EP0793715A1 - Procede de production de lignine-peroxydase et de manganese-peroxydase a partir d'une culture de phanerochaete chrysosporium - Google Patents
Procede de production de lignine-peroxydase et de manganese-peroxydase a partir d'une culture de phanerochaete chrysosporiumInfo
- Publication number
- EP0793715A1 EP0793715A1 EP95943287A EP95943287A EP0793715A1 EP 0793715 A1 EP0793715 A1 EP 0793715A1 EP 95943287 A EP95943287 A EP 95943287A EP 95943287 A EP95943287 A EP 95943287A EP 0793715 A1 EP0793715 A1 EP 0793715A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- peroxidase
- culture
- mic
- manganese
- lignin
- 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.)
- Withdrawn
Links
- 241000222393 Phanerochaete chrysosporium Species 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 21
- 238000004519 manufacturing process Methods 0.000 claims abstract description 28
- 108010054320 Lignin peroxidase Proteins 0.000 claims description 38
- 108010059896 Manganese peroxidase Proteins 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 30
- 150000003904 phospholipids Chemical class 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 150000003905 phosphatidylinositols Chemical class 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 230000009469 supplementation Effects 0.000 claims description 2
- LWGJTAZLEJHCPA-UHFFFAOYSA-N n-(2-chloroethyl)-n-nitrosomorpholine-4-carboxamide Chemical compound ClCCN(N=O)C(=O)N1CCOCC1 LWGJTAZLEJHCPA-UHFFFAOYSA-N 0.000 claims 1
- 239000002609 medium Substances 0.000 description 28
- 230000000694 effects Effects 0.000 description 26
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 10
- 229940041514 candida albicans extract Drugs 0.000 description 9
- 239000012138 yeast extract Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 238000013019 agitation Methods 0.000 description 6
- OJYLAHXKWMRDGS-UHFFFAOYSA-N zingerone Chemical compound COC1=CC(CCC(C)=O)=CC=C1O OJYLAHXKWMRDGS-UHFFFAOYSA-N 0.000 description 6
- 108700020962 Peroxidase Proteins 0.000 description 5
- 102000003992 Peroxidases Human genes 0.000 description 5
- 230000005587 bubbling Effects 0.000 description 5
- 238000009423 ventilation Methods 0.000 description 5
- OEGPRYNGFWGMMV-UHFFFAOYSA-N (3,4-dimethoxyphenyl)methanol Chemical compound COC1=CC=C(CO)C=C1OC OEGPRYNGFWGMMV-UHFFFAOYSA-N 0.000 description 4
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 235000019524 disodium tartrate Nutrition 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 239000011572 manganese Substances 0.000 description 4
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 4
- 239000011707 mineral Substances 0.000 description 4
- 235000010755 mineral Nutrition 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
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- 150000003839 salts Chemical class 0.000 description 4
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 4
- 239000001433 sodium tartrate Substances 0.000 description 4
- 229940095064 tartrate Drugs 0.000 description 4
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- 229940088594 vitamin Drugs 0.000 description 4
- 229930003231 vitamin Natural products 0.000 description 4
- 229910000368 zinc sulfate Inorganic materials 0.000 description 4
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 3
- 229920005610 lignin Polymers 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 3
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- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000222382 Agaricomycotina Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 235000009414 Elaeocarpus kirtonii Nutrition 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 239000005569 Iron sulphate Substances 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
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- 229920005830 Polyurethane Foam Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 244000236151 Tabebuia pallida Species 0.000 description 1
- 235000013584 Tabebuia pallida Nutrition 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
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- 230000000593 degrading effect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
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- 230000002349 favourable effect Effects 0.000 description 1
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- 239000007970 homogeneous dispersion Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
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- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000011496 polyurethane foam Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
Definitions
- the present invention relates to a process for the production of peroxidases from the fungus Phanerochaete chrysosporium.
- Phanerochaete chrysosporium is a fungus associated with "white wood rot". It is a hymenomycete, which is part of the order of aphylophorales and of the family of corticaceae. It has the property of degrading lignin until mineralization (final products: CO2 + H2O).
- This fungus produces exocellular peroxidases: these are in particular the manganese peroxidase isozymes (MnPs) [KUWAHARA et al. FEBS Let., 169, p. 247-250, (1984)], and those of lignin peroxidase (LiPs) [TIEN M. and KIRK. T.K., Science, 221, p. 661-663, (1983); GLENN et al. Biochem. Biophys. Res. Commun., 114, p. 1077-1083]. These enzymes are glycosylated hemoproteins, the average molecular mass of which is 40 Da [LEISOLA et al., J. Biol. Chem. , 262 p. 419-424, (1984)].
