WO2020108923A1 - Procede de production d'enzymes par une souche appartenant a un champignon filamenteux - Google Patents
Procede de production d'enzymes par une souche appartenant a un champignon filamenteux Download PDFInfo
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- WO2020108923A1 WO2020108923A1 PCT/EP2019/080130 EP2019080130W WO2020108923A1 WO 2020108923 A1 WO2020108923 A1 WO 2020108923A1 EP 2019080130 W EP2019080130 W EP 2019080130W WO 2020108923 A1 WO2020108923 A1 WO 2020108923A1
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- 101710112457 Exoglucanase Proteins 0.000 description 3
- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 3
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Classifications
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- 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- 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
-
- 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
- C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- 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
- C12P21/00—Preparation of peptides or proteins
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- 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
- C12R2001/885—Trichoderma
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- 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
Definitions
- the invention relates to a process for the production of cellulases by a filamentous fungus, necessary for the enzymatic hydrolysis of lignocellulosic biomass implemented, for example, in the processes for producing so-called second generation (2G) sweet juices.
- 2G second generation
- sugary juices can be used to produce other products chemically or biochemically / fermentatively (for example, alcohols such as
- ethanol-type biofuels or butanol or other molecules, for example solvents such as acetone and other bio-based molecules .
- Cellulases can also be used in other processes, especially in the chemical, paper and textile industries.
- 2nd generation biofuels (known as "2G"), if we take this particular example of implementation, is the subject of numerous studies.
- the latter are produced, in particular, from woody substrates such as different woods (hardwoods and conifers, miscanthus, or "TCR", which is the acronym for Quick Growth Coppice), by-products from agriculture (wheat straw , rice straw, corn cobs, etc ...) or other food industries, paper mills, etc ...
- TCR which is the acronym for Quick Growth Coppice
- biofuels first generation which are produced from sugar cane, corn, wheat or beet.
- Lignocellulosic biomass is characterized by a complex structure made up of three main fractions: cellulose (35 to 50%), which is a polysaccharide essentially made up of hexoses; hemicellulose (20 to 30%), which is a polysaccharide essentially consisting of pentoses; and lignin (15 to 25%), which is a polymer of complex structure and of high molecular weight, composed of aromatic alcohols linked by ether bonds. These different molecules are responsible for the intrinsic properties of the plant wall and are organized into a complex tangle.
- cellulose and hemicellulose are those which allow the production of 2G sweet juices.
- the process of its transformation into an ethanol-type biofuel comprises several stages:
- the pretreatment makes it possible to make the cellulose accessible to the enzymes which are cellulases.
- the enzymatic hydrolysis stage allows the transformation of cellulose into sugars, such as glucose, which are then transformed into ethanol during the fermentation stage by generally the yeast Saccharomyces cerevisiae.
- the stage of distillation will separate and recover the ethanol from the fermentation wort. Note, as mentioned above, that one can also choose to stop the process for obtaining sugars of the glucose type, in order to valorize them as such, or even treat them differently to obtain other alcohols or bio-based molecules.
- Lignocellulosic materials are cellulosic materials, that is to say made up of more than 90% by weight of cellulose, and / or lignocellulosic, that is to say made up of cellulose, hemicelluloses, which are polysaccharides essentially consisting of pentoses and hexoses as well as lignin, which is a macromolecule of complex structure and of high molecular weight, based on phenolic compounds.
- Wood, straw, corn cobs are the most used lignocellulosic materials, but other resources, dedicated forest crops, residues of alcohol-producing plants, sugar and cereals, products and residues of the paper industry and products processing lignocellulosic materials can be used. Most of them are made up of about 35 to 50% cellulose, 20 to 30% hemicellulose and 15 to 25% lignin.
- the process for the biochemical transformation of lignocellulosic materials into ethanol comprises a physico-chemical pretreatment step, followed by an enzymatic hydrolysis step using an enzymatic cocktail, a step of ethanolic fermentation of the released sugars, ethanolic fermentation and the enzymatic hydrolysis can be carried out simultaneously, and a step of purifying the ethanol.
