CN101407794B - Laccase inducer and use thereof for improving microbial laccase production ability - Google Patents
Laccase inducer and use thereof for improving microbial laccase production ability Download PDFInfo
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- CN101407794B CN101407794B CN200810233650.5A CN200810233650A CN101407794B CN 101407794 B CN101407794 B CN 101407794B CN 200810233650 A CN200810233650 A CN 200810233650A CN 101407794 B CN101407794 B CN 101407794B
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- laccase
- inducer
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- bagasse
- ethanol
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 67
- 239000000411 inducer Substances 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title claims description 13
- 230000000813 microbial effect Effects 0.000 title claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 241000609240 Ambelania acida Species 0.000 claims abstract description 32
- 239000010905 bagasse Substances 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 241000233866 Fungi Species 0.000 claims abstract description 9
- 235000015097 nutrients Nutrition 0.000 claims description 24
- 229960004756 ethanol Drugs 0.000 claims description 21
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 230000001939 inductive effect Effects 0.000 claims description 13
- 230000003068 static effect Effects 0.000 claims description 11
- 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 9
- 239000008103 glucose Substances 0.000 claims description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 8
- 239000002054 inoculum Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 abstract 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 22
- 229920005610 lignin Polymers 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 230000006698 induction Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 150000002989 phenols Chemical class 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 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 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- -1 phenylpropyl alcohol alkane Chemical class 0.000 description 2
- VOWZNBNDMFLQGM-UHFFFAOYSA-N 2,5-dimethylaniline Chemical group CC1=CC=C(C)C(N)=C1 VOWZNBNDMFLQGM-UHFFFAOYSA-N 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 241001290175 Coriolopsis trogii Species 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108010054320 Lignin peroxidase Proteins 0.000 description 1
- 108010059896 Manganese peroxidase Proteins 0.000 description 1
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 1
- 241000222350 Pleurotus Species 0.000 description 1
- 229920001131 Pulp (paper) Polymers 0.000 description 1
- 241000222645 Trametes cinnabarina Species 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 231100001234 toxic pollutant Toxicity 0.000 description 1
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Abstract
The invention discloses a compound inducer for improving the capacity of a microorganism strain for producing a laccase, which comprises bagasse, ethanol and a bluestone liquid; and a comparison research carried out on a whiterot fungi strain XG8 by utilizing the compound inducer shows the laccase capacity in a fermentation liquid is improved by 10 times than that of the strain not added with thecompound inducer. The inducer of the invention is quite suitable for being applied to the large-scale production of industrialized laccases.
Description
Technical field
The invention belongs to biological technical field, particularly, relate to laccase inducer, and the application in improving microbial laccase production ability.
Background technology
White-rot fungi is the most important lignin degradation bacterium of nature, its generation and secrete a series of can lignin degradings and the enzyme of xenobiontics, they mainly comprise lignin peroxidase, manganese peroxidase and laccase.Laccase mainly is by the basidiomycetes secretion that causes the timber white rot, it is important lignin degradation enzyme, a class polyphenoloxidase that belongs to cupric, it can make the phenolic compound oxidation by molecular oxygen, remove the lignin in timber, the paper pulp, make effluent decolouring and degraded in the pulp and paper industry, can significantly reduce the toxic pollutant that is discharged in the environment.
It is reported that phenols substrate, ethanol and some metal ion have inducing action to generation and the secretion of laccase.The phenols substrate all contains more-OH ,-NH usually
2, xylogen be the polymer that constituted by phenylpropyl alcohol alkane monomers such as tonquinol, lubanol and sinapyl alcohols as substrate analogue, can induce the generation of laccase in certain degree.Alcohols also has certain inducing action to the generation of laccase, and the abduction mechanism of ethanol mainly is divided into following 3 points (i) has increased membrane permeability, promotes the secretion of albumen; (ii) prevent single aromatic compound polymerization; (iii) pass through the synthetic of oxygenizement indirect induction laccase.The inducing action of metal ion has a lot of reports, wherein CuSO
4Be to use at most, induce best heavy metal inductor, Cu
2+Adding improved the activity of laccase from two aspects, promoted the synthetic of laccase, strengthened the stability of laccase in extracellular environment.
According to the domestic and foreign literature report, different culture condition has remarkable influence to the laccase vigor.Test shows that static cultivation is conducive to the XG8 strain enzyme-producing, and this may be relevant to the mechanical shear stress sensitivity that vibration produces with the laccase of some white rot fungus secretion, and visible shaking culture also is not suitable for the generation of part white-rot fungi laccase.
