CN103053876B - Biodegradation method of ginkgolic acid - Google Patents
Biodegradation method of ginkgolic acid Download PDFInfo
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- CN103053876B CN103053876B CN201210552048.4A CN201210552048A CN103053876B CN 103053876 B CN103053876 B CN 103053876B CN 201210552048 A CN201210552048 A CN 201210552048A CN 103053876 B CN103053876 B CN 103053876B
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Abstract
The invention discloses a biodegradation method of ginkgolic acid. Laccase LacC is utilized; and according to the proportion, when the ginkgolic acid with a certain concentration is contained in a degradation system, the concentration of the added Laccase LacC is 0.004 U/mL to 0.006 U/mL, the pH of the degradation system is 4.0-6.0, the degradation temperature is 45 DEG C to 60 DEG C, and the degrading treatment time lasts for 10-24 hours. When only the laccase is used for degrading the ginkgolic acid, the degradation efficiency is less than 30%; and after a mediator is added, and especially after ABTS is added, the degradation efficiency is highest to 100%.
Description
Technical field
The present invention is intended to set up a kind of method of laccase biodegradation ginkgolic acid, relates to enzyme engineering and field of food.
Background technology
The main component of ginkgo biloba p.e (EGB) is flavones and lactone compound, has the effect of anti-oxidant, antiplatelet activity factor (PAF) and the effect of antagonism PAF specific receptor.A large amount of pharmacology analysis and clinical trial confirm, ginkgo biloba p.e has significant prevention and result for the treatment of to coronary heart disease, hypertension, angina pectoris, artery sclerosis, Aging, senile dementia, Senile Memory Deficits, aging etc. and the cardiovascular and cerebrovascular relevant disease that circulates.Ginkgo biloba p.e is widely used in medicine, health products, food and cosmetics, and moral, Fa Deng state be a large amount of import ginkgo leaves of several countries (being mainly Korea S, China) from East Asia every year, as pharmaceutical raw material, and the sale of finished goods whole world.But ginkgolic acid is present in gingko episperm, ginkgo leaf and ginkgo nut, and in exosper, content is the highest.Its structure can regard that salicylic acid molecule has the series compound of longer side chain on phenyl ring C6 position as, this long-chain divides alkyl and the large class of thiazolinyl two, generally formed by 13-17 carbon atom, belong to cytotoxin, may with induction body sudden change and carcinogenic relevant, can cause that the allergic inflammation that skin, mucous membrane etc. are located reacts.Therefore, ginkgolic acid (Ginkgolic acids) is limit index crucial in ginkgo biloba p.e.But in extraction ginko leaves flavone and lactone compound isoreactivity substance process, ginkgolic acid also can be leached out simultaneously.Its content is different and different with extraction process, purifying process.But ginkgolic acid severe overweight in domestic most ginkgo biloba p.e, people's health in serious harm.At present, the strict control to ginkgolic acid content in ginkgo biloba p.e in the world, has promoted the research dynamics of people to selectively removing separating Ginkgo phenolic acids material.At present, the method for reporting is mainly to improve extraction process, strengthening purification link.The yield that has reduced undoubtedly active material, has increased preparation cost.
Laccase is a kind of polyphenol oxidase of cupric, and this enzyme ubiquity in whiterot fungi also produces in minority lower fungus and plant, mostly is secreting type glycoprotein.Because laccase is a kind of oxidoreducing enzyme, its effect is mainly catalytic oxidation-reduction reaction, oxidation reaction that can a lot of phenol type of catalysis compound, but because its redox potential is lower, non-phenols structural compounds but can not be degraded in direct oxidation, or not high to much phenol type degradation efficiency.If under some little molecular oxidation reducing medium helps, the substrate scope of laccase effect can further expand, and can be oxidized the organic compound of non-phenol type, also can improve significantly the degradation efficiency of phenol type compound.Laccase redox mediator system (LMS) refers to that laccase carries out the process of living things catalysis under the existence of amboceptor and oxygen.Amboceptor can form active high and have the intermediate of certain stability under the effect of enzyme, and these reactive intermediates can pass to substrate by electron gain from oxygen molecule, thereby make degradation of substrates.Laccase/redox mediator system more and more receives concern both domestic and external as a kind of system with very large potential using value.
