CN103055464A - Method for degrading octyl phenol by utilizing laccase - Google Patents
Method for degrading octyl phenol by utilizing laccase Download PDFInfo
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- CN103055464A CN103055464A CN2012105395906A CN201210539590A CN103055464A CN 103055464 A CN103055464 A CN 103055464A CN 2012105395906 A CN2012105395906 A CN 2012105395906A CN 201210539590 A CN201210539590 A CN 201210539590A CN 103055464 A CN103055464 A CN 103055464A
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 87
- DUIOKRXOKLLURE-UHFFFAOYSA-N 2-octylphenol Chemical compound CCCCCCCCC1=CC=CC=C1O DUIOKRXOKLLURE-UHFFFAOYSA-N 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000000593 degrading effect Effects 0.000 title abstract description 10
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- 238000000502 dialysis Methods 0.000 claims description 12
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for degrading octyl phenol by utilizing laccase. The method comprises the steps of: preparing a LacA enzyme solution; adjusting the concentration of the octyl phenol within 25-200 mg/L; adding the laccase having a concentration of 0.5-3 U/mL in the LacA enzyme solution; controlling the pH of a reaction system to 3.5-6; and degrading under the condition of keeping the temperature at 25-50 DEG C. As a bio-enzyme catalysis technology is adopted, the method for degrading the octyl phenol by utilizing the accase has outstanding characteristics of high removal efficiency, good convenience and safety, no pollution and the like. By comparing specifity of different isozymes to the octyl phenol, selecting the laccase having the strongest specifity to the octyl phenol and adding a medium substance, the method for degrading thhe octyl phenol by utilizing the laccase increases the reaction efficiency of the laccas, redudes reaction time, therefore reduces usage amount of the laccase, and lowers operating cost. The method for degrading the octyl phenol by utilizing the laccase can degrade octyl phenol efficiently and can obtain the degradation rate up to 97.2% after 12-hour reaction under a suitable catalysis condition when the concentration of the octyl phenol is 100 mg/L.
Description
Technical field
The present invention relates to bioengineering and environmental contaminants process field, be specifically related to a kind of method of utilizing laccase degraded octyl phenol.
Background technology
The use of OPEO starts from the forties in 20th century, to the eighties use and to increase rapidly, be the commercial second largest non-ionic surface active agent in the whole world at present.Alkyl phenol be OPEO in 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 very slow.Therefore, a large amount of enrichments of octyl phenol in environment have been caused.Octyl phenol by water body or biological accumulation, enters body through biological chain on the one hand, enters in the body by the food that pollutes on the other hand.Just once found that alkyl phenol had estrogen active as far back as researcher in 1938, numerous population degenerations that studies show that octyl phenol and some aquatiles and mammal and extinction, fertility decline and forfeiture since the nineties, the male biological effect such as feminize are relevant.Recently, people begin in depth to study the care between this class material and reproductive endocrine interference.Find that octyl phenol can disturbance endocrine and affect the sexual development level, thereby cause female sex premature and male sperm quantity to reduce and the illnesss such as Quality Down.So it is extremely urgent to seek a kind of method of the octyl phenol of effectively degrading.
The method of processing at present octyl phenol mainly contains: chemical oxidization method, extraction, physisorphtion, biological degradation method etc.And these methods all exist that efficient is low, the problem of secondary pollution, high in cost of production aspect.Bioanalysis mainly is the metabolism of utilizing microorganism, so the processing time is longer, and degradation efficiency is lower; The shortcoming of physico-chemical process various degrees and secondary pollution.Enzymatic process is to utilize the character of enzyme to act on efficiently pollutant, is the method for phenolic comp ' ds pollution in a kind of new processing environment.Because the method is effective, easy to operate, efficient is high, day by day be subject to people concern (the Agriculture of Anhui science, 2009,37(30): 14858-14861).
