DE19856928A1 - Enzymatic preparation of mangan ions, superoxide anions and hydrogen peroxide uses laccase - Google Patents
Enzymatic preparation of mangan ions, superoxide anions and hydrogen peroxide uses laccaseInfo
- Publication number
- DE19856928A1 DE19856928A1 DE1998156928 DE19856928A DE19856928A1 DE 19856928 A1 DE19856928 A1 DE 19856928A1 DE 1998156928 DE1998156928 DE 1998156928 DE 19856928 A DE19856928 A DE 19856928A DE 19856928 A1 DE19856928 A1 DE 19856928A1
- Authority
- DE
- Germany
- Prior art keywords
- laccase
- ions
- hydrogen peroxide
- reaction
- iii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 108010029541 Laccase Proteins 0.000 title claims abstract description 24
- -1 mangan ions Chemical class 0.000 title claims abstract description 17
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 7
- 150000007524 organic acids Chemical class 0.000 claims abstract description 5
- 235000005985 organic acids Nutrition 0.000 claims abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 4
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 18
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 150000002500 ions Chemical class 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 7
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims 1
- 235000011180 diphosphates Nutrition 0.000 claims 1
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 claims 1
- 229940049920 malate Drugs 0.000 claims 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 239000007787 solid Substances 0.000 claims 1
- 229940095064 tartrate Drugs 0.000 claims 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 abstract description 2
- 108010059896 Manganese peroxidase Proteins 0.000 description 13
- 239000011572 manganese Substances 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 229910052802 copper Inorganic materials 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 230000003647 oxidation Effects 0.000 description 9
- 238000007254 oxidation reaction Methods 0.000 description 9
- 102000019197 Superoxide Dismutase Human genes 0.000 description 6
- 108010012715 Superoxide dismutase Proteins 0.000 description 6
- MMIPFLVOWGHZQD-UHFFFAOYSA-N manganese(3+) Chemical compound [Mn+3] MMIPFLVOWGHZQD-UHFFFAOYSA-N 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 5
- 239000007800 oxidant agent Substances 0.000 description 5
- 241001360262 Hypholoma frowardii Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- NXYBZKHBQWRTLK-UHFFFAOYSA-B manganese(3+);phosphonato phosphate Chemical class [Mn+3].[Mn+3].[Mn+3].[Mn+3].[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O.[O-]P([O-])(=O)OP([O-])([O-])=O NXYBZKHBQWRTLK-UHFFFAOYSA-B 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 2
- 108700020962 Peroxidase Proteins 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000033558 biomineral tissue development Effects 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- IKCLCGXPQILATA-UHFFFAOYSA-N 2-chlorobenzoic acid Chemical class OC(=O)C1=CC=CC=C1Cl IKCLCGXPQILATA-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- YVGGHNCTFXOJCH-UHFFFAOYSA-N DDT Chemical compound C1=CC(Cl)=CC=C1C(C(Cl)(Cl)Cl)C1=CC=C(Cl)C=C1 YVGGHNCTFXOJCH-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 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 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000007824 aliphatic compounds Chemical class 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 150000004951 benzene Polymers 0.000 description 1
- 150000001555 benzenes Polymers 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000011280 coal tar Substances 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005202 decontamination Methods 0.000 description 1
- 230000003588 decontaminative effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 150000002013 dioxins Chemical class 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- JLYXXMFPNIAWKQ-GNIYUCBRSA-N gamma-hexachlorocyclohexane Chemical compound Cl[C@H]1[C@H](Cl)[C@@H](Cl)[C@@H](Cl)[C@H](Cl)[C@H]1Cl JLYXXMFPNIAWKQ-GNIYUCBRSA-N 0.000 description 1
- JLYXXMFPNIAWKQ-UHFFFAOYSA-N gamma-hexachlorocyclohexane Natural products ClC1C(Cl)C(Cl)C(Cl)C(Cl)C1Cl JLYXXMFPNIAWKQ-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 229960002809 lindane Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- KUDPGZONDFORKU-UHFFFAOYSA-N n-chloroaniline Chemical class ClNC1=CC=CC=C1 KUDPGZONDFORKU-UHFFFAOYSA-N 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 125000001997 phenyl group Polymers [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 238000010405 reoxidation reaction Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 238000009279 wet oxidation reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/08—Reclamation of contaminated soil chemically
