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• • 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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• 1995
  • 1995-01-02 FR FR9500002A patent/FR2728911A1/fr active Granted
  • 1995-12-28 US US08/849,732 patent/US5972672A/en not_active Expired - Fee Related
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