenol after reacting 24h under the rotating speed of 100rpm in shaking bath.Concrete result of the test is as shown in Figure 3: when enzyme liquid concentration is less than 2.0U/mL, degradation rate becomes large along with the increase of enzyme liquid addition gradually, and when enzyme dosage continues to increase, degradation rate rises slowly.
Embodiment 5 studies the impact of pH value on laccase LacA degraded octyl phenol
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, pH respectively to 3.5,4.0,4.5,5.0,5.5,6.0 of reaction system is regulated with citrate buffer solution, and control temperature is at 40 DEG C, to detect the residual quantity of octyl phenol after reacting 24h under the rotating speed of 100rpm in shaking bath.The concrete outcome obtained is as shown in Figure 4: react and have good degradation effect under the condition of pH4 ~ 5.5, wherein when pH4.5, the degradation efficiency of octyl phenol is up to 79.8%.
Embodiment 6 studies the impact of temperature on laccase LacA degraded octyl phenol
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, the pH regulating reaction system with citric acid-sodium citrate buffer solution is 4.5, control temperature is respectively 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C and 50 DEG C, to detect the concentration of octyl phenol after reacting 24h under the rotating speed of 100rpm in shaking bath.Concrete result of the test is as shown in Figure 5: laccase has good degradation effect to octyl phenol under the condition of 25 DEG C ~ 45 DEG C, and wherein under 35 DEG C of conditions, the degradation efficiency of octyl phenol is up to 81.3%.
Embodiment 7 studies the action effect of amboceptor material to laccase LacA degraded octyl phenol
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, the pH to 4.5 of reaction system is regulated with citrate buffer solution, and control temperature is at 35 DEG C, add amboceptor substance A BTS, HOBT, guaiacol and VA respectively to final concentration 0.1mM, set the contrast not adding amboceptor material, to detect the residual quantity of octyl phenol after reacting 12h under the rotating speed of 100rpm in shaking bath simultaneously.Concrete result of the test is as shown in Figure 6: the interpolation of four kinds of amboceptor materials all can promote that laccase is to the degraded of octyl phenol, wherein the effect of ABTS is best, after reaction 12h, the degradation rate of octyl phenol reaches 97.2%, is approximately 1.8 times that do not add ABTS, thus improves the reaction efficiency of laccase.
SEQUENCELISTING
<110> Nanjing Forestry University
<120> mono-kind utilizes the method for laccase degraded octyl phenol
<130>
<160>10
<170>PatentInversion3.3
<210>1
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<213> artificial sequence
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gcggaattcgctatcgggcctgtgacc27
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<213> artificial sequence
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gggtctagattagaggtcggatgagtc27
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<212>DNA
<213> artificial sequence
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cccatcgatagccattgggcccgtc25
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<211>25
<212>DNA
<213> artificial sequence
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ccctctagatcagaggtcggacgag25
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<213> artificial sequence
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ggggaattcatgggtctgcagcgatt26
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<213> artificial sequence
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gggtctagatcactggttagcctcgctca29
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ggggaattcatgggcagggtctcatctctctg32
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<212>DNA
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gggtctagattagaggtcggatgagtcaagagcg34
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<212>DNA
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ggggaattcatgggcaagtttcactcttttgtgaa35
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gggtctagatcagaggtcggacgagtccaaa31
Claims (2)
1. utilize a method for laccase degraded octyl phenol, it is characterized in that step is: regulate octyl phenol concentration at 100mg/L, add laccase and make enzyme liquid concentration be 0.5 ~ 2U/mL, control the pH4.5 of reaction system, temperature is degraded under remaining on 35 DEG C of conditions; 2,2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) of final concentration 0.1mM is also added with in degraded system; Described degradation time is 12 ~ 24h; Described laccase is LacA, LacA is obtained by following method: cultivate Trametes versicolor (Trametesversicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, 200mmol/L-pH5.0 citrate buffer solution, 1mmol/L guaiacol, 0.1mLTween80,0.3mmol/LCu
2+, condition of culture is 28 DEG C and cultivates 5d; Centrifugal 10min at having cultivated 1L zymotic fluid and being placed in the refrigerated centrifuge 4 DEG C of 10000rpm is collected supernatant and is laccase crude enzyme liquid; Then in supernatant, add ammonium sulfate to final concentration is 80%wt, places 24h and be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, precipitates and dissolves dialysis with 20mMpH7.5Tris-HClbuffer; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharoseTMCL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMpH7.5Tris-HClbuffer, elution buffer: 20mMpH7.0Tris-HClbuffer, the concentration gradient of NaCl is 0-0.6mol/L, elution volume is 800mL, elution speed: 1.5mL/min, every 4mL collects a pipe and detects laccase activity, obtains laccase LacA.