In view of ginkgolic acid severe overweight in domestic most ginkgo biloba p.es, people's health in serious harm.At present, the method for reporting is mainly to improve extraction process, strengthening purification link.The yield that has not only reduced active material, has increased preparation cost, and effect is obvious not, is difficult to reach the limit standard to ginkgolic acid content in ginkgo biloba p.e in the world.And up to the present, utilize laccase or laccase mediators system to carry out biodegradation to ginkgolic acid and yet there are no research report at home and abroad.
Summary of the invention
The technical problem solving:
The object of the invention is to set up a kind of method of biodegradation ginkgolic acid, be intended to some ginkgo goods effectively removing ginkgolic acid after laccase degraded.
Technical scheme:
A biodegradation method for ginkgolic acid, utilizes laccase LacC, and in degraded system, the concentration of laccase LacC is 0.004U/mL-0.006U/mL, the pH4.0-6.0 of degraded system, and degradation temperature is 45 DEG C～60 DEG C, the degradation treatment time is 10～24 hours.In degraded system, be added with 2 of final concentration 0.1～1mM, 2 '-Lian nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), I-hydroxybenzotriazole (HOBT), murexide (VA) or guaiacol.
Described laccase LacC is made 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 DEG C and cultivates 5d; 1L has been cultivated at 4 DEG C of the refrigerated centrifuges that zymotic fluid is placed in 10000rpm to centrifugal 10min to be collected supernatant and is laccase crude enzyme liquid; Then in supernatant, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, and precipitation is dissolved dialysis with 20mM pH 7.5Tris-HCl buffer; The enzyme liquid that dialysis treatment is crossed carries out DEAESepharose
tMcL-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, and every 4mL collects a pipe and detects laccase activity, three protein peaks detected by Protein Detection instrument, that last peak is collected is laccase LacC.
Described laccase LacC also can be made by following method:
(1) cultivation of bolt bacterium (Trametes versicolor): culture medium is PDA: glucose 20g/L, potato juice 20%wt, inoculates fresh bolt bacterium mycelia, and 180rpm cultivates to filter for 3-4 days and collects mycelium at 28 DEG C;
(2) extraction of the total RNA of bolt bacterium: get after the mycelium PBS cushioning liquid cleaning of bolt bacterium of collection, under liquid nitrogen, grind mycelium to Powdered, collect in 2mL centrifuge tube, add concuss after 1mL Trizol, at 4 DEG C, after the centrifugal 10min of 12000g, shift supernatant to 2mL centrifuge tube, add 200 μ L chloroform concussion 15s to mix at latter 30 DEG C and hatch 2-3min, at 4 DEG C, the centrifugal 10min of 12000g gets the upper strata stillness of night, add the isopropyl alcohol of 0.8 times of volume of supernatant to mix the centrifugal 10min of 12000g at latter 4 DEG C, remove clean 2 times with 75%wt ethanol water after supernatant after at 4 DEG C the centrifugal 5min of 7500g be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid, at-20 DEG C, preserve,
(3) bolt bacterium cDNA's is synthetic: taking the total RNA of bolt bacterium as template, utilize reverse transcription to synthesize cDNA the first chain:
In microcentrifugal tube, prepare following template ribonucleic acid/Primer reactant liquor:
50μM Oligo dT 1μL，
10mM dNTP Mixture 1μL，
Total RNA 1 μ g,
DEPC-H
2O 7μL；
Mix after being incubated 5min at latter 65 DEG C and place 1min on ice;
In above-mentioned microcentrifugal tube, prepare following cDNA synthesis reaction solution:
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;
After above-mentioned reactant liquor mixes, at 50 DEG C, be incubated 1h, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtaining synthetic for cDNA the second chain immediately;
(4) clone of bolt bacterium gene: design LacC primer, carrier is pPICZ α B:
P9:GGG
GAATTCATGGGCAAGTTTCACTCTTTTGTGAA；
P10:GGG
TCTAGATCAGAGGTCGGACGAGTCCAAA；
The clone of gene LacC utilizes primer P9 and P10, and reaction condition is 94 DEG C, 5min; Time out, adds Ex Taq 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 LacC, use EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, the carrier after gene LacC cuts with enzyme is connected and spends the night respectively, conversion Escherichia coli, obtain recombinant plasmid pPICZ alpha B-LacC;
(5) preparation of restructuring laccase LacC: by after recombinant plasmid pPICZ alpha B-LacC linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, recombinant yeast is lined on YPD flat board and activated, cultivate 2 days for 28 DEG C, after growing single bacterium colony, be inoculated in 10mL BMGY fluid nutrient medium, in 30 DEG C, 200r/min shaking table is cultivated 48h, OD
600reach the centrifugal 5min of 2 ~ 6,3000rpm and collect thalline, abandon supernatant, with sterile purified water washing thalline 1 ~ 2 time; Thalline is diluted to OD with BMMY inducing culture
600=1, with 4 layers of gauzes replacement cotton plug, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); Cultured culture medium being placed at 4 DEG C of the refrigerated centrifuges of 10000rpm to centrifugal 10min collects supernatant and is laccase crude enzyme liquid; First ultrafiltration apparatus is concentrated to laccase crude enzyme liquid; Then in concentrate, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, and precipitation is dissolved dialysis with 20mM pH 7.0Tris-HCl buffer; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH 7.0Tris-HCl buffer, elution buffer: containing 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 LacC obtains recombinating.