- the enzyme cocktail is a mixture of cellulolytic (also called cellulase) and / or hemicellulolytic enzymes.
- Cellulolytic enzymes exhibit three main types of activity: endoglucanases, exoglucanases and cellobiases, the latter also being called b-glucosidases.
- Hemicellulolytic enzymes in particular exhibit xylanase activities.
- the enzymatic hydrolysis is effective and takes place under mild conditions.
- the cost of enzymes remains very high, representing from 20 to 50% of the cost of transforming the lignocellulosic material into ethanol. Therefore, a lot of work has been done to reduce this cost: optimizing the production of enzymes first, by selecting hyperproductive microorganisms and improving the production processes of said enzymes, decreasing the amount of enzymes in hydrolysis then, by optimizing the pretreatment step, by improving the specific activity of these enzymes, and by optimizing the implementation of the enzymatic hydrolysis step.
- Trichoderma reesei is the most used microorganism for the production of cellulases. Wild strains have the ability to excrete, in the presence of an inducing substrate, cellulose for example, the enzyme complex considered to be best suited for the hydrolysis of cellulose.
- the enzymes in the enzyme complex contain three main types of activity: endoglucanases, exoglucanases and cellobiases and other proteins with properties essential for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, xylanases for example .
- the presence of an inducing substrate is essential for the expression of cellulolytic and / or hemicellulolytic enzymes.
- the nature of the carbon substrate has a strong influence on the composition of the enzyme complex. This is the case with xyloses, which, combined with a carbon-inducing substrate such as cellulose or lactose, makes it possible to significantly improve the so-called xylanase activity.
- xyloses which, combined with a carbon-inducing substrate such as cellulose or lactose, makes it possible to significantly improve the so-called xylanase activity.
- the regulation of cellulase genes on different carbon sources has been studied in detail. They are induced in the presence of cellulose, its hydrolysis products, such as cellobiose, or certain oligosaccharides such as lactose or sophorose (cf. Ilmén et al., 1997; Appl. Environ. Microbiol. 63: 1298-1306 .)
- the process for the production of cellulases by Trichoderma reesei has undergone significant improvements with a view to extrapolation to an industrial scale.
- To obtain good productivities in enzymes it is necessary to provide a source of carbon quickly assimilated for the growth of Trichoderma reesei, and an inducing substrate which allows the expression of cellulases and the secretion in the culture medium.
- Cellulose can play these two roles; however, it is difficult to use at the industrial stage and it has been proposed to replace it with soluble carbon sources, of the glucose, xylose or lactose type, the lactose also playing the role of an inducing substrate.
- Patent FR-B-2 555 603 proposes a protocol which makes it possible to achieve a protein concentration of the order of 35 to 40 g / L with a productivity of the order of 0.2 g / L / h and which consists of two stages: a first stage of growth in "batch" mode where it is necessary to provide a source of carbon quickly assimilated for the growth of Trichoderma reesei, then a stage of production in "fed-batch” mode using a inducing substrate (example: lactose) which allows the expression of cellulases and secretion in the culture medium.
- the optimal flux applied is between 35 and 45 mg.g Vh 1 (milligrams of inducing substrate per milligram of biomass and per hour).
- patent EP-B-2,744,899 proposes an improvement by selecting in particular a bioreactor having a coefficient of volumetric transfer of oxygen KLa associated with a particular selection both of the concentration of carbonaceous growth substrate in the first step and a limiting flux level of carbon source in the second step.
- a foam during the growth stage in particular. It may be a so-called “dry” foam, ie constituting a dispersion of gas in a liquid phase, and which therefore has a density close to that of a gas and which forms in the upper part of the bioreactor. It can also be a so-called “wet” foam, which is a foam for extending / increasing the reaction volume due to the trapping of gas bubbles (of air) in the liquid. Its density is higher than dry foam (because its gas / liquid ratio is lower). Whether it is one or the other, or a mixture of these two types of foam, they present a real problem of industrial process control.
- a first solution consisted in adding anti-foaming agents to the reaction medium during the growth step.
- anti-foam agents is certainly effective in resuspending the foam in liquid form, but it is not without its drawbacks.