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
- C02F1/5236—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using inorganic agents
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/72—Treatment of water, waste water, or sewage by oxidation
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Environmental & Geological Engineering (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Inorganic Chemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Soil Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Die Erfindung betrifft ein Verfahren zur enzymatischen Erzeugung von Mangan(III)-Ionen, Superoxid-Anionen und Wasserstoffperoxid als Oxidan tien mit hoher Oxidationskraft. Das Verfahren kann vielseitig im (umwelt-) technischen und gewerblichen Bereich sowie in der Abfallwirtschaft zur Eliminierung von organischen Stoffen in Industrie- und Prozeßabwässern, Böden, und festen Abfallstoffen eingesetzt werden. Weitere Anwendungen ergeben sich u. a. durch Laborverfahren zum Nachweis des enzymatischen Abbaus von nieder- und hochmolekularen Substanzen und Substanz gemischen.The invention relates to a process for the enzymatic production of Manganese (III) ions, superoxide anions and hydrogen peroxide as the oxidane high oxidation power. The process can be versatile in (environmental) technical and commercial area as well as in waste management Elimination of organic matter in industrial and process waste water, Soils, and solid waste materials are used. More applications arise u. a. through laboratory methods for the detection of the enzymatic Degradation of low and high molecular weight substances and substance mix.
Mn(III)-Ionen, die enzymatisch herkömmlicherweise durch Mangan- Peroxidasen aus Weißfäulepilzen gebildet werden, haben sich als effektive Oxidantien bei der Depolymerisierung und Mineralisierung von organischen, sowohl nieder- als auch hochmolekularen Natur- und Fremdstoffen erwiesen, darunter unterschiedlichste persistente Umweltschadstoffe [M. Hofrichter, K. Scheibner, I. Schneegaß and W. Fritsche: "Enzymatic combustion of aromatic and aliphatic compounds by manganese peroxidase from Nematoloma frowardii", (1998), Appl. Environ. Microbiol. 64, S. 399-404; oder M. Hofrichter, K. Scheibner, F. Bublitz, I. Schneegaß, D. Ziegenhagen, R. Martens and W. Fritsche: "Depolymerization of straw lignin by manganese peroxidase from Nematoloma frowardii is accompanied by release of carbon dioxide", (1998), Holzforschung 52, S. 1-6; oder M. Hofrichter, K. Scheibner, I. Schneegaß, D. Ziegenhagen and W. Fritsche: "Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii", (1998), Appl. Microbiol. Biotechnol. 49, S. 584-588]. Mittels Mangan-Peroxidase hergestellte Mangan(III)-Ionen können auch zur Delignifizierung und Bleichung bei der Papierherstellung und zur Behandlung von Abwässern aus der Holz- und Papierindustrie sowie der Textilindustrie eingesetzt werden [JP 09075079 A oder WO 93/20959].Mn (III) ions, which are enzymatically conventionally produced by manganese Peroxidases formed from white rot fungi have proven to be effective Oxidants in the depolymerization and mineralization of organic, both low and high molecular weight natural and foreign substances have been proven, including various persistent environmental pollutants [M. Court judge, K. Scheibner, I. Schneegaß and W. Fritsche: "Enzymatic combustion of aromatic and aliphatic compounds by manganese peroxidase from Nematoloma frowardii ", (1998), Appl. Environ. Microbiol. 64, pp. 399-404; or M. Hofrichter, K. Scheibner, F. Bublitz, I. Schneegaß, D. Ziegenhagen, R. Martens and W. Fritsche: "Depolymerization of straw lignin by manganese peroxidase from Nematoloma frowardii is accompanied by release of carbon dioxide ", (1998), Holzforschung 52, pp. 1-6; or M. Hofrichter, K. Scheibner, I. Schneegaß, D. Ziegenhagen and W. Fritsche: "Mineralization of synthetic humic substances by manganese peroxidase from the white-red fungus Nematoloma frowardii ", (1998), Appl. Microbiol. Biotechnol. 49, pp. 584-588]. Made with manganese peroxidase Manganese (III) ions can also be used for delignification and bleaching Paper manufacture and for the treatment of waste water from the wood and Paper industry and the textile industry are used [JP 09075079 A or WO 93/20959].