Described degradation temperature is 50 DEG C.
Described 2, the concentration of 2 '-Lian nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts is 0.6mM.
The degradation reaction time is 12h.
The application of laccase LacC in degraded ginkgolic acid.
Concrete scheme content for: the method for a kind of ginkgolic acid of degrading that the present invention sets up is, with laccase, it is carried out to biodegradation, in order to improve degradation efficiency, adds amboceptor, form laccase-mediator system, the ginkgolic acid of can degrading efficiently.
PH4.5 citrate buffer solution+laccase+amboceptor that degraded system of the present invention is ginkgolic acid+100mM, altogether 5mL.50 DEG C of reaction temperatures, shaking bath rotating speed 100rmp, 12 hours reaction time.The n-hexane extraction of 10mL for reaction system after degraded, coextraction three times is drawn supernatant 1mL in small test tube at every turn, merges three extracts (3mL) and uses N
2dry up, add the chromatogram methyl alcohol of 1mL, excessively after the organic filter membrane of 0.45 μ m, be HPLC.The condition of HPLC is: Agilent 1260, Eclipse XDB-C18 chromatographic column (4.6 × 250mm, 5 μ are m), mobile phase is V(methyl alcohol): V(3% glacial acetic acid)=92:8, column temperature is 40 DEG C, detection wavelength is 310nm, flow velocity is 1.5mL/min, sample size 20 μ L, running time 10min.
The assay method of laccase activity of the present invention is: measure with ultraviolet specrophotometer, detection wavelength is 420nm, buffer solution is the citrate buffer solution of the pH4.5 of 100mM, reaction system is: 1980 μ L buffer solution+1mL1mMABTS+20 μ L laccases, buffer solution and ABTS add the preheating 5min in 50 DEG C of water-baths of rear elder generation, after enzyme-added liquid, timing 3min(comprises the 30s that vibrates in water-bath after enzyme-added), the A value of writing down per minute, computing formula is: laccase activity (U/L)={ [(A
3-A
1)/2] × 5000/12} × extension rate.
Beneficial effect: simple degrades to ginkgolic acid with laccase, and its degradation efficiency, less than 30%, adds after amboceptor material, particularly adds after ABTS, and its degradation efficiency reaches as high as 100%.
Brief description of the drawings
Fig. 1 is the result of different restructuring Isozymes of Laccase degraded ginkgolic acids;
Fig. 2 adds the impact of different little molecule amboceptors on laccase LacC degraded ginkgolic acid;
Fig. 3 is the impact on laccase LacC degraded ginkgolic acid of different ABTS concentration;
Fig. 4 is the impact on laccase LacC degraded ginkgolic acid of different enzyme dosage;
Fig. 5 is the impacts of different pH values on laccase LacC degraded ginkgolic acid;
Fig. 6 is the impact of temperature on laccase LacC degraded ginkgolic acid.
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
The preparation of 1.1 Coriolus Versicolors (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 occurring in nature screening, also can buy from commercial channels and obtain (for example can purchased 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, 180rpm cultivates to filter for 3-4 days and collects mycelium at 28 DEG C.