Laccase is a kind of oxidoreducing enzyme of cupric, belongs to oxidasic blue copper family.Have widely Substratspezifitaet, sphere of action comprises phenols, Polyphenols, amine, aryl amine etc.Laccase is corresponding free radical by capturing the substrate molecule single electron with substrate conversion such as phenols, fragrance or fatty amines, by the electronics transmission, at three nuclears bunch with oxygen reduction Cheng Shui.And when having some little molecules as redox mediator, the laccase oxidability strengthens.Therefore this patent utilizes laccase degraded octyl phenol, obtaining a kind of method of the octyl phenol of degrading efficiently, thereby alleviates the pollution of octyl phenol in the environment.
Summary of the invention
The technical problem that solves: the method for utilizing laccase removal octyl phenol pollutant that the objective of the invention is to propose a kind of good degrading effect, catalytic efficiency height, convenience, safety non-pollution.
Technical scheme: a kind of method of utilizing laccase degraded octyl phenol, step is: regulates octyl phenol concentration at 25～200mg/L, adds the enzyme liquid concentration 0.5～3U/mL of laccase, the pH3.5 of control reaction system～6, temperature remains under 25～50 ℃ of conditions and degrades.
Described laccase is LacA, LacA obtains by following method: cultivate bolt bacterium (Trametes versicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, 200mmol/L-pH 5.0 citrate buffer solutions, 1mmol/L guaiacol, 0.1mL Tween 80,0.3mmol/L Cu
2+, condition of culture is 28 ℃ and cultivates 5d; Place 4 ℃ of lower centrifugal 10min collection supernatants of refrigerated centrifuge of 10000rpm to be the laccase crude enzyme liquid with having cultivated the 1L zymotic fluid; Then adding ammonium sulfate to final concentration in supernatant is 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mM pH 7.5Tris-HCl buffer dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
TMThe CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH 7.5Tris-HClbuffer, elution buffer: 20mM pH 7.0Tris-HCl buffer, the concentration gradient of NaCl is 0-0.6mol/L, elution volume is 800mL, elution speed: 1.5mL/min, every 4mL collect a pipe and detect laccase activity, obtain laccase LacA.
Described laccase LacA obtains by following method:
(1) cultivation of bolt bacterium (Trametes versicolor)
The culture medium of bolt bacterium is the PDA culture medium, includes glucose 20g/L, and potato juice 20%wt inoculates fresh bolt bacterium mycelia, and 28 ℃ of lower 180rpm cultivate to filter in 3-4 days and collect mycelium;
(2) extraction of the total RNA of bolt bacterium
After the mycelium that gets the bolt bacterium of collection cleans with PBS cushioning liquid, under liquid nitrogen, grind mycelium to Powdered, collect in the 2mL centrifuge tube, shake after adding 1mL Trizol, shift supernatant behind 4 ℃ of centrifugal 10min of lower 12000g to the 2mL centrifuge tube, add behind the 200 μ L chloroforms concussions 15s mixing and hatch 2-3min under 30 ℃, 4 ℃ of centrifugal 10min of lower 12000g get the upper strata stillness of night, 4 ℃ of centrifugal 10min of lower 12000g behind the isopropyl alcohol mixing of 0.8 times of volume of adding supernatant, remove and clean rear 4 ℃ of centrifugal 5min of lower 7500g with the 75%wt ethanol water behind the supernatant and be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid ,-20 ℃ of lower preservations;
(3) cDNA's is synthetic
Take the total RNA of bolt bacterium as template, utilize reverse transcription to synthesize cDNA the first chain:
Preparation following template ribonucleic acid/Primer reactant liquor in microcentrifugal tube:
50μM Oligo dT 1μL，
10mM dNTP Mixture 1μL，
DEPC-H
2O 7μL；
Place 1min on ice behind 65 ℃ of lower insulation 5min behind the mixing;
The following cDNA synthesis reaction solution of preparation in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScript Buffer 4μL，
40U/μL RNase Inhibiter 0.5μL，
200U/μL PrimeScript RTase 1μL，
RNase free H
2O 4.5μL;
Under 50 ℃, be incubated 1h behind the above-mentioned reactant liquor mixing, cooled on ice behind 70 ℃ of lower insulation 15min, the reactant liquor that obtains is used for synthesizing of cDNA the second chain immediately;
(4) LacA gene cloning
Design of primers:
LacA primer, carrier are pPICZ α B:
P5:GGGGAATTCATGGGTCTGCAGCGATT；
P6:GGGTCTAGATCACTGGTTAGCCTCGCTCA；
