EP2852665A1 - Laccase aus podospora anserina und dessen verwendungen - Google Patents

Laccase aus podospora anserina und dessen verwendungen

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Publication number
EP2852665A1
EP2852665A1 EP13735432.0A EP13735432A EP2852665A1 EP 2852665 A1 EP2852665 A1 EP 2852665A1 EP 13735432 A EP13735432 A EP 13735432A EP 2852665 A1 EP2852665 A1 EP 2852665A1
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EP
European Patent Office
Prior art keywords
laccase
seq
enzyme
laccase according
host cells
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Withdrawn
Application number
EP13735432.0A
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English (en)
French (fr)
Inventor
Nicolas Mano
Fabien Durand
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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Publication of EP2852665A1 publication Critical patent/EP2852665A1/de
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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.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/783—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/88—Taste or flavour enhancing agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q15/00—Anti-perspirants or body deodorants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/08—Preparations for bleaching the hair
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00—Preparations for care of the hair
    • A61Q5/10—Preparations for permanently dyeing the hair
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present invention relates to a novel laccase isolated from Podospora anserina, its preparation process and its use in particular for the delignification of paper, as a bleaching agent, depolluting and deodorizing or for reducing oxygen.
  • Laccases are copper enzymes that oxidize polyphenols using oxygen as the final acceptor of electrons. They are present in plants, in many fungi (degradation of lignin) as well as in some bacteria.
  • laccases are such that their active site is composed of 4 atonies of copper, one of type 1 ( ⁇ ), isolated, responsible for the oxidation of phenols and a custer of 3 atoms of copper (one of type 2, T2 , and two of type 3, T3) responsible for the activation! we have (3 ⁇ 4.
  • laccases The mechanism of action of laccases, and in particular the role of the metallic center, remains poorly understood; a mechanism is admitted in two stages:
  • Copper T1 pulls an electron off the substrate
  • the electron is transferred to the center T2 / T3 over a distance of about 12.5 ⁇ , after complete reduction of the nuclear center, the reduction of the molecular oxygen intent.
  • the inventors have now identified a new laccase produced by Podospora anserina which has more advantageous characteristics than commercially available laccases, in particular that of Trametes versicolor.
  • the invention relates to the purified laccase isolated from Podospora anserina from SEQ. ID. No. i; this enzyme corresponds to a protein predicted by the sequencing of the genome of Podospora anserina (translated from the gene having the number of B2ANK.8 accession in the UniProt database, the complete gene is SEQ. ID. No. 2) except for the 30 amino acids positioned at the N-terminus of the protein; in particular, the purified laccase (purity> 95%) according to the invention has an identity percentage of less than 90%, and in order of preference increasing at least 95%, 97%, 98% and 99% of identity, compared to the laccase of Podospora anserina from SEQ. ID. No.
  • nucleic acid sequence SEQ. JD. No. 4 (corresponding to SEQ ID NO: 2 excluding the fragment coding for the 30 amino acids positioned at the N-terminus) codes for this laccase of SEQ. ID. No. 1.
  • the laccase of SEQ, ID. N 3 is also an object of the present invention, the laccase of SEQ. ID. No. 3 differs from the laccase of SEQ. fD. No. 1 in that it comprises an additional amino acid, a serine, in N-terminal positio; this laccase is obtained by expressing in the yeast Pichia postons the nucleic acid molecule of SEQ. ID. No. 4 cloned into the expression vector pFD56 whose construction is such that it allows the expression of a protein carrying an additional serine N ⁇ terminal.
  • the laccase of SEQ. ID. No. 5, which corresponds to the polypeptide encoded by SEQ ID NO. No. 2 and which has the 30 amino acids of N-terminus, is also an object of the present invention.
  • a variant of the invention relates to a laccase of SEQ. ID. No. 1 comprising at least one alteration (substitution, deletion or insertion) within the sequence of 30 amino acids positioned at the N-terminus, the other amino acids not in this sequence being preferably unchanged and the identity of this enzyme being at least 90%, preferably 95%, 97%, 98% and 99%, with SEQ ID NO. No. l.
  • sequence with respect to the sequence of the laccase of Podospora anserina is assessed according to the percentage of amino acid residues that are identical, when the two sequences are aligned, so as to obtain the maximum of correspondence between them.
  • the percent identity can be calculated by those skilled in the art using a computer program for sequence comparison such as, for example, that of the BLAST suite (Altschul et al., NAR, 25, 3389-3402).