1.1.2 the extraction of the total RNA of Coriolus Versicolor (Coriolus versicolor)
Get after the mycelium PBS cushioning liquid cleaning once of Coriolus Versicolor (Coriolus versicolor) of collection, under liquid nitrogen, grind mycelium to Powdered, collect in 2mL centrifuge tube, add concuss after 1mL Trizol, at 4 DEG C, after the centrifugal 10min of 12000g, shift supernatant to 2mL centrifuge tube, add 200 μ L chloroform concussion 15s to mix at latter 30 DEG C and hatch 2-3min, at 4 DEG C, the centrifugal 10min of 12000g gets the upper strata stillness of night, add the isopropyl alcohol of 0.8 times of volume of supernatant to mix the centrifugal 10min of 12000g at latter 4 DEG C, after removing supernatant, with 75%wt ethanol water, (DEPC processed, remove mRNA enzyme) clean after 2 times the centrifugal 5min of 7500g at 4 DEG C and be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid, at-20 DEG C, preserve.
1.1.3 Coriolus Versicolor (Coriolus versicolor) cDNA's is synthetic
Taking the total RNA of Coriolus Versicolor (Coriolus versicolor) as template, utilize reverse transcription to synthesize cDNA the first chain (following reverse transcription agents useful for same all comes from kit " PrimeScriptTM1stStrand cDNA Synthesis Kit ", purchased from Takara company).
In microcentrifugal tube, prepare following template ribonucleic acid/Primer reactant liquor:
50μM Oligo dT 1μL，
10mM dNTP Mixture 1μL，
Total RNA 1 μ g,
DEPC-H
2O 7μL；
Mix after being incubated 5min at latter 65 DEG C and place 1min on ice
In above-mentioned microcentrifugal tube, prepare following cDNA synthesis reaction solution:
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;
After above-mentioned reactant liquor mixes, at 50 DEG C, be incubated 1h, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtaining synthetic for cDNA the second chain immediately.
1.1.4 the clone of Coriolus Versicolor (Coriolus versicolor) gene
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 represents EcoR I site;
P2:GGG
tCTAGAtTAGAGGTCGGATGAGTC, underscore represents Xba I site;
P3:CCC
aTCGATaGCCATTGGGCCCGTC, underscore represents Cla I site;
P4:CCC
tCTAGAtCAGAGGTCGGACGAG, underscore represents Xba I 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 Ex Taq 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 respectively EcoR I, 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, carrier pPICZ α A after Lcc2 cuts with enzyme is connected and spends the night respectively, transforms Escherichia coli, obtains recombinant plasmid pPICZ alpha A-Lcc1 and pPICZ α A-Lcc2.
1.1.5 the recombinate preparation of laccase
By after recombinant plasmid pPICZ alpha A-Lcc1 linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, and recombinant yeast is lined on YPD flat board and activated, cultivate 2 days, after growing single bacterium colony, be inoculated in 10mL BMGY fluid nutrient medium, in 30 DEG C for 28 DEG C, 200r/min shaking table is cultivated 48h, OD
600reach the centrifugal 5min of 2 ~ 6,3000rpm and collect thalline, abandon supernatant, with sterile purified water washing thalline 1 ~ 2 time.Thalline is diluted to OD with BMMY inducing culture
600=1, with 4 layers of gauzes replacement cotton plug, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V).
The culture medium that (is cultured to the 13rd day for best) by cultivating is placed at 4 DEG C of the refrigerated centrifuges of 10000rpm centrifugal 10min and collects supernatant and be laccase crude enzyme liquid.First ultrafiltration apparatus is concentrated to laccase crude enzyme liquid; Then in concentrate, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C, 20mM Tris-HCl buffer(pH 7.0 for precipitation at 4 DEG C) dissolve dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM Tris-HCl buffer(pH 7.0), elution buffer: containing 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.
By above-mentioned same method, laccase Lcc2 obtains recombinating.
The original bacterium bolt of 1.2 use bacterium (Trametes versicolor) directly cultivation and fermentation is prepared laccase LacA and LacB and LacC
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), 1mmol/L guaiacol, 0.1mL Tween 80,0.3mmol/L Cu
2+, condition of culture is 28 DEG C and cultivates 5d.Collect supernatant and be laccase crude enzyme liquid having cultivated zymotic fluid (1L) and be placed at 4 DEG C of the refrigerated centrifuges of 10000rpm centrifugal 10min.Then in supernatant, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C, 20mMTris-HCl buffer(pH 7.5 for precipitation at 4 DEG C) dissolve dialysis; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
tMcL-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, three protein peak figure detected by Protein Detection instrument, collect respectively three kinds of laccases, and called after laccase LacA and LacB and LacC.
The preparation of 1.3 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 purchased 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.
1d3.3 bolt bacterium Trametes versicolor cDNA's is synthetic
The same 1.1.3 of method.