Reaction condition is 94 ℃, 5min; Time out adds Ex Taq polymerase, adds the sealing of 40 μ L paraffin oils; 30 circulations (94 ℃, 30s; 58 ℃, 30s; 72 ℃, 90s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations; Obtain gene LacA, use EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, gene LacA connects with carrier after enzyme is connected and spends the night, the conversion Escherichia coli obtain 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 lined on the YPD flat board activate, cultivated 2 days, and after growing single bacterium colony, be inoculated in the 10mL BMGY fluid nutrient medium for 28 ℃, in 30 ℃, the 200r/min shaking table is cultivated 48h, and OD600 reaches the centrifugal 5min of 2 ~ 6,3000rpm and collects thalline, abandon supernatant, wash thalline 1 ~ 2 time with sterile purified water; Thalline is diluted to OD600=1 with the BMMY inducing culture, replaces cotton plug with 4 layers of gauze, in 28 ℃, the 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); Place 4 ℃ of lower centrifugal 10min of refrigerated centrifuge of 10000rpm to collect supernatant cultured culture medium and be the laccase crude enzyme liquid; At first ultrafiltration apparatus is concentrated to the laccase crude enzyme liquid; Then adding final concentration in concentrate is the ammonium sulfate of 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mM pH 7.0Tris-HCl buffer dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE SepharoseTM CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH 7.0Tris-HCl buffer, elution buffer: contain 0.4,0.6, the 20mM pH 7.0Tris-HCl buffer of 1.0M, elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, laccase LacA obtains recombinating.
Also be added with 2 of final concentration 0.1mM in the above-mentioned degraded system, 2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), I-hydroxybenzotriazole (HOBT), murexide (VA) or guaiacol.
Described octyl phenol concentration is at 100mg/L.
Described enzyme liquid addition is less than 2.0U/mL.
Described degradation time is 12～24h.
Beneficial effect:
What the present invention adopted is biological enzyme technique, and it is high to have the efficient of removal, convenient, the outstanding features such as safety non-pollution;
The more different isodynamic enzyme of the present invention is chosen the strongest laccase of octyl phenol selectivity the selectivity of octyl phenol, adds the amboceptor material, can improve the reaction efficiency of laccase, reduces the reaction time, thereby reduces the use amount of laccase, has reduced operating cost.
The present invention's octyl phenol of degrading efficiently, when octyl phenol concentration was 100mg/L, reaction 12h degradation rate can reach 97.2%.
Description of drawings
Fig. 1 represents among the present invention that five kinds of Isozymes of Laccases are to the degradation efficiency of octyl phenol;
Fig. 2 represents that the efficient of LacA enzymatic degradation octyl phenol among the present invention and average degradation rate are with the situation of change of octyl phenol concentration;
Fig. 3 represents that the efficient of LacA enzymatic degradation octyl phenol among the present invention is with the situation of change of laccase addition;
Fig. 4 represents that the efficient of LacA enzymatic degradation octyl phenol among the present invention is with the situation of change of pH value;
Fig. 5 represents the efficient variation with temperature situation of LacA enzymatic degradation octyl phenol among the present invention;
Fig. 6 represents that mediator material of the present invention is on the impact of the efficient of LacA enzymatic degradation octyl phenol.
The specific embodiment
The present invention is described in more detail below in conjunction with specific embodiment, and listed embodiment only is the present invention is described and does not limit the present invention.
The preparation of embodiment 1 laccase
1.1 the preparation of Coriolus Versicolor (Coriolus versicolor) restructuring laccase Lcc1 and Lcc2
1.1.1 the cultivation of Coriolus Versicolor (Coriolus versicolor)
Coriolus Versicolor (Coriolus versicolor, this microorganism can obtain from the occurring in nature screening, also can buy from commercial channels and obtain (for example can available from Chinese industrial microorganism fungus kind preservation administrative center)) culture medium be PDA: glucose 20g/L, potato juice 20%wt, inoculate fresh Coriolus Versicolor mycelia, 28 ℃ of lower 180rpm cultivate to filter in 3-4 days and collect mycelium.