Auch der Einsatz von unterschiedlichen mangan-unabhängigen Peroxidasen, beispielsweise Meerrettich-Peroxidase oder Peroxidasen und Lignin-Peroxi dasen aus Pilzen, zur Delignifizierung und Bleichung bei der Papier herstellung und zur Behandlung von Abwässern aus der Holz- und Papier industrie, wie auch der Textilindustrie, ist bekannt [WO 94/12619, EP 0418201 A2, oder DE 195 23 389 A1, oder WO 93/24618]. Diese Enzyme werden weiterhin zum Abbau von durch Kohlenteer verursachten Kontaminationen eingesetzt [WO 88/01255]. Peroxidase-Reaktionen verlan gen entsprechend dem Charakter der katalysierten Reaktion die Anwesenheit von Wasserstoffperoxid.The use of different manganese-independent peroxidases, for example horseradish peroxidase or peroxidases and lignin peroxi made from mushrooms, for delignification and bleaching on paper production and treatment of waste water from wood and paper industry, as well as the textile industry, is known [WO 94/12619, EP 0418201 A2, or DE 195 23 389 A1, or WO 93/24618]. These enzymes continue to be mined by coal tar Contamination used [WO 88/01255]. Peroxidase reactions required presence depending on the nature of the catalyzed reaction of hydrogen peroxide.
Wasserstoffperoxid kann auch als Oxidationsmittel bei der in situ-Sanierung von Grundwässern, die mit polyzyklischen aromatischen Kohlenwasser stoffen (PAK), Chlorbenzoaten, Phenolen, Chloranilinen, polychlorierten Benzenen, Pestiziden, wie DDT oder Lindan, Dioxinen, oder Farbstoffen kontaminiert sind, eingesetzt werden [NL 1000372].Hydrogen peroxide can also act as an oxidant in in situ remediation of groundwater containing polycyclic aromatic hydrocarbons substances (PAH), chlorobenzoates, phenols, chloroanilines, polychlorinated Benzene, pesticides, such as DDT or lindane, dioxins, or dyes are contaminated, are used [NL 1000372].
In chemisch-physikalischen Verfahren zur Wasserdekontamination dient Wasserstoffperoxid als Oxidationsmittel zur Initiierung des Abbaus der Fremdstoffe [K. E. Koeppke und G. von Hagel: "Wastewater treatment by hydrogen peroxide oxidation", (1994), GWF, Gas-, Wasserfach: Wasser/Ab wasser 132, S. 313-317; oder K. E. Koeppke: "Disposal site leachate water treatment by chemical wet oxidation", (1994), Muell Abfall 23, S. 273-280]. Used in chemical-physical processes for water decontamination Hydrogen peroxide as an oxidizing agent to initiate the breakdown of the Foreign substances [K. E. Koeppke and G. von Hagel: "Wastewater treatment by hydrogen peroxide oxidation ", (1994), GWF, Gas-, Wasserfach: Wasser / Ab water 132, pp. 313-317; or K. E. Koeppke: "Disposal site leachate water treatment by chemical wet oxidation ", (1994), Muell Abfall 23, pp. 273-280].
Problematisch bei der Anwendung von Wasserstoffperoxid in enzymatischen oder chemisch-physikalischen Verfahren ist:Problematic when using hydrogen peroxide in enzymatic or chemical-physical process is:
Peroxidasen sind im allgemeinen bei zu hohen Wasserstoffperoxid konzentrationen instabil. Bestimmte Konzentrationsbereiche dürfen somit nicht überschritten werden. Bei Zufuhr externen Wasserstoffperoxids ist daher eine entsprechende Dosierung nötig, die nur durch apparativen Aufwand erreichbar ist. Zusätzlicher Aufwand entsteht durch die notwendige Anlieferung und Lagerung des Wasserstoffperoxids. Letztgenanntes gilt auch für chemisch-physikalische Verfahren.Peroxidases are generally when hydrogen peroxide is too high concentrations unstable. Certain concentration ranges are allowed not be exceeded. When external hydrogen peroxide is added Therefore an appropriate dosage is necessary, which can only be achieved by apparatus Effort is achievable. Additional effort arises from the necessary Delivery and storage of the hydrogen peroxide. The latter also applies for chemical-physical processes.