1.3.4 the clone of bolt bacterium Trametes versicolor gene
The bolt bacterium Trametes versicolor laccase gene sequence (LccA:AB212732 announcing according to NCBI; LccB:AB212722; LccC:AB212734) design respectively primer:
LacA primer: (carrier pPICZ α B)
P5:GGG
gAATTCaTGGGTCTGCAGCGATT; Underscore represents EcoR I site
P6:GGG
tCTAGAtCACTGGTTAGCCTCGCTCA; Underscore represents Xba I 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 Ex Taq 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 EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, carrier after gene LacA, LacB cut with enzyme with LacC is connected and spends the night respectively, transform Escherichia coli, obtain recombinant plasmid pPICZ alpha B-LacA, pPICZ α C-LacB and pPICZ α B-LacC.
1.3.5 the recombinate preparation of laccase LacA, LacB and LacC
The same 1.1.5 of method, the pure enzyme of obtain recombinating laccase LacA, LacB and LacC.
The result of the different restructuring Isozymes of Laccase of embodiment 2 degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, in the reaction system that contains this substrate, add respectively five kinds of laccases to 0.5U/mL; With citrate buffer solution adjusting pH to 4.5, control temperature at 50 DEG C; Under rotating speed with 100rpm in shaking bath, react the residual quantity that detects ginkgolic acid after 12h.
The detection method of ginkgolic acid is as follows: the first reaction system to 5mL, add the n-hexane of 2 times of volumes to carry out liquid-liquid extraction, each sample extraction three times, to ensure that the ginkgolic acid in reaction system extracts completely, standing 30min gets respectively upper strata hexane solution 1mL after the complete layering of solution, and Nitrogen evaporator dries up rear with 1mL methyl alcohol dissolution process sample.Ginkgolic acid residual quantity in above-mentioned reaction system is carried out to analyzing and testing with HPLC.Liquid chromatograph is Agilent 1260, Eclipse XDB-C18 chromatographic column (4.6 × 250mm, m), mobile phase is V(methyl alcohol to 5 μ): V(3% glacial acetic acid))=92:8, flow velocity is 1.5mL/min, sample size 20 μ L, running time 10min.
The computing formula of ginkgolic acid degradation rate of the present invention is:
Degradation rate (%)=[(initial concentration-residual concentration)/initial concentration] × 100%.
Concrete outcome as shown in Figure 1, has carried out five kinds of laccase LacA, LacB, LacC, Lcc1 and Lcc2 after 12h in reaction the degradation rate of ginkgolic acid has been respectively to 15.5%, 8.7%, 27.0%, 11.7%, 4.1%.Show with these five kinds of laccases, ginkgolic acid to be degraded separately, degradation effect is laccase LacC relatively preferably.
Embodiment 3 researchs add the impact of different little molecule amboceptors on laccase LacC degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, in the reaction system that contains this substrate, add laccase LacC to 0.5U/mL, add respectively small molecular mediator ABTS, guaiacol, HOBT and VA to 1mM; With citrate buffer solution adjusting pH to 4.5, control temperature at 50 DEG C; Under rotating speed with 100rpm in shaking bath, react the residual quantity that detects ginkgolic acid after 12h.Concrete outcome as shown in Figure 2, has carried out adding after 12h the laccase of ABTS, guaiacol, HOBT and VA to be respectively 100%, 86.6%, 54.4%, 57.7% to the degradation rate of ginkgolic acid in reaction.Showing to add the degradation effect of ginkgolic acid after small molecular mediator to significantly improve, add the degradation effect of ABTS best, reach 100%, is secondly guaiacol, and HOBT and VA's is relative lower.
Embodiment 4 studies the impact of different enzyme dosages on laccase LacC degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, adopt the citric acid-sodium citrate buffer solution of pH 4.5, add ABTS to concentration be 0.2mM, add respectively laccase LacC to concentration be 0.002U/mL, 0.004U/mL, 0.006U/mL, 0.008U/mL, 0.01U/mL, 0.025U/mL, 0.05U/mL, 0.075U/mL, 0.1U/mL, 0.125U/mL, control temperature at 50 DEG C, under the rotating speed with 100rpm in shaking bath, react the residual quantity that detects ginkgolic acid after 12h.Specifically result of the test as shown in Figure 3: the degradation rate corresponding to enzyme amount that can obtain above-mentioned concentration is respectively 49.4%, 58.1%, 63.4%, 63.5%, 64.5%, 64.0%, 61.3%, 59.2%, 59.9%, 57.7%.Enzyme dosage is more suitable at 0.004U/mL-0.006U/mL.