- MnPs manganese peroxidase isozymes
- LiPs lignin peroxidase
- These enzymes are glycosylated hemoproteins,
- the manganese peroxidases and lignin peroxidases are capable of catalyzing the oxidation of numerous aromatic substrates, in particular li ⁇ gnine, by using hydrogen peroxide as cosubstrate. These properties find their main applications in the field of stationery and that of waste treatment. So far, it is mainly lignin peroxidases which have been used in this type of application.
- French Patent 2,574,427 describes two strains of Phanerochaete chrysosporium, having a particularly high lignin-peroxidase activity, and their culture on a medium. containing a source of assimilable nitrogen, as well as a source of assimilable carbon, and a source of assimilable mineral salts.
- This process comprises several successive stages, each of them being carried out under different culture conditions;
- the first step is carried out on a synthetic medium, comprising mineral salts, a carbon source and a nitrogen source, in the presence of yeast extract, a source of phospholipids, and emulsified fatty acids;
- the mycelium formed is then cultivated in a partially renewed culture medium, supplemented with veratrylic alcohol, but not comprising emulsified fatty acids, and whose phospholipid content represents only 1/7 to 1/8 of that of the medium culture used in the first step;
- the medium is entirely replaced by a new medium, comprising the same proportion of phospholipids and veratrylic alcohol as that of the second stage, and the carbon source, the nitrogen source, and the yeast extract, at 1/4 of their content in the middle of the first stage;
- a fourth culture step is finally carried out in a medium devoid of yeast extract, carbon source, and emulsified fatty acids, but comprising the same proportion of phospholipids and veratry
- Lignin peroxidase can then be recovered from the culture medium.
- the implementation of this process makes it possible to very significantly increase the enzyme content; the lignin-peroxidase activity in the medium is approximately 240 U per liter per day.
- manganese peroxidases are appearing more and more as likely to play a key role in the biotransformation of aromatic polymeric compounds of lignin type. Indeed, unlike lignin-peroxidases whose activity is limited by the low penetration of the lignified walls by the enzyme, manganese-peroxidases act through species of low molecular weight, diffusing easily.
- the catalytic cycle of manganese-peroxidases involves the oxidation of Mn (II) to Mn (III) which, after complexation with organic acids, generates oxidizing diffusing species capable of depolymerizing natural lignin [WARIISHI K., et al. , Biochem. Biophys. Res. Comm. 176, p. 269-276, (1991)].
- the inventors set themselves the goal of increasing the production of exocellular peroxidases of Phanerochaete chrysosporium from cultures of this fungus, as well as increasing the MnP / LiP ratio.
- MIC 390 new strains
- MIC 249 new strains
- MIC 396 new strains
- MIC 390 new strains
- MIC 249 new strains
- MIC 396 new strains
- MIC 396 hypersecretory of the exocellular peroxidases lignin-peroxidase and manganese-peroxidase.
- MIC 396 new strains which can be implemented either with free cells, or with cells immobilized on a support.
- the cultures obtained in accordance with the invention can produce approximately 2 times more lignin peroxidase than those of Patent EP 0 437 500 and 10 times more manganese peroxidase than those obtained previously by BONNARME and JEFFRIES.
- the implementation of the present invention also makes it possible to control and modify, as necessary, the manganese-peroxidase / lignin-peroxidase ratio.
- the present invention relates to the strains of Phanerochaete chrysosporium MIC 249, MIC 390, and MIC 396, which were deposited on December 20, 1994, with the CNCM (National Collection of Cultures of Microorganisms) held by the Institut Pasteur , 26 rue du Doctor Roux in Paris, under the respective numbers 1-1511, 1-1512, and 1-1513.
- CNCM National Collection of Cultures of Microorganisms
- a subject of the present invention is also a process for the production of lignin-peroxidase and / or manganese-peroxidase from a culture of Phanerochaete chrysosporium, which process is characterized in that it comprises the cultivation of at least a strain of Phanerochaete chrysosporium chosen from the group consisting of strains MIC 249, MIC 390, and MIC 396 mentioned above.
- the culture medium comprises at least one source of carbon, at least one source of nitrogen, mineral salts, trace elements, vitamins and it is supplemented with yeast extract.