1.1.2 the extraction of the total RNA of Coriolus Versicolor (Coriolus versicolor)
After the mycelium that gets the Coriolus Versicolor (Coriolus versicolor) of collection cleans once with PBS cushioning liquid, under liquid nitrogen, grind mycelium to Powdered, collect in the 2mL centrifuge tube, concuss behind the adding 1mL Trizol, shift supernatant behind 4 ℃ of centrifugal 10min of lower 12000g to the 2mL centrifuge tube, add behind the 200 μ L chloroforms concussions 15s mixing and hatch 2-3min under 30 ℃, 4 ℃ of centrifugal 10min of lower 12000g get the upper strata stillness of night, 4 ℃ of centrifugal 10min of lower 12000g behind the isopropyl alcohol mixing of 0.8 times of volume of adding supernatant, (DEPC processed with the 75%wt ethanol water after removing supernatant, removed the mRNA enzyme) clean 2 times after 4 ℃ of centrifugal 5min of lower 7500g be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid ,-20 ℃ of lower preservations.
1.1.3 Coriolus Versicolor (Coriolus versicolor) cDNA's is synthetic
Take the total RNA of Coriolus Versicolor (Coriolus versicolor) as template, utilize reverse transcription to synthesize cDNA the first chain (following each reverse transcription agents useful for same all comes from kit " PrimeScriptTM1stStrand cDNA Synthesis Kit ", available from Takara company).
Preparation following template ribonucleic acid/Primer reactant liquor in microcentrifugal tube:
50μM Oligo dT 1μL，
10mM dNTP Mixture 1μL，
DEPC-H
2O 7μL；
Place 1min on ice behind 65 ℃ of lower insulation 5min behind the mixing
The following cDNA synthesis reaction solution of preparation in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScript Buffer 4μL，
40U/μL RNase Inhibiter 0.5μL，
200U/μL PrimeScript RTase 1μL
RNase free H
2O 4.5μL;
Under 50 ℃, be incubated 1h behind the above-mentioned reactant liquor mixing, cooled on ice behind 70 ℃ of lower insulation 15min, the reactant liquor that obtains is used for synthesizing of cDNA the second chain immediately.
1.1.4 Coriolus Versicolor (Coriolus versicolor) gene cloning
Coriolus Versicolor (Coriolus versicolor) the laccase gene sequence (lcc1:HM137002 and lcc2:D84235) of announcing according to NCBI designs respectively primer:
P1:GC
GGAATTCGCTATCGGGCCTGTGACC, underscore represent the EcoRI site;
P2:GGG
TCTAGATTAGAGGTCGGATGAGTC, underscore represent the XbaI site;
P3:CCC
ATCGATAGCCATTGGGCCCGTC, underscore represent Cla I site;
P4:CCC
TCTAGATCAGAGGTCGGACGAG, underscore represent the 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 ℃, 5min; Time out adds Ex Taq polymerase, adds the sealing of 40 μ L paraffin oils; 35 circulations (94 ℃, 50s; 55 ℃, 90s; 72 ℃, 80s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations.Obtain gene lcc1 and lcc2, use respectively EcoRI, XbaI and Cla I and Xba I enzyme are cut, simultaneously expression vector pPICZ α A also with identical enzyme respectively enzyme cut, gene lcc1, lcc2 connects respectively with carrier pPICZ α A after enzyme is connected and spends the night, and transforms Escherichia coli, obtains recombinant plasmid pPICZ alpha A-lcc1 and pPICZ α A-lcc2.
1.1.5 the preparation of restructuring laccase
After recombinant plasmid pPICZ alpha A-Lcc1 linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, recombinant yeast is lined on the YPD flat board activate, cultivated 2 days, and after growing single bacterium colony, be inoculated in the 10mL BMGY fluid nutrient medium, in 30 ℃ for 28 ℃, the 200r/min shaking table is cultivated 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 is diluted to OD with the BMMY inducing culture
600=1, replace cotton plug with 4 layers of gauze, in 28 ℃, the 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V).