- the resuspension of the liquid in liquid causes a sharp increase in pH, largely disrupting the pH regulation that must be carried out, or even an unwanted changeover from the growth stage to the production. This can be caused by the massive supply of reagents (sugars) blocked on the surface due to the foam, which, when it collapses under the effect of the anti-foam, suddenly come into contact with the biomass. large quantity.
- anti-foaming agents also causes a decrease in the concentration of dissolved oxygen in the medium (because it coalesces the air bubbles), which can have an impact on microorganisms which are strict aerobes or on their productivity.
- these agents are often based on oils, which cannot be eliminated on their own: once the production of enzymes is complete, if the separation is carried out between the enzymes and the rest of the biomass (the fungi ), in particular by conventional technologies using membrane filtration means, these defoaming agents are capable of clogging the membranes, and it may therefore be necessary to have to add a step of separation of these agents once the production is complete , without which poor separation performance results. Their addition also has an additional production cost.
- the invention therefore aims to develop an improved enzyme production process, avoiding or at least limiting the foaming phenomenon, without causing at least some of the above drawbacks, and in particular without causing any complication in the conduct of the nor compel them to make specific genetic modifications to microorganisms.
- the invention firstly relates to a process for the production of enzymes by a strain belonging to a filamentous fungus, said process comprising two stages:
- step (b) a second step of producing enzymes, from the culture medium obtained in the first step (a), in the presence of at least one inducing carbon substrate, at a pH less than or equal to 4.6.
- the pH of the growth stage is regulated to keep it within the required range.
- the pH of the production stage is also regulated.
- the regulation is done in a known manner, in particular by monitoring the pH continuously or sequentially with ad hoc sensors, and adding (acid or base during the step to remain at the set point.
- the pH of one and / or the other of the steps using a buffer solution.
- the solution of the invention is surprisingly simple, since there was nothing to suggest that modifying, towards a higher acidity, in reasonable proportions (without going down, preferably below 3.5 or 3.6 or 3.7), the pH during growth was going to have consequences on the complex phenomenon of foaming. It is very advantageous in terms of industrial production management: - the bioreactor in which the growth step is carried out is fully equipped to regulate the pH to these values, so there is no difficulty in implementing it the invention with conventional bioreactors; - since there is no or very little foam formed, it is possible to calculate the size of the bioreactor as accurately as possible, and to increase its useful volume (it is no longer necessary to provide an additional “lost” volume intended to contain possible excessive foam overflows);
- the pH in stage (a) of growth and / or in stage (b) of production is at least 3.5, and in particular less than or equal to 4.4, it is in particular between 3.5 and 4.4 or between 3.8 and 4.4: this brings the pH of the growth stage closer to the pH of the production stage.
- the pH in the growth stage is maintained at at least 3.6, in particular at least 3.7 or at least 3.8.
- the pH in the growth stage is maintained at at most 4.4.
- the pH in stage (a) of growth is substantially identical to the pH in stage (b) of production. If, in particular, the two stages are carried out in the same bioreactor, choosing the same pH thus simplifies the regulation of the pH over the entire duration of the process. It is thus possible to have the same regulation instruction on the two stages or to use the same buffer solution.
- the pH in stage (b) of production can be chosen more acidic than the pH in stage (a) of growth, for example at least 0.3 to 0.6, in particular a more acidic pH (lower therefore) from 0.4 to 0.6.
- the pH, during stage (a) of growth is regulated by controlled addition of a nitrogenous compound, in particular of ammonia, which plays both the role of basic agent and of source agent d for the growth of microorganisms.
- step (b) of production takes place in batch, fed-batch or continuous mode or according to several of these modes successively.
- the method according to the invention may comprise an intermediate step (c) between step (a) and step (b), this intermediate step (c) being a step of diluting the culture medium obtained at growth stage (a).
- stage (a) of growth and stage (b) of production can be carried out in the same bioreactor or in two separate reactors, with transfer of the reaction medium from one reactor to the other.
- the first case is the simplest, with a single reactor we avoid having to transfer the reaction medium.