  • the BLAST programs are implemented on the comparison window consisting of the entire SEQ. ID. No. 1 indicated as a reference sequence.
  • a peptide having an amino acid sequence having at least X% identity with a reference sequence is defined, in the present invention, as a peptide whose sequence may include up to 100-X alterations per 100 amino acids of the sequence. reference, while retaining the functional properties of said reference peptide.
  • alteration includes deiét ⁇ ons, substitutions or insertions consecutive or dispersed amino acids in the reference sequence.
  • the new laccase according to the invention has improved properties compared to. laccases commercially available from Tramet.es versicolor, Rhtts vernicifera, Amaricus bisporus or Pleurotus ostreat s.
  • the present invention also relates to a nucleic acid molecule encoding the laccase according to the invention; preferably, it is a nucleic acid molecule of sequence chosen from SEQ. ID, No. 2 or preferentially, it is about SEQ. ID. No. 4 coding for the laccase of Podospora anserina cleaved at the first 30 amino acids positioned at the N-terminus of the. protein.
  • the molecule of nucleic acid encoding the laccase according to the invention can be cloned into an expression vector such as a plasmid and transformed into an appropriate host such as a bacterium, a yeast or a cell culture.
  • Expression vector means a vector having a region allowing the insertion of a coding nucleotide sequence between the signals essential for its expression, in particular, a promoter (constitutive or inducible), a ribosome binding site, a transcription termination signal and, optionally, a selection marker such as an antibiotic resistance gene.
  • the present invention also relates to an expression vector comprising said nucleic acid molecule and to a host cell transformed with said expression vector and expressing an iacca.se according to the invention.
  • the introduction of the expression vector into the host cell can be carried out by any method known to those skilled in the art, in particular by modifying the membrane permeability of the host cell, for example in the presence of calcium ions, or by eleciroporation.
  • said cells After culturing the transformed host cells to express the laccase according to the invention, said cells can be recovered by centrifugation, iysées in order to release the enzymes of which said laccase according to the invention.
  • the laccase according to the invention is produced by the yeast Pichia pas to ris.
  • the nucleic acid molecule of SEQ. Id. ° 4 coding for the laccase sequence SEQ. Id. No. 3 when cloned into the vector pFD56 as described below, is introduced by homologous recombination into the yeast genome at the AOX1 gene.
  • the plasmid pFD56 once linearized by digestion with the enzyme pme1, is introduced into the yeast by electroporarion and the positive clones are selected on YPD + agar medium containing zeocin at 100 ⁇ g / ml.
  • a preculture of 200 ml of YPD medium supplemented with zeocin (100 ⁇ g / ml) is inoculated. After stirring overnight at 220 rpm and at 30 ° C, this preculture is then centrifuged for 10 minutes at 4000 rpm and the pellet is taken up in 200 ml of sterile water to eliminate any presence of glucose. After a second centrifugation, a culture of 2L in MMH medium containing 1MM of C SO 4 in a 5L Erlenmeyer flask is then seeded with this pellet. The yeasts are incubated at 25 ° C with stirring (220 rpm) for 2 hours before adding 0.5% methanol to start the induction. This induction step will be repeated for 5 days to obtain the maximum of enzymes.
  • vector for expression in Pichia postons pFD56
  • plasmid pPICZa plasmid pPICZa containing the DNA sequence coding for the laccase of Podospora anserina in phase with the Saccharomyces cerevisi ⁇ -factor secretion factor and containing the methanol-inducible AOX.1 promoter .
  • yeast strain Pichia pastoris GS1 used for the production of the laccase according to the invention after integration of the cassette resulting from the vector pFD56 containing the AOXI promoter, the peptide signai ⁇ -factor and the DNA sequence encoding the laccase of Podospora anserina.
  • Escherichia coli can be chosen as host microorganism, the plasmids which can then be used are in particular the plasmids pBluescript, pUC18, pET, pGEX, pGS. pMAL-e2 or the like.
  • the laccase is advantageously expressed by an E. coli bacterium transformed with a pET21a expression vector coding for an enzyme contiguous to a 6HIS label in the C-terminal position.
  • This process is fast and simple; indeed, the induction of the expression of the laccase of Podospora anserina in E. coli bacterium takes place in 4 to 24 hours.
  • 6HIS label allows the purification of laccase of Podospora anserina by affinity chromatography on a nickel resin in a single step to obtain a pure enzyme.