The culture medium that to cultivate (being cultured to the 13rd day for best) places 4 ℃ of lower centrifugal 10min of refrigerated centrifuge of 10000rpm to collect supernatant and is the laccase crude enzyme liquid.At first ultrafiltration apparatus is concentrated to the laccase crude enzyme liquid; Then adding final concentration in concentrate is the ammonium sulfate of 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mM Tris-HCl buffer(pH 7.0) the dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
TMThe CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM Tris-HCl buffer(pH 7.0), elution buffer: contain 0.4,0.6, the 20mM Tris-HCl buffer(pH 7.0 of 1.0M), elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, laccase Lcc1 obtains recombinating.
With above-mentioned same method, laccase Lcc2 obtains recombinating.
1.2 prepare laccase LacA and LacB and LacC with the direct cultivation and fermentation of original bacterium bolt bacterium (Trametes versicolor)
Cultivate bolt bacterium (Trametes versicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, citrate buffer solution (200mmol/L, pH 5.0), the 1mmol/L guaiacol, 0.1mL Tween 80,0.3mmol/L Cu2+, condition of culture are 28 ℃ and cultivate 5d.To cultivate zymotic fluid (1L) places 4 ℃ of lower centrifugal 10min collection supernatants of refrigerated centrifuge of 10000rpm to be the laccase crude enzyme liquid.Then adding final concentration in supernatant is the ammonium sulfate of 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mMTris-HCl buffer(pH 7.5) the dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
TMThe CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM Tris-HCl buffer(pH 7.5), elution buffer: 20mM Tris-HCl buffer(pH 7.0), 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, detect three protein peak figure by the Protein Detection instrument, collect respectively three kinds of laccases, and called after laccase LacA and LacB and LacC.
1.3 the preparation of bolt bacterium Trametes versicolor restructuring laccase LacA, LacB and LacC
1.3.1 the cultivation of bolt bacterium Trametes versicolor
This microorganism of bolt bacterium Trametes versicolor can screen from occurring in nature, mutagenic obtained, also can buy from commercial channels and obtain (for example can available from Chinese industrial microorganism fungus kind preservation administrative center), the same 1.1.1 of cultural method.
1.3.2 the extraction of the total RNA of bolt bacterium Trametes versicolor
The same 1.1.2 of method.
1.3.3 bolt bacterium Trametes versicolor cDNA's is synthetic
The same 1.1.3 of method.
1.3.4 bolt bacterium Trametes versicolor gene cloning
Bolt bacterium Trametes versicolor laccase gene sequence (lccA:AB212732 according to the NCBI announcement; LccB:AB212722; LccC:AB212734) design respectively primer:
LacA primer: (carrier pPICZ α B)
P5:GGG
GAATTCATGGGTCTGCAGCGATT; Underscore represents the EcoRI site
P6:GGG
TCTAGATCACTGGTTAGCCTCGCTCA; Underscore represents the 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 ℃, 5min; Time out adds Ex Taq polymerase, adds the sealing of 40 μ L paraffin oils; 30 circulations (94 ℃, 30s; 58 ℃, 30s; 72 ℃, 90s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations.Obtain gene lacA, lacB and lacC, all use EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, gene lacA, lacB and lacC connect respectively with carrier after enzyme is connected and spend the night, transform Escherichia coli, obtain 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, the pure enzyme of obtain recombinating laccase LacA, LacB and LacC.
Embodiment 2 different restructuring Isozymes of Laccases are to the selectivity of octyl phenol
In the reaction system that contains octyl phenol 50mg/L, add respectively five kinds of laccases to 2U/mL; Regulate pH to 4.5 with citrate buffer solution, the control temperature is at 40 ℃; In shaking bath to detect the residual quantity of octyl phenol behind the reaction 24h under the rotating speed of 100rpm.Concrete outcome has carried out behind the 24h five kinds of laccase Lcc1, Lcc2, LacA, LacB and LacC in reaction the degradation rate of octyl phenol has been respectively 43.6%, 0%, 80.7%, 75.3%, 61.1% as shown in Figure 1.Show that laccase Lcc2 can not act on octyl phenol, and laccase LacA is best to the degradation effect of octyl phenol.Respectively Lcc1, LacB, LacC 1.9,1.1,1.3 times.