- the carbon source concentration is preferably between 5 and 20 g / l.
- a carbon source preferentially used comprises glycerol, but other sources of carbon which can be assimilated more slowly or more quickly, can be used, alone or as a mixture; these are, for example, carbon sources such as maltose, raffinose, starch, xylose, rhamnose, arabinose, fructose, sorbitol, mannose, cellobiose, cellulose.
- the carbon source comprises at least one phospholipid chosen from the group consisting of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE) , acylphosphatidylethanolamine (APE), phosphatidylinositol (PI), phosphatidic acid (AP), or a mixture of said phospholipids; in this case it may be a mixture of reconstituted phospholipids, or else a mixture of phospholipids originating from a natural source, for example soy phospholipids.
- a mixture of phospholipids comprises more than 25% of phosphatidylinositol and less than 15% of PC.
- the concentration of the phospholipid or of the mixture of phospholipids (reconstituted, or derived from soya) is between 0.1 and 10 g / liter.
- the nitrogen source can for example consist of amino acids, sodium nitrate or a mixture of these different sources of nitrogen, in combination with the yeast extract.
- the nitrogen source concentration is preferably between 0.5 and 20 g / i.
- the assimilable mineral salts include the potassium, calcium and magnesium salts, and are used at a concentration of between 0.5 and 100 mM.
- the trace elements are mainly composed of iron sulphate, zinc sulphate, manganese sulphate, and copper sulphate.
- concentrations of iron, zinc and copper sulphates are the concentrations usually used for the cultivation of Phanerochaete chrysosporium (see for example European Patent 0 437 500).
- concentration of Mn 2+ has no influence on the production of manganese peroxidase under the culture conditions in accordance with the invention, and can vary within a relatively wide range, • it is preferably between 1 and 600 mM.
- the medium is also supplemented with a source of vitamins; we can for example use a mixture of vitamins whose composition respects that given by [TATUM et al. Am. J. Bot., 37: 38-46 (1950)].
- the vitamin mixture is used at a concentration between 0.001 g and 1 g per liter.
- the culture medium is further supplemented, during cultivation, by adding, at a rate of 0.001 to 10 grams per liter, of at least one production activating element.
- enzymes and / or protective thereof; this activating and / or protective element may consist of one of the constituents of the initial medium, or of a mixture of several of them.
- this activating and / or protective element comprises veratrylic alcohol, which in this case is used at a concentration of between 0.1 and 1 gram per liter.
- veratrylic alcohol which in this case is used at a concentration of between 0.1 and 1 gram per liter.
- This addition can be carried out, in the form of a solution, an emulsion, or liposomes, by adding a mixture of surfactant (TWEEN 80 for example), with fatty acids (C18: 12, C18: 2) and phospholipids rich in phosphatidylinositol.
- a mixture of surfactant TWEEN 80 for example
- fatty acids C18: 12, C18: 2
- phospholipids rich in phosphatidylinositol phospholipids rich in phosphatidylinositol.
- the medium can be supplemented continuously (pump) or discontinuously. Supplementation with activating element (s) and / or protector (s) is carried out when the culture has reached a growth stage such that one is at the start of the expression of the genes coding for the peroxidases (production messenger RNAs coding for manganese peroxidases and lignin peroxidases. This stage, which has been defined experimentally, has been reached after a period which may vary according to the strains and the culture conditions, but which generally represents approximately 2 days of culture, at a temperature between 28 ° C and 40 ° C.
- the cultures can be carried out in a manner known per se, either with cells immobilized on a support, or with free cells.
- these are hung or adsorbed on one or more hydrophobic, hydrophilic or neutral supports, the surface of which is preferably rough, and comprising for example cells, meshes (grids or skein), hollow, holes.
- These supports, hollow or not, are arranged in an orderly or not, fixed or mobile in the liquid phase constituted by the appropriate nutritive medium, and can have various shapes.
- non-ordered fixed supports mention may be made of RASHIG cylindrical rings, of sandstone, glass, metal, plastic, - polyurethane, polyester, polyamide foams, • metallic filings, or plastic, plastic wires.
- fixed ordered supports By way of nonlimiting examples of fixed ordered supports, mention may be made of grids or meshes of stainless steel, glass, plastic, polyurethane, polyester, Nylon, polyacrylate, polyamide, etc.
- supports which are mobile in the liquid phase
- supports made of polyurethane, polyester, polyamide, extruded plastic.