  • the skilled person chooses the host cell according to the expression vector used.
  • the expression vector pET21a when used, ors chooses a host cell expressing T7 RNA polymerase such as strains of E. coli BL 21 D.E3, BL 21 -SI, BL2 1 pLys, Novablue (DE3 ) or BL 2 i Star.
  • a host cell expressing T7 RNA polymerase such as strains of E. coli BL 21 D.E3, BL 21 -SI, BL2 1 pLys, Novablue (DE3 ) or BL 2 i Star.
  • the present invention also relates to a process for preparing a laccase according to the invention comprising the steps of:
  • step a a.) host cell preparation, expressing the laccase according to the invention; b) culturing the host cells prepared in step a);
  • step d) treatment of the culture medium obtained in step e) by hydrophobic interaction chromatography;
  • the process according to the invention is such that:
  • yeast strain Pichia pastoris used is the GS115 strain
  • the expression vector, in Pichia pastoris is the plasmid pPICZa containing the DNA sequence coding for the laccase of Podospora anserina in phase with the Saccharomyces cerevisiae ⁇ -factor secretion factor and containing the methanolic inducing AOX1 promoter;
  • the culture carried out in step b) comprises at least one step of culture in the liquid phase, with stirring, at a temperature of between 18 and 37 ° C., preferably 25 ° C., during which the expression of laccase is induced by the addition of methanol; induction by addition of methanol can optionally be renewed.
  • the process When the process is carried out according to these preferred conditions, it allows the production of laccase with a short induction time, of the order of 3 to 7 days; the purification of laccase is carried out in. a single chromatography step hydrophobic interactions and the laecase thus produced includes the four copper atoms necessary for. his activity.
  • the method for preparing a laecase according to the invention comprises the steps of:
  • step d) treating, the lysate obtained in step c) by affinity cliromaiography;
  • the laecase according to the invention has electrochemical properties that are better than the laccases sold commercially (see point 10 of the experimental part), in particular that of Trametes versicoior.
  • laccases according to the invention are of particular interest in the following applications;
  • laccases concern the delignil cation and / or the whiteness of the payroll.
  • the laccases according to the invention offer an advantageous alternative for achieving the delignification but also the bleaching of the paperless chlorine-free paper.
  • laccases according to the invention can also be used to remove the ink from the paper and / or discolor, especially for its recycling ; they may also be used for the treatment of fabrics, in particular the bleaching of cotton or to produce the discolored indigo color of jeans,
  • isaccase Another very frequent application of isaccase is their use as a depolluting agent; this enzyme can indeed degrade a broad spectrum of undesirable environmental contaminants including phenols (possibly chlorinated phenolic pollutant) and plastics.
  • Advantageous use of the Iaccases according to the invention thus relates to the depollution of phenol products.
  • iaccases Thanks to their ability to degrade pollutants, iaccases also deodorize materials such as tissues; they are thus useful in detergent compositions for washing clothes or dishes.
  • Iaccases are used in the food industry as a food product preservative, particularly as an additive in order to eliminate oxidizing reagents (deoxygenation), for example for the stabilization of fruit juices and wine.
  • They are also used as a flavor enhancer of a food product; for example, they are involved in the process of preparing cocoa to enhance its taste (soaking in a laccase solution before drying and grilling),
  • the Iaccases according to the invention also have the advantage of permitting the crosslinking of the whey proteins, in oligo- or polymers, and thus lead to the formation of gels (Faergemand et al, 1998 J, Agric Food Chem.46, 1326-1333 Mattinen et al, 2005 FEBS Journal 272, 3640-3650).
  • Iaccases are involved in the process of producing ethanol from recycled raw material.
  • iaccases are useful for the synthesis of anesthetic, anti-inflammatory, antibiotic or iodine compounds.
  • the laccase according to the invention is of particular interest for the manufacture of electrodes on which the enzyme is immobilized.
  • the present invention also relates to laccase electrodes comprising a conductive material such as a conductive metal, especially platinum, copper, silver, aluminum, gold or aluminum. steel or carbon, such as glassy carbon, carbon fibers, carbon nanotube fibers or diamond ... said conductive material is covered with a deposit comprising at least one laccase according to the invention; said deposit may further comprise a redox polymer to improve the electrical conduction between the enzyme and the electrode as well as the stability of the system.
  • a conductive material such as a conductive metal, especially platinum, copper, silver, aluminum, gold or aluminum.