The detection method of octyl phenol is as follows: at first to the reaction system of 10mL, add isopyknic benzinum and carry out liquid-liquid extraction, each sample extraction three times, to guarantee that the octyl phenol in the reaction system extracts fully, leave standstill 30min and get respectively upper strata petroleum ether solution 2mL after the complete layering of solution, Nitrogen evaporator dries up rear with 2mL methyl alcohol dissolution process sample.Octyl phenol residual quantity in the above-mentioned reaction system is carried out analyzing and testing with HPLC.Liquid chromatograph is Agilent 1260, and Eclipse XDB-C18 chromatographic column (4.6 * 250mm, 5 μ m), mobile phase is the 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
The concentration of the octyl phenol of reaction system be respectively under 25mg/L, 50mg/L, 75mg/L, 100mg/L, 150mg/L, the 200mg/L add laccase LacA to concentration be 2U/mL, regulate pH to 4.5 with citrate buffer solution, the control temperature is at 40 ℃, in shaking bath to detect the residual quantity of octyl phenol behind the reaction 24h under the rotating speed of 100rpm.Result of the test is as shown in Figure 2: degradation rate is along with the increase of octyl phenol concentration reduces gradually; When the concentration of octyl phenol was lower than 100mg/L, the average degradation rate of octyl phenol was linear growth trend, and when concentration continued to increase, the ascendant trend of the average degradation rate of octyl phenol was slow.Therefore, concentration of substrate should be controlled within the specific limits (octyl phenol concentration is controlled at 100mg/L) to obtain better degradation effect.
Choosing octyl phenol concentration is 100mg/L, adopt the citric acid-sodium citrate buffer solution of pH 4.5, adding respectively laccase to concentration is 0.5U/mL, 1.0U/mL, 1.5U/mL, 2.0U/mL, 2.5U/mL, 3.0U/mL, the control temperature is at 40 ℃, in shaking bath to detect the residual quantity of octyl phenol behind the reaction 24h under the rotating speed of 100rpm.Concrete result of the test is as shown in Figure 3: when enzyme liquid concentration during less than 2.0U/mL, it is large that degradation rate becomes gradually along with the increase of enzyme liquid addition, and when enzyme dosage continues to increase, degradation rate rises slowly.
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, with the pH of citrate buffer solution conditioned reaction system respectively to 3.5,4.0,4.5,5.0,5.5,6.0, and the control temperature is at 40 ℃, in shaking bath to detect the residual quantity of octyl phenol behind the reaction 24h under the rotating speed of 100rpm.The concrete outcome that obtains is as shown in Figure 4: reaction has preferably degradation effect under the condition of pH4 ~ 5.5, wherein the degradation efficiency of octyl phenol is up to 79.8% when pH 4.5.
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, pH with citric acid-sodium citrate buffer solution conditioned reaction system is 4.5, the control temperature is respectively 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, in shaking bath to detect the concentration of octyl phenol behind the reaction 24h under the rotating speed of 100rpm.Specifically result of the test as shown in Figure 5: laccase has preferably degradation effect to octyl phenol under 25 ℃～45 ℃ condition, wherein the degradation efficiency of octyl phenol is up to 81.3% under 35 ℃ of conditions.
Embodiment 7 research amboceptor materials are to the action effect of laccase LacA degraded octyl phenol
Choosing octyl phenol concentration is 100mg/L, adding laccase to concentration is 2.0U/mL, pH to 4.5 with citrate buffer solution conditioned reaction system, and the control temperature is at 35 ℃, add respectively amboceptor substance A BTS, HOBT, guaiacol and VA to final concentration 0.1mM, set simultaneously the contrast do not add the amboceptor material, in shaking bath to detect the residual quantity of octyl phenol behind the reaction 12h under the rotating speed of 100rpm.Specifically result of the test as shown in Figure 6: the interpolation of four kinds of amboceptor materials all can promote laccase to the octyl group Phenol degradation, wherein the effect of ABTS is best, the degradation rate of octyl phenol reaches 97.2% behind the reaction 12h, is approximately 1.8 times that do not add ABTS, thereby has improved the reaction efficiency of laccase.