2. utilize a method for laccase degraded octyl phenol, it is characterized in that step is: regulate octyl phenol concentration at 100mg/L, add laccase and make enzyme liquid concentration be 0.5 ~ 2U/mL, control the pH4.5 of reaction system, temperature is degraded under remaining on 35 DEG C of conditions; 2,2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) of final concentration 0.1mM is also added with in degraded system; Described degradation time is 12 ~ 24h; Described laccase LacA is obtained by following method:
(1) cultivation of Trametes versicolor (Trametesversicolor)
The culture medium of bolt bacterium is PDA culture medium, includes glucose 20g/L, potato juice 20%wt, inoculates fresh bolt bacterium mycelia, and at 28 DEG C, 180rpm cultivates 3-4 days collecting by filtration mycelium;
(2) extraction of bolt bacterium total serum IgE
After getting the mycelium PBS cushioning liquid cleaning of the bolt bacterium of collection, at pulverized under liquid nitrogen mycelium to Powdered, collect in 2mL centrifuge tube, shake after adding 1mLTrizol, supernatant is shifted to 2mL centrifuge tube after the centrifugal 10min of 12000g at 4 DEG C, 2-3min is hatched at adding latter 30 DEG C of 200 μ L chloroform concussion 15s mixing, at 4 DEG C, the centrifugal 10min of 12000g gets supernatant liquor, add supernatant 0.8 times of volume isopropyl alcohol mixing latter 4 DEG C at the centrifugal 10min of 12000g, be precipitated with the centrifugal 5min of 7500g at latter 4 DEG C of 75%wt ethanol water cleaning after removing supernatant, after making its natural drying, add DEPC water-soluble solution RNA to precipitate, as template ribonucleic acid, preserve at-20 DEG C,
(3) synthesis of cDNA
With bolt bacterium total serum IgE for template, reverse transcription is utilized to synthesize cDNA first chain:
Following template ribonucleic acid/Primer reactant liquor is prepared in microcentrifugal tube:
50μMOligodT 1μL，
10mMdNTPMixture1μL，
Total serum IgE 1 μ g,
DEPC-H
2O7μL；
1min is placed on ice after being incubated 5min at mixing latter 65 DEG C;
Following cDNA synthesis reaction solution is prepared in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScriptBuffer4μL，
40U/μLRNaseInhibiter0.5μL，
200U/μLPrimeScriptRTase1μL，
RNasefreeH
2O4.5μL;
At 50 DEG C, be incubated 1h after the mixing of above-mentioned reactant liquor, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtained is immediately for the synthesis of cDNA second chain;
(4) clone of LacA gene
Carrier is pPICZ α B, and design of primers is:
LacA primer:
P5:GGGGAATTCATGGGTCTGCAGCGATT；
P6:GGGTCTAGATCACTGGTTAGCCTCGCTCA；
Reaction condition is 94 DEG C, 5min; Time out, adds ExTaq polymerase, adds 40 μ L paraffin oil sealings; 30 circulations, each loop parameter is: " 94 DEG C, 30s; 58 DEG C, 30s; 72 DEG C, 90s "; 72 DEG C, 10min; Reaction stops, 4 DEG C of insulations; Obtain gene LacA, with EcoRI, XbaI enzyme cutting, simultaneously expression vector also with identical enzyme respectively enzyme cut, the carrier after gene LacA cuts with enzyme is connected and spends the night, and transformation of E. coli, obtains recombinant plasmid pPICZ alpha B-LacA;
(5) preparation of restructuring laccase LacA
After recombinant plasmid pPICZ alpha B-LacA linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, recombinant yeast is lined on YPD flat board and activate, cultivate 2 days, be inoculated in 10mLBMGY fluid nutrient medium after growing single bacterium colony for 28 DEG C, in 30 DEG C, 200r/min shaking table is cultivated 48h, OD600 and is reached 2 ~ 6,3000rpm centrifugal 5min collection thalline, abandon supernatant, wash thalline 1 ~ 2 time with sterile purified water; Thalline BMMY inducing culture is diluted to OD600=1, replaces cotton plug with 4 layers of gauze, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); At cultured culture medium being placed in the refrigerated centrifuge 4 DEG C of 10000rpm, centrifugal 10min collects supernatant and is laccase crude enzyme liquid; First ultrafiltration apparatus concentrates laccase crude enzyme liquid; Then in concentrate, add the ammonium sulfate that final concentration is 80%wt, at 4 DEG C, place 24h to be placed in the refrigerated centrifuge of 10000rpm centrifugal 30min at 4 DEG C, precipitate and dissolve dialysis with 20mMpH7.0Tris-HClbuffer; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharoseTMCL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mMpH7.0Tris-HClbuffer, elution buffer: containing 0.4,0.6, the 20mMpH7.0Tris-HClbuffer of 1.0M, elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, obtain restructuring laccase LacA.
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Application publication date: 20130424 Assignee: Nanjing Suxin International Trade Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2023320000227 Denomination of invention: A Method for Degradation of Octylphenol by Laccase Granted publication date: 20160302 License type: Common License Record date: 20231110 |