Bei Bildung von Wasserstoffperoxid im Reaktionssystem durch Wasser stoffperoxid-generierende Enzyme, wie z. B. Glucose-Oxidase, muß ein zusätzliches Enzymsubstrat, wie beispielsweise Glucose, eingesetzt werden. Neben dem damit verbundenen Aufwand erhöht sich auch die Gefahr mikrobieller Fremdinfektionen. Die Langzeitstabilität von Enzymen, wie Glucose-Oxidase, unter konkreten Reaktionsbedingungen ist darüber hinaus begrenzt. Nach einer Woche werden selten mehr als 50% Restaktivität erhalten.When water forms hydrogen peroxide in the reaction system peroxide-generating enzymes, such as. B. glucose oxidase, must additional enzyme substrate, such as glucose, can be used. In addition to the effort involved, the risk also increases external microbial infections. The long-term stability of enzymes, such as Glucose oxidase, under specific reaction conditions, is beyond that limited. After a week there is rarely more than 50% residual activity receive.
Aufgabe der Erfindung ist es, die Oxidantien möglichst langzeitstabil, umweltfreundlich und aufwandgering, insbesondere mit begrenztem Kosten- und Energieeinsatz, bereitzustellen. Die Gefahr von mikrobiellen Fremd infektionen soll dabei weitgehend vermieden werden.The object of the invention is to keep the oxidants stable for as long as possible, environmentally friendly and effortless, especially with limited costs and energy input. The danger of microbial foreign infections should be largely avoided.
Die Aufgabe wird erfindungsgemäß dadurch gelöst, daß die Oxidantien durch enzymatische Aktivierung von Luftsauerstoff erzeugt werden. Als neuartige enzymatische Reaktion wurde überraschend gefunden, daß Laccase in der Lage ist, die Oxidation von Mangan(II) zu Mangan(III) zu katalysieren, und darüber hinaus bei Anwesenheit von niedermolekularen organischen Säuren und Luftsauerstoff die Bildung von Superoxid-Anionen und Wasserstoff peroxid initiiert. Versuche haben ergeben, daß mit diesem Verfahren unter Reaktionsbedingungen nach einem Monat noch ca. 70% Aktivität der Laccase erhalten sind.The object is achieved in that the oxidants by enzymatic activation of atmospheric oxygen are generated. As a novel enzymatic reaction was surprisingly found that laccase in the Able to catalyze the oxidation of manganese (II) to manganese (III), and moreover in the presence of low molecular weight organic acids and atmospheric oxygen the formation of superoxide anions and hydrogen initiated peroxide. Experiments have shown that with this method under Reaction conditions after a month still about 70% activity of the Laccase are preserved.
Das Verfahren kann einzeln oder in Kombination mit existierenden biologischen oder chemisch-physikalischen Verfahren eingesetzt werden.The method can be used individually or in combination with existing ones biological or chemical-physical processes can be used.
Die Erfindung soll nachstehend anhand dreier in den Zeichnungen dargestellter Ausführungsbeispiele näher erläutert werden:The invention is based on three in the drawings illustrated embodiments are explained in more detail:
Es zeigen:Show it:
Fig. 1a: Extinktion von spektralphotometrisch verfolgten Mangan(III)- Pyrophosphat-Komplexen in Abhängigkeit der Reaktionszeit Fig. 1a: Absorbance of spectrophotometric manganese (III) - pyrophosphate complexes depending on the reaction time
Fig. 1b: Extinktion der gemäß Fig. 1a gebildeten Mangan(III)-Pyro phosphat-Komplexe in Abhängigkeit von der Wellenlänge nach vier Stunden Reaktionszeit FIG. 1b: absorbance of Figure 1a formed manganese (III) pyro-phosphate complexes as a function of wavelength, after four hours of reaction time Fig.