So far, do not utilize bagasse, ethanol and CuSO in the prior art
4The report of the laccase inducer that solution is combined into.
Summary of the invention
The present invention is intended to provide a kind of new prescription for whiterot fungi produces laccase development of new inductor.Utilize bagasse to induce the generation of laccase will be conducive to contained a large amount of lignin molecules in the laccase degraded bagasse in addition, thereby will be separated from each other by the baggasse fiber of lignin molecule adhesion, realize that the delignification of bagasse is handled, the bagasse paper industry is had significant application value.In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
Laccase inducer comprises bagasse, ethanol and copper-bath.
Laccase inducer, the inductor composition is in the 100ml liquid nutrient medium: bagasse dry weight: 10-15g.L
-1, CuSO
4: 5-50mg.L
-1, ethanol: 20-50g.L
-1
Laccase inducer, the inductor composition is in the 100ml liquid nutrient medium: bagasse dry weight: 10-15g.L
-1, CuSO
4: 20-30mg.L
-1, ethanol: 40-50g.L
-1
Laccase inducer is used for improving microbial laccase production ability.
Laccase inducer is used for the cultural method of microbial laccase production ability:
(1) preparation of inoculum:
The microorganism strains lawn is inoculated in the liquid of glucose substratum, in 10 days results of 25 ℃ of static cultivations thalline, with the thalline fracture, as inoculum;
(2) inducing culture of bacterial strain:
Inoculum is inoculated in the liquid basic medium, and static cultivation is cultivated and is added laccase inducer after 4 days in nutrient solution, carries out inducing culture and detects and extracted laccase in 15 days.
In the described method, seed culture medium consists of: MgSO
47H
2O, 0.5g, NaCl0.5g, KH
2PO
41.0g, NH
4SO
41.0g, yeast powder 1.0g, glucose 15g adds water and is settled to 1 liter, transfers pH5~6.
The preparation of inductor and employing:
CuSO
4Solution: preparation 1g.L
-1CuSO
4Original solution, 115 ℃ of sterilization 20min; In the nutrient solution of 100ml, add 1g.L respectively
-1CuSO
4Solution makes CuSO in the nutrient solution
4Final concentration is 5-50mg.L
-1
Ethanolic soln: dehydrated alcohol is with the membrane filtration degerming of 0.2 μ m; In the nutrient solution of 100ml, add 2.5ml respectively, 3.875ml, 5.375ml, the 6.25ml dehydrated alcohol makes that the ethanol final concentration is ethanol: 20-50g.L in the nutrient solution
-1
Solid bagasse: take by weighing 1g and be about the bagasse (dry weight) of 2cm in 115 ℃ of sterilization 20min.
Culture condition is: lawn is inoculated in the 100ml liquid of glucose substratum, in 10 days results of 25 ℃ of static cultivations thalline, with bacterial cell disruption, equivalent is inoculated in the 100ml liquid base substratum, in static, vibration and add under the different condition such as inductor and cultivate, add inductor bagasse dry weight: 10-15g.L after 4 days at yeast culture respectively
-1, CuSO
4: 5-50mg.L
-1, ethanol: 20-50g.L
-1In nutrient solution, carry out inducing culture and extracted laccase and detection in 15 days.
Excellent beneficial effect of the present invention is:
Lignin is one of effect substrate of laccase, adds the generation that a certain amount of lignin solution is conducive to laccase in nutrient solution, and lignin, bagasse, ethanol and CuSO have been adopted in this test
4The inductor of these several cheapnesss of solution, test-results show, bagasse induce effect best, bagasse has certain effect of inducing may be relevant with the inducing action that wherein contains a certain amount of lignin and phenols inductor such as forulic acid, coumaric acid.With domestic and foreign literature report with expensive ABTS and 2,5-xylidene(s) as inductor, the effect of inducing of bagasse has more the advantage of industrial applications.