  • steel or carbon such as glassy carbon, carbon fibers, carbon nanotube fibers or diamond ...
  • said conductive material is covered with a deposit comprising at least one laccase according to the invention; said deposit may further comprise a redox polymer to improve the electrical conduction between the enzyme and the electrode as well as the stability of the system.
  • the redox polymer may for example be chosen from polymers based on ferrocene, osmium and ruthenium and conductive polymers such as, for example, polypyrrole and polyananilline.
  • the methods for immobilizing the laccase on said conductive material may be chosen from the standard methods available to those skilled in the art which include in particular the inclusion of the laccase of a polymeric matrix, the adsorption of the laccase to the polymeric membrane surface, covalent bonding, electrodeposition (Gao et al., Chem Int.Ed. 2002, 41, No. 5, 810-813) or the technique described in the US patent application 2009/0053582.
  • the laccase electrode on which the laccase is immobilized is also covered with a membrane which prevents detachment of said enzyme from the electrode.
  • said membrane may consist of a nation, cellulose or any biocompatible material, that is to say compatible with a physiological vironcession.
  • the present invention thus also relates to an oxygen biosensor, phenolic compounds or aromatic amines consisting of a laccase electrode according to the invention.
  • a biosensor consists of an electrode on which is immobilized a bioreceptor capable of recognizing a biological target; the fixation of the biological target on the bioreceptor leads to physico-chemical modifications of the membrane and the production of an electrical signal by an electrochemical transducer (amperometric, potentiometric, conductimetric, ...) attached to the electrode.
  • the bioreceptor is a laccase according to the invention and the biological target is a compound selected from oxygen, phenolic compounds or aromatic amines.
  • the present invention also relates to an oxygen sensor consisting of an electrode according to the invention.
  • the laccase electrode according to the invention can also be advantageously used as a cathode in an enzymatic biopic;
  • Figure 1A schematically represents the operating principle of an enzymatic biopia.
  • the biopi IES 3 enzyme according to the invention are devices comprising an electrode laccase (lacca) as cathode and an anode where an oxidation reaction of a substrate occurs (catalyzed F "enzyme X");
  • the substrate may be glucose and the "enzyme X" glucose oxidase, such a cell is of particular interest when the biopic is implanted in an individual for a medical application;
  • the substrate may also be chosen, for example, from nitrites, nitrates, sulphides, urates, ascorbates, glutamates, pyruvates, lactates, cellulose ...
  • glucose oxidase glucose oxidase (glucose or all the sugars being oxidized by this enzyme)
  • lactate oxidase lactate
  • pyruvate oxidase pyruvay
  • alcohol oxidase alcohol
  • cholesterol oxidase cholesterol oxidase
  • glutamate oxidase glutamate
  • pyranose oxidase pyranose
  • choline oxidase cellobiose dehydrogenase (glucose)
  • glucose dehydrogenase glucose dehydrogenase or all sugars being oxidized by this enzyme
  • pyranose dehydrogenase fructose dehydrogenase (fructose)
  • FIG. 1B more specifically illustrates enzymatic glucose biopia; such an enzymatic biopic consists of two electrodes modified by the immobilization of enzymes.
  • a glucose oxidase (GOx) is attached to the anode (1) via a conductive polymer "/” and a laccase (LAC) is attached to the cathode (2) by means of a conductive polymer "ZI".
  • GOx glucose oxidase
  • LAC laccase
  • the electrons are transferred from the glucose present in the physiological fluid to the GOx, then from the GOx to the conductive polymer "/" and the conductive polymer "I" to the anode, to the cathode, the electrons are transferred from the cathode to the conductive polymer "27", then to the laccase and finally the laccase to oxygen present in the physiological fluid.
  • a biopile can also possibly operate by modifying the electrodes with their respective enzymes and by adding soluble mediators, such as ferrocenemethanol for the anode and potassium ferricyanide for the cathode, and adding if necessary a membrane separating the anode and the cathode.
  • soluble mediators such as ferrocenemethanol for the anode and potassium ferricyanide for the cathode
  • Such a biopile can be used as a miniaturized energy source and implanted in a living organism.
  • Figure 1A shows schematically the operating principle of an enzymatic biopile
  • Figure 1B shows a glucose enzyme biopile.
  • Figure 2 shows the plasmid map of the vector pFD56.