- the mycelium is in the form of balls which can have a diameter from 0.5 to 5 mm.
- the cultures are carried out with aeration and agitation of the medium.
- the aeration of the medium is carried out by the introduction of air, pure oxygen or any other mixture of gases ensuring a sufficient supply of oxygen to the microorganism, by means of a device allowing a homogeneous dispersion of this gas. (sintered glass, cane).
- the agitation of the medium can be carried out mechanically. It can also be obtained pneumatically by direct action of the ventilation system, or by an equivalent system used simultaneously.
- the level of agitation and / or aeration is chosen so as to allow a homogeneous initial occupation of the support in the case of immobilized cells, and the formation of mycelial balls with an average diameter of 0.5 to 5 mm in the case of free cells, while limiting the shear stresses undergone by the mycelial hyphae.
- This level may be variable over the duration of the culture.
- the initial incubation period (approximately 2 days) is carried out at a temperature of approximately 37 ° C., and can be followed by a temperature change favorable to the production of peroxidases, as described for example in Patent EP 0 437,500.
- the production of manganese peroxidase and / or that of lignin peroxidase, and consequently, the MnP / LiP ratio can be controlled according to the age of the culture and / or the presence of activators, as well as strains used. We can thus obtain majority enzyme cocktails either by manganese-peroxidase, either as lignin-peroxidase (activity greater than or equal to 60% of all the manganese-peroxidase and lignin-peroxidase activities).
- the MnP / LiP ratio it is also possible to control the MnP / LiP ratio, and preferably to increase either the production of manganese-peroxidase or the production of lignin-peroxidase.
- This variant is characterized in that: to modify the MnP / LiP ratio in favor of the production of manganese-peroxidase, the culture of Phanerochaete chrysosporium is carried out for a period greater than 24 hours and less than 90 hours, in presence of a mixture of phospholipids rich in phosphatidylinositol, as defined above, at a concentration of between 0.5 and 5 g / 1, preferably of the order of 1.5 g / 1;
- the strains which were selected in accordance with the invention are the strains of Phanerochaete chrysosporium MIC 249 (CNCM 1-1511), MIC 390 (CNCM 1-1512), and MIC 396 (CNCM 1-1513).
- EXAMPLE 1 COMPARISON OF THE PRODUCTION OF LIGNIN-PEROXYDASES AND MANGANESE-PEROXYDASES BY DIFFERENT STRAINS
- composition of the medium Glycerol 10 g / 1
- Veratrylic alcohol 0.42 g / 1
- the medium is distributed under sterile conditions at the rate of 100 ml per Erlenmeyer flask; each Erlenmeyer flask contains cubes of polyurethane foam.
- the inoculation is carried out using mycelial fragments of a preculture.
- the Erlenmeyer flasks are oxygenated at the start of the culture (TO), then incubated at 37 ° C. with shaking, at 90 or 120 rpm.
- lignin-peroxidase and manganese-peroxidase activities are measured after 4 days of culture according to the following protocols: a) determination of the lignin-peroxidase activity: this activity is determined by measuring the oxidation rate of veratryl alcohol in corresponding aldehyde, in the presence of hydrogen peroxide [TIEN and KIRK, Proc Natl. Acad. Sci. USA, 81: 2280-2284 (1984)]. The reaction is followed, at 30 ° C., by spectrophotometry at 310 nm. The molar extinction coefficient of veratrylic aldehyde at this wavelength is 9300 M -1 .cm "1 .
- the activity in the medium is expressed in nkatal.ml -1 or else in units / liter (U / l): one lignin-peroxidase unit corresponds to one micromole of veratrylic aldehyde formed per minute.
- this activity is determined by measuring the oxidation rate of vanillylacetone in the presence of MnS0 [PASZCZYNSKI et al. FEMS Microbiol. Lett, 29: 37-41 (1985)]. The reaction is followed, at 30 ° C., by spectrophotometry at 334 nm. The molar extinction coefficient of vanillylacetone at this wavelength is 18,300 M -1 .cm -1 .
- the activity in the medium is expressed in nkatal.ml -1 or in units / liter (U / l): one unit manganese peroxidase corresponds to one micromole of vanillylacetone formed per minute.