The laccase production ability of edible mushrooms white rot enzyme XG8 bacterial strain self is not as good as the bright red samguineus of reporting at present (Pycnoporus cinnabarinus), hair bolt bacterium (Trametes trogii) etc., but after it adds bagasse, ethanol and copper sulfate, laccase induces effect remarkable, and a kind of new prescription that provides that other whiterot fungi bacterial strain produces laccase development of new inductor is provided.Utilize bagasse to induce the generation of laccase will be conducive to contained a large amount of lignin molecules in the laccase degraded bagasse in addition, thereby will be separated from each other by the baggasse fiber of lignin molecule adhesion, realize the delignification processing of bagasse, had important use to be worth to the bagasse paper industry.Improve edible mushrooms high No. 8 bacterial strains in Hunan (pleurotus) laccase production ability, this combination inductor both can be applicable to other whiterot fungi bacterial strain and had been used for improving its laccase production ability.
Description of drawings
Fig. 1 is that the laccase activity under lignin, bagasse are induced compares;
The laccase activity that Fig. 2 induces for the different concns cupric ion compares;
The laccase activity that Fig. 3 induces for different concentration ethanol compares;
Fig. 4 compares for the synergistic laccase activity of combination inductor.
Embodiment
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiment, but do not limit the present invention with this.
Embodiment 1:
1. bacterial strain
Edible mushrooms XG8 bacterial strain: purchase in Kunming, Kunming, Yunnan Province edible mushrooms institute.
2. substratum, inductor and culture condition
2.1 substratum
Substratum is formed: MgSO
47H
2O, 0.5g, NaCl0.5g, KH
2PO
41.0g, NH
4SO
41.0g, yeast powder 1.0g, glucose 15g adds water and is settled to 1 liter, transfers pH5~6.This substratum is mainly used in the preservation bacterial classification and reaches in induction experiment as basic medium.
2.2 inductor
CuSO
4Solution: preparation 1g.L
-1CuSO
4Original solution, 115 ℃ of sterilization 20min.In induction experiment, treat that the mycelium cultivation after four days, adds 1g.L respectively in the nutrient solution of 100ml
-1CuSO
4Solution makes CuSO in the nutrient solution
4Final concentration is respectively 5mg.L
-1, 8mg.L
-1, 10mg.L
-1, 20mg.L
-1, 50mg.L
-1
Ethanolic soln: dehydrated alcohol is with the membrane filtration degerming of 0.2 μ m.In induction experiment, treat that mycelium cultivates after four days, in the nutrient solution of 100ml, add 2.5ml respectively, 3.875ml, 5.375ml, the 6.25ml dehydrated alcohol makes that the ethanol final concentration is respectively 20g.L in the nutrient solution
-1, 31g.L
-1, 43g.L
-1, 50g.L
-1
Solid bagasse: take by weighing 1g and be about the bagasse (dry weight) of 2cm in 115 ℃ of sterilization 20min.In induction experiment, treat that the mycelium cultivation was added in the nutrient solution after four days.
2.3 culture condition
An amount of lawn is inoculated in the 100ml liquid of glucose substratum, in 10 days results of 25 ℃ of static cultivations thalline, the back with 30 sterilized granulated glass spherees (diameter 2mm) with bacterial cell disruption, the thalline equivalent of fragmentation is inoculated in the triangular flask of the 250ml that 100ml liquid base substratum is housed, respectively in static, vibration and add under the different condition such as inductor and cultivate, wherein inductor adds in the nutrient solution after 4 days at yeast culture, carries out inducing culture.
Add the combination inductor in the liquid nutrient medium of cultivating the whiterot fungi bacterial strain, to improve its enzymatic productivity, the component proportions of adding the combination inductor is: bagasse dry weight: 10-15g.L
-1, CuSO
4: 5-50mg.L
-1, ethanol: 20-50g.L
-1
Preferably: bagasse (dry weight): 10-15g.L
-1, CuSO
4: 20-22mg.L
-1, ethanol: 43-45g.L
-1Continue to cultivate detection in about 15 days and extract laccase after adding the combination inductor.
3 laccase vitality tests
Laccase measures with 2,2 '-azine-two (3-ethyl benzothiazoles-6-sulfonic acid) (being called for short ABTS) are substrate, contain 0.15mmol.L in the reaction system
-1ABTS, 0.1mol.L
-1Sodium-acetate buffer (pH5.2) and an amount of crude enzyme liquid, reaction 5min under 30 ℃, assaying reaction liquid is at the increased value (ε of 420nm place light absorption value
420=36000M
-1Cm
-1).It is 1 unit of activity (U) that the definition per minute makes 1 μ mol ABTS transform required enzyme amount.