  • Figure 3 is a graph showing the catalytic activity of the laccase of SEQ. he). # 3 according .'invention depending on the concentration of ABTS at 37 ° C,
  • Figure 4 is a graph showing the catalytic activity of SEQ ID laccase. No. 3 according to the invention as a function of the concentration of SGZ at 37 ° C.
  • Figure 5 is a graph showing the relative activity of the laccase of SEQ. ID. No. 3 according to the invention as a function of the pH on the oxidation of ABTS.
  • Figure é illustrates the stability of the laccase of SEQ. ID. No. 3 according to the invention at different pH at 4 ° C.
  • Figure 7 is a graph showing the relative activity of the laccase of
  • SEQ. ID. No. 3 according to the invention as a function of the temperature on the oxidation of the ABTS.
  • Figure 8 is a graph showing the stability of the laccase of SEQ.
  • Figure 9 shows the effect of NaCl on the activity of SEQ laccase. ID. No. 3 according to the invention at pH 7.
  • This strain is used for plasmid amplification during the steps of constructing the protein expression vector.
  • GS1 15 Pichia pastoris yeast strain used for the production of Laccase after integration of the cassette from vector PFD56 containing the AOX1 promoter, the signal peptide-factor and the DNA sequence coding for the laccase of SEQ. ID, No. 3 according to the invention derived from Podospora anserina.
  • Bacteria DH has $ supercompetent are prepared using the method inoue (Sambrook and Russian!).
  • the DNA is introduced into the yeast Pichia pastoris GS1 by electroporation on an Eppendorf Eporator (Eppendorf, France).
  • a plasmid DNA purification kit (Qiagen) is used for small and large amounts of DNA preparations.
  • the double-stranded DNA is sequenced by the company Millegen (Toulouse, France).
  • the SED sequence gene. ID. No. 4 corresponding to the sequence coding for the truncated Podospora anserina laccase (B2ANKS accession) of the portion coding for the first 30 amino acids of the N-terminus was synthesized by. Genecust Europe (Luxembourg), The NheI and NotI restriction sites were respectively added at 3 'and 5' of the sequence to facilitate cloning.
  • the plasmid pPICZa as well as the synthesized gene were then treated with the two restriction enzymes Nbe1 and NoIl and the digests were gel purified with the "nucleospia" kit.
  • the laccase gene is then ligated into the plasmid by co-incubation with T4 DNA Ligase at 37 ° C overnight.
  • the neoformed plastrids (pFD56) are then selected and amplified by transformation of DH5 ⁇ bacteria on a box containing zeocin at 25 ⁇ g / ml.
  • the corresponding gene is introduced by homologous recombination at the AOX1 gene.
  • the plasmid pFD56 once linearized by digestion with PmeI enzyme is introduced into yeast by electroporation and positive clones are selected on YPD medium - agar containing Zeocin to iOOpg / 'ml.
  • the sequence of the plasmid thus obtained is SEQ. 1D. # 6.
  • the laccase enzyme of SEQ ID. No. 3 is produced by the yeast Pichia pastoris via methanoi induction. To do this, a preculture of 200 ml of zeocin-supplemented YPD medium (100 ⁇ g / ml) is inoculated with strain GS1 having integrated the cassette contained on the plasmid pFD56. After stirring overnight at 220 rpm and at 30 ° C. C, this preculture is then centrifuged for 10 min at 4000 rpm and the pellet is taken up in 200 ml of sterile water in order to eliminate any presence of glucose.
  • a 2L culture in MMH medium containing 2 mM CuSO 4 in an Erlenmeyer flask of 51 is then seeded with this pellet.
  • the yeasts are incubated at 25 ° C with stirring (220 rpm) for 2hrs before adding 0.5% methanol to start the induction. This induction step will be repeated during days to obtain the maximum of enzymes.
  • the 2L culture is centrifuged and the supernatant containing the enzyme of interest is concentrated on a stirring cell with a YM10 membrane with a cutoff of 1 OkDa to reach a volume of 4. 5mI final.
  • 1.7M of ammonium sulfate is added to the 4-5ml of the culture supernatant before being filtered through a 0.22 ⁇ filter to be injected onto a hydrophobic interaction chromatography column, a 60mJ PhenyiHP (GE Healthcare *), coupled to the AT system purifying (GE Healthcare *), equilibrated in a Potassium phosphate 50r.oM buffer, (NH ⁇ SC 1.7, pB 6.