SEQUENCE LISTING
＜110〉Nanjing Forestry University
＜120〉a kind of method of utilizing laccase degraded octyl phenol
<130>
<160> 10
<170> PatentIn version 3.3
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<212> DNA
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gcggaattcg ctatcgggcc tgtgacc 27
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gggtctagat tagaggtcgg atgagtc 27
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cccatcgata gccattgggc ccgtc 25
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ccctctagat cagaggtcgg acgag 25
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ggggaattca tgggtctgca gcgatt 26
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ggggaattca tgggcagggt ctcatctctc tg 32
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gggtctagat tagaggtcgg atgagtcaag agcg 34
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ggggaattca tgggcaagtt tcactctttt gtgaa 35
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gggtctagat cagaggtcgg acgagtccaa a 31
Claims (7)
1. method of utilizing laccase degraded octyl phenol is characterized in that step is: regulates octyl phenol concentration at 25～200mg/L, adds the enzyme liquid concentration 0.5～3U/mL of laccase, and the pH3.5 of control reaction system～6, temperature remains under 25～50 ℃ of conditions and degrades.
2. the method for utilizing laccase degraded octyl phenol according to claim 1, it is characterized in that described laccase is LacA, LacA obtains by following method: cultivate bolt bacterium (Trametes versicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, 200mmol/L-pH 5.0 citrate buffer solutions, the 1mmol/L guaiacol, 0.1mLTween 80,0.3mmol/L Cu
2+, condition of culture is 28 ℃ and cultivates 5d; Place 4 ℃ of lower centrifugal 10min collection supernatants of refrigerated centrifuge of 10000rpm to be the laccase crude enzyme liquid with having cultivated the 1L zymotic fluid; Then adding ammonium sulfate to final concentration in supernatant is 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mM pH 7.5Tris-HCl buffer dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharose
TMThe CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH 7.5Tris-HCl buffer, elution buffer: 20mM pH 7.0Tris-HCl buffer, the concentration gradient of NaCl is 0-0.6mol/L, elution volume is 800mL, elution speed: 1.5mL/min, every 4mL collect a pipe and detect laccase activity, obtain laccase LacA.
3. the method for utilizing laccase degraded octyl phenol according to claim 1 is characterized in that described laccase LacA obtains by following method:
(1) cultivation of bolt bacterium (Trametes versicolor)
The culture medium of bolt bacterium is the PDA culture medium, includes glucose 20g/L, and potato juice 20%wt inoculates fresh bolt bacterium mycelia, and 28 ℃ of lower 180rpm cultivate to filter in 3-4 days and collect mycelium;
(2) extraction of the total RNA of bolt bacterium
After the mycelium that gets the bolt bacterium of collection cleans with PBS cushioning liquid, under liquid nitrogen, grind mycelium to Powdered, collect in the 2mL centrifuge tube, shake after adding 1mL Trizol, shift supernatant behind 4 ℃ of centrifugal 10min of lower 12000g to the 2mL centrifuge tube, add behind the 200 μ L chloroforms concussions 15s mixing and hatch 2-3min under 30 ℃, 4 ℃ of centrifugal 10min of lower 12000g get the upper strata stillness of night, 4 ℃ of centrifugal 10min of lower 12000g behind the isopropyl alcohol mixing of 0.8 times of volume of adding supernatant, remove and clean rear 4 ℃ of centrifugal 5min of lower 7500g with the 75%wt ethanol water behind the supernatant and be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid ,-20 ℃ of lower preservations;
(3) cDNA's is synthetic
Take the total RNA of bolt bacterium as template, utilize reverse transcription to synthesize cDNA the first chain:
Preparation following template ribonucleic acid/Primer reactant liquor in microcentrifugal tube:
50μM Oligo dT 1μL，
10mM dNTP Mixture 1μL，
Total RNA 1 μ g,
DEPC-H
2O 7μL；
Place 1min on ice behind 65 ℃ of lower insulation 5min behind the mixing;
The following cDNA synthesis reaction solution of preparation in above-mentioned microcentrifugal tube:
Above-mentioned RNA/Primer mixed liquor 10 μ L,
5×PrimeScript Buffer 4μL，