Claims (5)
1. a laccase inducer is characterized in that for the cultural method that improves microbial laccase production ability:
(1) preparation of inoculum: whiterot fungi bacterial strain tongue is inoculated in the liquid of glucose substratum, in 10 days results of 25 ℃ of static cultivations thalline, with the thalline fracture, as inoculum;
(2) inducing culture of bacterial strain: inoculum is inoculated in the liquid base substratum, and static cultivation is cultivated and is added laccase inducer after 4 days in nutrient solution, carries out inducing culture and extracts laccase in 15 days and detected laccase content;
Described laccase inducer comprises bagasse, ethanol and copper-bath, wherein is bagasse dry weight 1.0-1.5g in the 100ml liquid nutrient medium; CuSO
40.5-5.0mg; Ethanol 2.0-5.0g.
2. laccase inducer as claimed in claim 1 is used for improving the cultural method of microbial laccase production ability, it is characterized in that preferably bagasse dry weight 1.0-1.5g in the 100ml liquid nutrient medium; CuSO
42.0-3.0mg; Ethanol 4.0-5.0g.
3. laccase inducer as claimed in claim 1 is used for improving the cultural method of microbial laccase production ability, it is characterized in that the liquid of glucose substratum of described inoculum preparatory phase consists of:
MgSO
47H
2O, 0.5g, NaCl0.5g, KH
2PO
41.0g, (NH
4)
2SO
41.0g, yeast powder 1.0g, glucose 15g adds water and is settled to 1 liter, transfers pH5~6.
4. laccase inducer is characterized in that preparation and the employing of inductor for the cultural method that improves microbial laccase production ability according to claim 1:
CuSO
4Solution: preparation 1g.L
-1CuSO
4Original solution, 115 ℃ of sterilization 20min; Adding concentration in the nutrient solution of 100ml respectively is 1g.L
-1CuSO
4Solution makes CuSO in the nutrient solution
4Ultimate density is 5-50mg.L
-1
Ethanolic soln: dehydrated alcohol is with the membrane filtration degerming of 0.2 μ m; In the nutrient solution of 100ml, add 2.5ml respectively, 3.875ml, 5.375ml, the 6.25ml dehydrated alcohol makes that the ethanol final concentration is ethanol 20-50g.L in the nutrient solution
-1
Solid bagasse: take by weighing dry weight 1g, be about the bagasse of 2cm, at 115 ℃ of sterilization 20min.
5. laccase inducer as claimed in claim 1 is used for improving the cultural method of microbial laccase production ability, it is characterized in that culture condition is: lawn is inoculated in the 100ml liquid of glucose substratum, in 10 days results of 25 ℃ of static cultivations thalline, with bacterial cell disruption, equivalent is inoculated in the 100ml liquid base substratum, under static and interpolation inductor condition, cultivate respectively, after 4 days, add inductor bagasse dry weight: 10-15g.L at yeast culture
-1, CuSO
4: 5-50mg.L
-1, ethanol: 20-50g.L
-1In nutrient solution, carry out inducing culture and extracted laccase in 15 days and detected laccase content.
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BR112013003725B1 (en) * | 2010-08-17 | 2020-05-26 | Council Of Scintific & Industrial Research | METHOD FOR OBTAINING LACASE ENZYME FROM ARTHROGRAPHIS SP |
CN103834695B (en) * | 2012-05-07 | 2016-06-08 | 安徽大学 | Fungus laccase inducing active compound and fermentation preparation method and application thereof |
CN102719410B (en) * | 2012-06-29 | 2016-01-20 | 浙江农林大学 | A kind of culture medium prescription being exclusively used in laccase and preparation method thereof |
CN103571801A (en) * | 2012-08-01 | 2014-02-12 | 深圳市绿微康生物工程有限公司 | Fermentation production method of fungal laccase and application of laccase |
CN104726425B (en) * | 2015-02-15 | 2018-08-03 | 东华大学 | A kind of method that candida tropicalis DK2 bacterial strains prepare laccase |
CN105624133A (en) * | 2016-01-20 | 2016-06-01 | 徐向群 | Technique for producing Inonotus obliquus lignocellulose catabolic enzyme by liquid submerged fermentation |
CN112359026B (en) * | 2020-11-18 | 2022-08-19 | 山东农业大学 | Method for increasing laccase content in auricularia polytricha |
CN113388660B (en) * | 2021-06-29 | 2023-05-19 | 白银赛诺动物保健技术有限公司 | Liquid color development method for rapidly screening laccase-producing bacteria |
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