- the MIC 249 strain is cultured under conditions identical to those indicated above in Example 1. The incubation is carried out at 37 ° C. with shaking at 90 rpm. The lignin-peroxidase and manganese-peroxidase activities are measured at different culture times, and the MnP / LiP ratio is calculated. The results are summarized in Table II below. TABLE II
- the MIC 390 strain is cultured under the following conditions, on a base medium the composition of which is as follows:
- This basic medium is supplemented with a mixture of phospholipids, NAT89, at concentrations of between 0.5 g / 1 and 1.89 g / 1.
- NAT 89 is supplied by NATTERMAN PHOSPHOLIPID Gmbh (Cologne, Germany), and its composition is as follows:
- the lignin-peroxidase and manganese-peroxidase activities are measured at the optimum production in the culture medium, namely 4.5 days of culture.
- the body of the bioreactor contains the immobilization support, which here consists of 2 concentric cylinders made of metal mesh (wire 0.15 mm in diameter) having a mesh of 0.5 mm, respective diameters of 70 mm (outer cylinder) and 30 mm (inner cylinder) for a height of 290 mm.
- the immobilization support here consists of 2 concentric cylinders made of metal mesh (wire 0.15 mm in diameter) having a mesh of 0.5 mm, respective diameters of 70 mm (outer cylinder) and 30 mm (inner cylinder) for a height of 290 mm.
- composition of the basic medium is as follows:
- the bioreactor is then thermostatically controlled at 37 ° C. and is aerated with filtered atmospheric air, introduced at a flow rate of 40 1 / h using a circular ventilation cane or a frit forming a regular bubbling located at the base of the metal cylinders, ensuring both agitation and uniform ventilation necessary for the formation of an adequate mycelial film.
- Inoculation is carried out using mycelial fragments from a preculture.
- the medium is sterile supplemented with a phospholipid mixture (NAT89) added with veratrylic alcohol (AVe).
- NAT89 phospholipid mixture
- AVe veratrylic alcohol
- composition of the basic medium is as follows: Glycerol 6.8 g / 1
- Disodium tartrate 1.565 g / 1
- the inoculation is carried out using mycelial fragments of a preculture or a spore solution with 2 ⁇ 10 5 spores / ml.
- the mycelial balls of approximately 0.5 to 1 mm in diameter are formed and the air flow is reduced to 20 l / h.
- the medium is sterile supplemented with a phospholipid mixture comprising veratrylic alcohol (AVe).
- AVe veratrylic alcohol
- Air bubbling is replaced by pure oxygen bubbling at a rate of 20 l / h.
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FR9500002 | 1995-01-02 | ||
FR9500002A FR2728911A1 (fr) | 1995-01-02 | 1995-01-02 | Procede de production de lignine-peroxydase et de manganese-peroxydase a partir d'une culture de phanerochaete chrysosporium |
PCT/FR1995/001746 WO1996021008A1 (fr) | 1995-01-02 | 1995-12-28 | Procede de production de lignine-peroxydase et de manganese-peroxydase a partir d'une culture de phanerochaete chrysosporium |
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CA (1) | CA2209082A1 (enrdf_load_stackoverflow) |
FI (1) | FI972773L (enrdf_load_stackoverflow) |
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DE102004026259A1 (de) * | 2004-05-28 | 2005-12-15 | Schwarze, Francis W.M.R., Dr. | Substrat zur Holzverrottung |
US8753844B2 (en) | 2011-05-06 | 2014-06-17 | The Regents Of The University Of California | Overproduction of ligninolytic enzymes |
EP2677030A1 (en) | 2012-06-21 | 2013-12-25 | Latvijas Valsts Koksnes kimijas instituts | Polyurethane rigid and flexible foams as composite obtained from wood origin raw materials and used as support for immobilization of microorganisms that produce ligninolytic enzymes |
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FR2637292B1 (fr) * | 1988-10-03 | 1992-06-05 | Agronomique Inst Nat Rech | Procede de production de lignine-peroxydase par des cellules non-proliferantes de phanerochaete chrysosporium |
US5342765A (en) * | 1991-08-01 | 1994-08-30 | Occidental Chemical Corporation | Method of producing extracellular products from aerobic microorganisms |
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- 1995-12-28 US US08/849,732 patent/US5972672A/en not_active Expired - Fee Related
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FR2728911A1 (fr) | 1996-07-05 |
FI972773A7 (fi) | 1997-06-27 |
CA2209082A1 (fr) | 1996-07-11 |
FI972773A0 (fi) | 1997-06-27 |
FI972773L (fi) | 1997-06-27 |
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