Fig. 2: wellenlängenabhängige Extinktion einer auf Grund der Oxidation von Mn(II)-Ionen ablaufenden Reduktion des ersten Kupferzen trums der Laccase Fig. 2: Wavelength-dependent absorbance of a reduction of the first copper center of the laccase due to the oxidation of Mn (II) ions
Fig. 3: durch Laccase katalysierte Bildung von Mn(III)-Komplexen und Wasserstoffperoxid nach 4 Stunden Reaktionszeit in Abhängigkeit unterschiedlicher organischer Säuren Fig. 3: Laccase-catalyzed formation of Mn (III) complexes and hydrogen peroxide after 4 hours reaction time varies depending different organic acids
Fig. 4: Bildung von Mn(III)-Malonat-Komplexen unter Einfluß unter schiedlicher Enzyme Fig. 4: Formation of Mn (III) -Malonat complexes under the influence of different enzymes
Mangan(II)-Sulfat einer Konzentration von 0,5 mmol/l wurde durch säulenchromatografisch gereinigte Laccase (eingesetzte Ausgangsaktivität: 0,2 U/ml) in Gegenwart von 100 mmol/l Natriumpyrophosphat bei einem pH- Wert von 4,5 und einer Temperatur von 35°C oxidiert. Die Bildung der dabei entstehenden Mangan(III)-Pyrophosphat-Komplexe wurde spektral photometrisch verfolgt (der kinetische Reaktionsverlauf ist in Fig. 1a dargestellt). Fig. 1b zeigt dabei das nach einer Reaktionszeit von vier Stunden erhaltene UV-Spektrum, das für die Mangan(III)-Pyrophosphat- Komplexe charakteristisch ist und zu deren Identifizierung dient.Manganese (II) sulfate at a concentration of 0.5 mmol / l was purified by laccase purified by column chromatography (starting activity used: 0.2 U / ml) in the presence of 100 mmol / l sodium pyrophosphate at a pH of 4.5 and one Oxidized temperature of 35 ° C. The formation of the manganese (III) -pyrophosphate complexes formed was monitored spectrally photometrically (the kinetic course of the reaction is shown in FIG. 1a). FIG. 1b shows the obtained after a reaction time of four hours UV spectrum that is characteristic of the manganese (III) complexes and -Pyrophosphat- used to identify it.
Die Messungen wurden an einem Zweistrahl-Spektralphotometer gegen einen Blindwert, der den kompletten Reaktionsansatz mit thermisch inaktiviertem Enzym enthielt, durchgeführt.The measurements were taken on a two-beam spectrophotometer against one Blank value that shows the complete reaction batch with thermally inactivated Enzyme contained carried out.
Zum Nachweis der Oxidation der Mangan(II)-Ionen (Ausgangskonzentration: 0,1 mmol/l) durch Reduktion des ersten Kupferzentrums der Laccase wurde diese in säulenchromatografisch gereinigter Form mit einer Ausgangsaktivität von 17,5 U/ml eingesetzt.To detect the oxidation of the manganese (II) ions (initial concentration: 0.1 mmol / l) by reducing the first copper center of the laccase this in a form purified by column chromatography with a starting activity of 17.5 U / ml.
Fig. 2 zeigt die spektralphotometrisch im sichtbaren Bereich verfolgte und
auf Grund der Oxidation von Mn(II)-Ionen ablaufenden Reduktion des ersten
Kupferzentrums der Laccase für unterschiedliche Reaktionsbedingungen:
Kurve 1: erstes Kupferzentrum in oxidierter Form (Peak bei 605 nm) zu
Reaktionsbeginn
Kurve 2: erstes Kupferzentrum in durch die Oxidation von Mn(II)-Ionen
bewirkter teilweise reduzierter Form (Reaktionszeit 5 min.)
Kurve 3: erstes Kupferzentrum in durch die Oxidation von Mn(II)-Ionen
bewirkter teilweise reduzierter Form (Reaktionszeit 20 min.)
Kurve 4: erstes Kupferzentrum in vollständig reduzierter Form, bewirkt
durch die Zugabe von Natriumdithionit FIG. 2 is followed spectrophotometrically in the visible region and due to the oxidation of Mn (II) ions running reduction shows the first copper center of laccase for different reaction conditions:
Curve 1: first copper center in oxidized form (peak at 605 nm) at the start of the reaction
Curve 2: first copper center in a partially reduced form caused by the oxidation of Mn (II) ions (reaction time 5 min.)
Curve 3: first copper center in a partially reduced form caused by the oxidation of Mn (II) ions (reaction time 20 min.)