40U/μL RNase Inhibiter 0.5μL，
200U/μL PrimeScript RTase 1μL，
RNase free H
2O 4.5μL;
Under 50 ℃, be incubated 1h behind the above-mentioned reactant liquor mixing, cooled on ice behind 70 ℃ of lower insulation 15min, the reactant liquor that obtains is used for synthesizing of cDNA the second chain immediately;
(4) LacA gene cloning
Design of primers:
LacA primer, carrier are pPICZ α B:
P5:GGGGAATTCATGGGTCTGCAGCGATT；
P6:GGGTCTAGATCACTGGTTAGCCTCGCTCA；
Reaction condition is 94 ℃, 5min; Time out adds Ex Taq polymerase, adds the sealing of 40 μ L paraffin oils; 30 circulations (94 ℃, 30s; 58 ℃, 30s; 72 ℃, 90s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations; Obtain gene LacA, use EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, gene LacA connects with carrier after enzyme is connected and spends the night, the conversion Escherichia coli obtain 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 lined on the YPD flat board activate, cultivated 2 days, and after growing single bacterium colony, be inoculated in the 10mL BMGY fluid nutrient medium for 28 ℃, in 30 ℃, the 200r/min shaking table is cultivated 48h, and OD600 reaches the centrifugal 5min of 2 ~ 6,3000rpm and collects thalline, abandon supernatant, wash thalline 1 ~ 2 time with sterile purified water; Thalline is diluted to OD600=1 with the BMMY inducing culture, replaces cotton plug with 4 layers of gauze, in 28 ℃, the 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); Place 4 ℃ of lower centrifugal 10min of refrigerated centrifuge of 10000rpm to collect supernatant cultured culture medium and be the laccase crude enzyme liquid; At first ultrafiltration apparatus is concentrated to the laccase crude enzyme liquid; Then adding final concentration in concentrate is the ammonium sulfate of 80%wt, places 4 ℃ of lower centrifugal 30min in the refrigerated centrifuge that 24h are placed on 10000rpm down at 4 ℃, and precipitation is with 20mM pH 7.0 Tris-HCl buffer dissolving dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE SepharoseTM CL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH 7.0Tris-HCl buffer, elution buffer: contain 0.4,0.6, the 20mM pH 7.0 Tris-HCl buffer of 1.0M, elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, laccase LacA obtains recombinating.
4. the method for utilizing laccase degraded octyl phenol according to claim 1, it is characterized in that also being added with in the degraded system 2 of final concentration 0.1mM, 2 '-Lian nitrogen-two (3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), I-hydroxybenzotriazole (HOBT), murexide (VA) or guaiacol.
5. the method for utilizing laccase degraded octyl phenol according to claim 1 is characterized in that described octyl phenol concentration is at 100mg/L.
6. the method for utilizing laccase degraded octyl phenol according to claim 1 is characterized in that described enzyme liquid addition is less than 2.0U/mL.
7. the method for utilizing laccase degraded octyl phenol according to claim 1 is characterized in that described degradation time is 12～24h.
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| CN108164005A (en) * | 2018-01-15 | 2018-06-15 | 湖北民族学院 | A kind of method using Cortex Magnoliae Officinalis blade crude enzyme liquid degradation nonyl phenol |
| CN109574255A (en) * | 2019-01-03 | 2019-04-05 | 重庆工商大学 | A kind of method of laccase continuous processing phenol wastewater |
| CN110845018A (en) * | 2019-11-07 | 2020-02-28 | 桂林理工大学 | Method for improving degradation rate of laccase for degrading diethylstilbestrol |
| CN111302499A (en) * | 2019-11-07 | 2020-06-19 | 桂林理工大学 | Method for rapidly catalyzing and degrading diethylstilbestrol |
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| CN111302499B (en) * | 2019-11-07 | 2022-04-08 | 桂林理工大学 | Method for rapidly catalyzing and degrading diethylstilbestrol |
| CN114606151A (en) * | 2022-04-27 | 2022-06-10 | 南京工业大学 | Recombinant pichia pastoris with surface displaying beta-galactosidase and construction method and application thereof |
| CN114606151B (en) * | 2022-04-27 | 2023-05-19 | 南京工业大学 | Recombinant pichia pastoris with beta-galactosidase displayed on surface, construction method and application |
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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 |