  • the elution is carried out by a gradient from 0% to 100% d Potassium phosphate buffer 50mM pH 6 at a flow rate of 2.5 mL min
  • the fractions containing the laccase protein are identified by an activity test with ABTS and are pooled, concentrated and stored in 50mM Potassium phosphate buffer pH 6 by Arrsicon YM10 membrane centrifugation At this stage, the protein is pure and can be stored at -20 ° C in soluble form.
  • the enzyme concentration of a solution is calculated from a BSA range according to the Bradford technique [2].
  • Enzymatic tests were performed using a Varian spectrophotometer in a 0.1 M citrate / phosphate buffer at 37 ° C in a volume of 3 mL following the oxidation of different substrates at a given wavelength as a function of time.
  • the specific activity of the enzyme is expressed in pmol of oxidized substrates per minute and per rng of protein.
  • the experiments are carried out at 37 ° C. on a Varian spectrophotometer in a 0.1 M citrate / phosphate buffer pH 3.4.
  • the concentration of ABTS varies in the test from 0 to 15 mM.
  • the test is triggered by the addition of enzyme.
  • the experimental points are analyzed by non-linear regression according to the model of Miehaelis-Menten using the Sigma-plot 6.0 software according to the equation below:
  • Figure 3 shows the catalytic activity of the accase of SEQ ID NO. No. 3 as a function of ABTS concentration at 37 ° C
  • the experiments are carried out at 37 ° C. on a Varian spectrophotometer in a 50 mM citrate-phosphate buffer pH 7.
  • the concentration of SGZ, diluted in methanol, varies in the 0 to 50 ⁇ test.
  • the test triggered by the addition of enzyme, is to monitor the oxidation of SGZ at 530 nm by colorimetric changes (ss 3 0n m -. 84 mM 1 cm "1).
  • the study of the change in the rate constant of the reaction as a function of pH is carried out over a pH range of from 3 to 7 in a 0.1 M citrate / phosphate buffer using FABTS at 1 nm as a substrate.
  • the experiments are carried out at 37 ° C. using a Varian spectrophotometer.
  • the activity is followed by the oxidation of ⁇ BTS resulting in a colorimetric change measured at 420 nm.
  • the test is triggered by adding the enzyme.
  • Figure 5 shows the relative activity of the laccase of SEQ. ÎD, No. 3 as a function of pH on oxidation of ABTS.
  • the enzyme at a concentration of 0.15 mg / ml is preincubated in 50 mM phosphate-citrate buffer (or 50 mM Tris-H 2 S0 4 for pH 8 and 9) at a given pH at 4 ° C.
  • samples of 5 ⁇ are taken and the residual activity of the enzyme incubated at different folds is determined using a Varian spectrophotometer at 44 () nm in a 0.1M citrate / phosphate pH buffer. 3.4 at 37 ° C, in the presence of 150 ⁇ ABTS, The test is triggered by adding the enzyme. The results obtained are shown in Figure 6.
  • the "enzyme is preincubated at a concentration of 0.35 mg MI in a bath to dryness at 60 ° C and 37 ° C in a potassium phosphate buffer 50 mM. PH 6.
  • Residual activity of the enzyme incubated at these temperatures is determined using a Varian spectrophotometer at 440 nm in 0.1 M citrate / phosphate buffer pH 3.4 at 37 ° C., in the presence of 150 ⁇ l of ABTS. is triggered by adding the enzyme
  • the results obtained are shown in Figure S. . Eiisde of the eBzymatii activity and the presence of NaCi
  • two enzymatic electrodes were prepared comprising either the Iaccase of SEQ. ID. No. 3 according to the present invention, the Iaccase of Trametes versicolor (the iaccase commonly used in electrochemistry) and a redox polymer.
  • the catalytic current for C reduction is 40% higher for the electrode modified with the new enzyme.
  • the stability over time is 200% greater.

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EP13735432.0A 2012-05-21 2013-05-21 Laccase aus podospora anserina und dessen verwendungen Withdrawn EP2852665A1 (de)

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FR1254615A FR2990698B1 (fr) 2012-05-21 2012-05-21 Laccase de podospora anserina et ses applications
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JP6816536B2 (ja) * 2017-01-30 2021-01-20 アイシン精機株式会社 乳酸酸化酵素、乳酸酸化酵素をコードする核酸分子、乳酸酸化酵素を用いた乳酸測定方法、乳酸センサー、及びバイオ燃料電池
CN110699395A (zh) * 2019-11-05 2020-01-17 常熟浸大科技有限公司 一种漆酶催化制备聚苯胺的方法
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