Curve 4: first copper center in a completely reduced form, caused by the addition of sodium dithionite
Das erste Kupferzentrum der Laccase zeigt in oxidierter Form ein charak teristisches Spektrum mit einem Peak bei 605 nm, der bei vollständiger Reduktion desselben verschwindet. Diese Reduktion kann, wie in Fig. 2 gezeigt, auch chemisch durch Zugabe von Natriumdithionit demonstriert werden. Zur Verhinderung der Reoxidation des ersten Kupferzentrums der Laccase, die im intakten Enzym durch einen Elektronenfluß vom ersten zum zweiten Kupferzentrum und die dort stattfindende Übertragung der Elektro nen auf molekularen Sauerstoff erfolgt, wurde das zweite Kupferzentrum durch Zugabe von 2 mmol/l Natriumfluorid zum Reaktionsansatz spezifisch gehemmt. Die Messungen wurden wiederum an einem Zweistrahl-Spektral photometer gegen einen Blindwert, der den kompletten Reaktionsansatz mit durch Zugabe von Natriumdithionit vollständig reduziertem Enzym enthielt, durchgeführt.The first copper center of the laccase shows in oxidized form a characteristic spectrum with a peak at 605 nm, which disappears when the latter is completely reduced. As shown in FIG. 2, this reduction can also be demonstrated chemically by adding sodium dithionite. To prevent reoxidation of the first copper center of laccase, which takes place in the intact enzyme by an electron flow from the first to the second copper center and the transfer of the electrons to molecular oxygen taking place there, the second copper center was specifically added to the reaction mixture by adding 2 mmol / l sodium fluoride inhibited. The measurements were again carried out on a double-beam spectrometer against a blank value, which contained the complete reaction mixture with enzyme completely reduced by adding sodium dithionite.
Die durch Laccase katalysierte Bildung von Mangan(III)-Komplexen sowie von Wasserstoffperoxid in Gegenwart von unterschiedlichen organischen Säuren, die jeweils in einer Ausgangskonzentration von 100 mmol/l eingesetzt wurden, ist tabellarisch in Fig. 3 dargestellt. Die Reaktion wurde jeweils in Gegenwart von säulenchromatografisch gereinigter Laccase mit einer Ausgangsaktivität von 0,2 U/ml sowie in Anwesenheit von Mangan(II)- Sulfat mit einer Ausgangskonzentration von 0,1 mmol/l bei einem pH-Wert von 5,0 und einer Temperatur von 35°C über einen Zeitraum von 4 Stunden verfolgt. Als Kontrollansätze dienten jeweils die kompletten Reaktions gemische mit thermisch inaktiviertem Enzym.The formation of manganese (III) complexes catalyzed by laccase and of hydrogen peroxide in the presence of different organic acids, each of which was used in an initial concentration of 100 mmol / l, is shown in a table in FIG. 3. The reaction was carried out in the presence of column chromatography-purified laccase with an initial activity of 0.2 U / ml and in the presence of manganese (II) sulfate with an initial concentration of 0.1 mmol / l at a pH of 5.0 and at a temperature of 35 ° C over a period of 4 hours. The complete reaction mixtures with thermally inactivated enzyme served as control approaches.
Die Wirkung der durch Laccase initiierten Bildung von Superoxid-Anionen
und Wasserstoffperoxid auf eine Peroxidase soll am Beispiel der Mangan-
Peroxidase-Reaktion erläutert werden. Fig. 4 zeigt dabei als Vergleich die
Bildung von Mangan(III)-Malonat-Komplexen unter dem Einfluß der
folgenden Enzyme:
Kurve 1: Mangan-Peroxidase
Kurve 2: Laccase
Kurve 3: Laccase + Mangan-Peroxidase
Kurve 4: Laccase + Mangan-Peroxidase + Superoxid-DismutaseThe effect of the formation of superoxide anions and hydrogen peroxide initiated by laccase on a peroxidase will be explained using the example of the manganese peroxidase reaction. Fig. 4 shows as a comparison the formation of manganese (III) malonate complexes under the influence of the following enzymes:
Curve 1: manganese peroxidase
Curve 2: laccase
Curve 3: laccase + manganese peroxidase
Curve 4: laccase + manganese peroxidase + superoxide dismutase
Die Mangan-Peroxidase-Reaktion katalysiert die Oxidation von Mangan(II)
zu Mangan(III) unter Verbrauch von Wasserstoffperoxid nach der folgenden
Reaktionsgleichung:
The manganese peroxidase reaction catalyzes the oxidation of manganese (II) to manganese (III) using hydrogen peroxide according to the following reaction equation:
2 Mn(II) + H2O2 + 2H+ → 2 Mn(III) + 2 H2O (1)2 Mn (II) + H 2 O 2 + 2H + → 2 Mn (III) + 2 H 2 O (1)
In Gegenwart von Malonat-Ionen werden die entstehenden Mangan(III)- Ionen durch Komplexbildung stabilisiert. Die gebildeten Mangan(III)- Malonat-Komplexe verfügen über ein Absorptionsmaximum bei 270 nm und können spektralphotometrisch erfaßt werden.In the presence of malonate ions, the manganese (III) - Ions stabilized by complex formation. The manganese (III) formed - Malonate complexes have an absorption maximum at 270 nm and can be recorded spectrophotometrically.