Embodiment 5 studies the impact of pH value on laccase LacC degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, add ABTS to concentration be 0.2mM, adding laccase to concentration is 0.01U/mL, regulate respectively to 3.0 of pH, 4.0,4.5,5.0,5.5,6.0 of reaction system with citrate buffer solution, control temperature at 50 DEG C, under the rotating speed with 100rpm in shaking bath, react the residual quantity that detects ginkgolic acid after 12h.Specifically result of the test as shown in Figure 4: reaction has good degradation effect at pH4.0-6.0.
Embodiment 6 studies the impact of temperature on laccase LacC degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, adopt the citric acid-sodium citrate buffer solution of pH 4.5, add ABTS to concentration be 0.2mM, adding laccase to concentration is 0.01U/mL, control temperature and be respectively 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, under the rotating speed with 100rpm in shaking bath, react the concentration that detects ginkgolic acid after 12h.Specifically result of the test as shown in Figure 5: laccase has good degradation effect to ginkgolic acid under the condition of 45 DEG C～60 DEG C, and wherein under 50 DEG C of conditions, the degradation efficiency of ginkgolic acid is best.
Embodiment 7 studies the impact of different ABTS concentration on laccase LacC degraded ginkgolic acid
Using 0.02%wt ginkgolic acid as substrate, adopt the citric acid-sodium citrate buffer solution of pH 4.5, add laccase LacC to concentration be 0.5U/mL, add respectively ABTS to concentration be 0.1,0.2,0.3,0.4,0.5,0.6mM, control temperature at 50 DEG C, under the rotating speed with 100rpm in shaking bath, react the residual quantity that detects ginkgolic acid after 12h.Specifically result of the test as shown in Figure 6: when the concentration of ABTS is 0.1～0.6mM, the degradation rate of ginkgolic acid is respectively 55.3%, 58.7%, 73.2%, 88.1%, 96.8%, 99.7%, the visible increase along with amboceptor ABTS concentration, ginkgolic acid degradation rate increases thereupon, just has the degraded of being close to completely when 0.6mM.
SEQUENCE LISTING
<110> Nanjing Forestry University
The biodegradation method of a <120> ginkgolic acid
<130>
<160> 10
<170> PatentIn version 3.3
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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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Claims (5)
1. the biodegradation method of a ginkgolic acid, it is characterized in that utilizing laccase LacC, in degraded system, the concentration of laccase LacC is 0.004U/mL-0.006U/mL, the pH4.0-6.0 of degraded system, degradation temperature is 45 DEG C～60 DEG C, the degradation treatment time is 10～24 hours, in degraded system, be added with 2 of final concentration 0.1～1mM, 2 '-Lian nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), described laccase LacC is made by following method: cultivate bolt bacterium (Trametes versicolor), culture medium is: wheat bran 20g/L, glucose 5g/L, ammonium tartrate 120mmol/L, 200mmol/L pH5.0 citrate buffer solution, 1mmol/L guaiacol, 0.1mL Tween80, 0.3mmol/L Cu
2+, condition of culture is 28 DEG C and cultivates 5d, 1L has been cultivated at 4 DEG C of the refrigerated centrifuges that zymotic fluid is placed in 10000rpm to centrifugal 10min to be collected supernatant and is laccase crude enzyme liquid, then in supernatant, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, and precipitation is dissolved dialysis with 20mM pH7.5Tris-HCl buffer, the enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH7.5Tris-HCl buffer, elution buffer: 20mM pH7.0Tris-HCl buffer, the concentration gradient of NaCl is 0-0.6mol/L, elution volume is 800mL, elution speed: 1.5mL/min, and every 4mL collects a pipe and detects laccase activity, three protein peaks detected by Protein Detection instrument, that last peak is collected is laccase LacC,
Or, made by following method:
(1) cultivation of bolt bacterium (Trametes versicolor): culture medium is PDA: glucose 20g/L, potato juice 20%wt, inoculates fresh bolt bacterium mycelia, and 180rpm cultivates to filter for 3-4 days and collects mycelium at 28 DEG C;