Die Entstehung von Superoxid-Anionen kann mit Hilfe der Superoxid-
Dismutase nachgewiesen werden, die folgende Reaktion katalysiert:
The formation of superoxide anions can be demonstrated with the help of superoxide dismutase, which catalyzes the following reaction:
2 O2 - + 2 H+ → H2O2 + O2 (2)2 O 2 - + 2 H + → H 2 O 2 + O 2 (2)
Die Bildung von Wasserstoffperoxid wird chemisch ebenfalls durch Reaktion
des Superoxid-Anions mit Mangan(II)-Ionen nach folgender Gleichung
katalysiert:
The formation of hydrogen peroxide is also chemically catalyzed by reaction of the superoxide anion with manganese (II) ions according to the following equation:
2 O2 - + 4 H+ + 2 Mn(II) → 2 H2O2 + 2 Mn(III) (3)2 O 2 - + 4 H + + 2 Mn (II) → 2 H 2 O 2 + 2 Mn (III) (3)
Entsprechend den Gleichungen (2) und (3) führt die Reaktion (3) im Vergleich zur Reaktion (2) zu einer doppelten Menge Wasserstoffperoxid. Die Anwesenheit von Superoxid-Dismutase führt zur Konkurrenz der Reaktionen (2) und (3). Es wird daher im Resultat weniger Wasserstoff peroxid (Substrat für die Mangan-Peroxidase) als bei Abwesenheit von Superoxid-Dismutase gebildet, was in Fig. 4 durch eine verlangsamte Mangan-Peroxidase-Reaktion bei Anwesenheit von Superoxid-Dismutase zum Ausdruck kommt.According to equations (2) and (3), reaction (3) leads to double the amount of hydrogen peroxide compared to reaction (2). The presence of superoxide dismutase leads to competition between reactions (2) and (3). As a result, less hydrogen peroxide (substrate for the manganese peroxidase) is formed as a result than in the absence of superoxide dismutase, which is shown in FIG. 4 by a slowed down manganese peroxidase reaction in the presence of superoxide dismutase.
Alle Reaktionen wurden in Gegenwart von Mangan(II)-Sulfat einer Ausgangskonzentration von 0,5 mmol/l und Na-Malonat einer Ausgangs konzentration von 100 mmol/l bei einem pH-Wert von 4,5 und einer Tem peratur von 35°C in einem Zweistrahl-Spektralphotometer verfolgt. Als Blindwerte dienten jeweils die kompletten Reaktionsansätze mit themisch inaktiviertem Enzym. Laccase und Manganperoxidase wurden in säulen chromatografisch gereinigter Form mit einer Ausgangsaktivität von 0,2 U/ml eingesetzt, Superoxid-Dismutase mit einer Ausgangsaktivität von 20 U/ml.All reactions were carried out in the presence of manganese (II) sulfate Initial concentration of 0.5 mmol / l and Na malonate from an initial concentration of 100 mmol / l at a pH of 4.5 and a tem temperature of 35 ° C in a two-beam spectrophotometer. As Blank values served the complete reaction approaches with thematic inactivated enzyme. Laccase and manganese peroxidase were in columns chromatographically purified form with an initial activity of 0.2 U / ml used, superoxide dismutase with an initial activity of 20 U / ml.
Claims (9)
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