(2) extraction of the total RNA of bolt bacterium: get after the mycelium PBS cushioning liquid cleaning of bolt bacterium of collection, under liquid nitrogen, grind mycelium to Powdered, collect in 2mL centrifuge tube, add concuss after 1mL Trizol, at 4 DEG C, after the centrifugal 10min of 12000g, shift supernatant to 2mL centrifuge tube, add 200 μ L chloroform concussion 15s to mix at latter 30 DEG C and hatch 2-3min, at 4 DEG C, the centrifugal 10min of 12000g gets the upper strata stillness of night, add the isopropyl alcohol of 0.8 times of volume of supernatant to mix the centrifugal 10min of 12000g at latter 4 DEG C, remove clean 2 times with 75%wt ethanol water after supernatant after at 4 DEG C the centrifugal 5min of 7500g be precipitated, after making it natural drying, add the water-soluble solution of DEPC RNA precipitation, as template ribonucleic acid, at-20 DEG C, preserve,
(3) bolt bacterium cDNA's is synthetic: taking the total RNA of bolt bacterium as template, utilize reverse transcription to synthesize cDNA the first chain:
In microcentrifugal tube, prepare following template ribonucleic acid/Primer reactant liquor:
After above-mentioned reactant liquor mixes, at 50 DEG C, be incubated 1h, be incubated cooled on ice after 15min at 70 DEG C, the reactant liquor obtaining synthetic for cDNA the second chain immediately;
(4) clone of bolt bacterium gene: design LacC primer, carrier is pPICZ α B:
P9:GGG
GAATTCATGGGCAAGTTTCACTCTTTTGTGAA；
P10:GGG
TCTAGATCAGAGGTCGGACGAGTCCAAA；
The clone of gene LacC utilizes primer P9 and P10, and reaction condition is 94 DEG C, 5min; Time out, adds Ex Taq 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 LacC, use EcoR I, Xba I enzyme is cut, simultaneously expression vector also with identical enzyme respectively enzyme cut, the carrier after gene LacC cuts with enzyme is connected and spends the night respectively, conversion Escherichia coli, obtain recombinant plasmid pPICZ alpha B-LacC;
(5) preparation of restructuring laccase LacC: by after recombinant plasmid pPICZ alpha B-LacC linearisation, electricity transforms Pichia pastoris and obtains recombinant bacterium, recombinant yeast is lined on YPD flat board and activated, cultivate 2 days for 28 DEG C, after growing single bacterium colony, be inoculated in 10mL BMGY fluid nutrient medium, in 30 DEG C, 200r/min shaking table is cultivated 48h, OD
600reach the centrifugal 5min of 2～6,3000rpm and collect thalline, abandon supernatant, with sterile purified water washing thalline 1～2 time; Thalline is diluted to OD with BMMY inducing culture
600=1, with 4 layers of gauzes replacement cotton plug, in 28 DEG C, 180r/min shaking table is cultivated, and adding methyl alcohol to final concentration every day is 0.6% (V/V); Cultured culture medium being placed at 4 DEG C of the refrigerated centrifuges of 10000rpm to centrifugal 10min collects supernatant and is laccase crude enzyme liquid; First ultrafiltration apparatus is concentrated to laccase crude enzyme liquid; Then in concentrate, adding final concentration is the ammonium sulfate of 80%wt, places in the refrigerated centrifuge that 24h is placed on 10000rpm centrifugal 30min at 4 DEG C at 4 DEG C, and precipitation is dissolved dialysis with 20mM pH7.0Tris-HCl buffer; The enzyme liquid that dialysis treatment is crossed carries out DEAE Sepharose
tMcL-6B ion-exchange chromatography, adopt equilibrium liquid: 20mM pH7.0Tris-HCl buffer, elution buffer: containing 0.4,0.6, the 20mM pH7.0Tris-HCl buffer of 1.0M, elution speed: 0.5mL/min, every 6min collects a pipe, detect laccase activity, laccase LacC obtains recombinating.
2. the biodegradation method of ginkgolic acid according to claim 1, is characterized in that described degradation temperature is 50 DEG C.
3. the biodegradation method of ginkgolic acid according to claim 1, is characterized in that describedly 2, and the concentration of 2 '-Lian nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts is 0.6mM.
4. the biodegradation method of ginkgolic acid according to claim 1, is characterized in that the described degradation reaction time is 12h.
5. the application of preparation-obtained laccase LacC in degraded ginkgolic acid in the biodegradation method of ginkgolic acid described in claim 1.
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