- the second case makes it possible to precisely adapt the characteristics and equipment of each of the bioreactors according to the needs of each of the stages.
- the concentration of carbonaceous growth substrate is chosen to be between 15 and 60 g / l.
- the second stage (b) of production is carried out with a limiting flux of inducing carbon substrate, in particular between 30 and 140 mg.g Vh 1 , (that is to say between 30 and 140 grams per gram of biomass and per hour), preferably between 35 and 45 mg.g Vh 1 , and preferably with an aqueous solution of inducing carbon substrate at a concentration of between 200 and 600 g / l.
- a limiting flux of inducing carbon substrate in particular between 30 and 140 mg.g Vh 1 , (that is to say between 30 and 140 grams per gram of biomass and per hour), preferably between 35 and 45 mg.g Vh 1 , and preferably with an aqueous solution of inducing carbon substrate at a concentration of between 200 and 600 g / l.
- the strain used in the process according to the invention is preferably a strain of Trichoderma reesei or of Trichoderma reesei modified by selection mutation or genetic recombination. But there is no need to genetically modify it in order to prevent the formation of hydrophobins during its growth. These may include strains CL847, RutC30, MCG77, or MCG80 mentioned above.
- the process according to the invention preferably produces cellulolytic and / or hemicellulolytic enzymes (cellulases).
- the method according to the invention is operated in the absence of anti-foaming agents, in particular during step (a) of production. No longer having to use anti-foaming agents is very advantageous economically.
- the addition of these agents can cause problems in limiting the transfer of oxygen from the air supplied to the bioreactor towards the liquid phase comprising the fungi, which harms their growth. These agents can also pose problems during the filtration of the culture medium at the end of the production stage.
- the subject of the invention is also the use of the enzymes obtained by the process described above for the enzymatic hydrolysis of terrestrial or marine cellulosic / hemicellulosic biomass.
- FIG. 1 represents photos of the bioreactors used according to examples conforming and not conforming to the process of the invention with a strain, photos taken during the growth step of the process.
- FIG. 2 represents photos of the bioreactors used according to examples conforming and not conforming to the process of the invention with a different strain, photos taken during the growth step of the process.
- Figure 3 is a graph showing the concentration in grams per liter of biomass and protein produced according to a comparative example.
- FIG. 4 is a graph representing the concentration in grams per liter of biomass and proteins produced according to an exemplary embodiment of the invention.
- FIG. 5 is a graph representing the concentration in grams per liter of biomass and proteins produced according to an exemplary embodiment of the invention.
- the present invention has developed a process for the production of enzymes, in particular cellulases, which makes it possible to prevent the appearance of foam. It has surprisingly appeared that working during growth at low pH (less than 4.6, in particular less than or equal to 4.4) does not slow the growth of the microorganism and makes it possible to avoid the appearance of the foam.
- the method of conducting the process has 2 phases:
- phase in batch mode which preferably lasts between 30 and 50 hours, with a pH advantageously less than or equal to 4.4 and with a concentration of carbon substrate of 15 to 60 g / L preferably
- a phase in fed-batch mode which preferably lasts between 100 and 200 hours, in particular between 100 and 150 hours, advantageously at a pH less than or equal to 4.4 also, and with a limiting flux of carbon source from 35 to 140mg.g 1 .h 1 , and preferably between 35 and 45 mg.g Vh 1 .
- the industrial strains used belong to the species Trichoderma reesei, modified to improve the cellulolytic and / or hemicellulolytic enzymes by mutation-selection processes, such as for example the CL847 strain ((one of these processes is described in particular in US Pat. No. 4,762,788 Strains improved by genetic recombination techniques can also be used, which are cultivated in agitated and aerated fermenters under conditions compatible with their growth and the production of enzymes.
- the main sources of carbon can be soluble sugars such as lactose, glucose or xylose:
- the carbonaceous growth substrate is preferably chosen from lactose, glucose, xylose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass.
- the inducing carbon substrate is preferably chosen from lactose, cellobiose, sophorose, the residues obtained after ethanolic fermentation of the monomer sugars of the enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a biomass cellulosic.
- This type of residue / extract can therefore also be used as a total carbon source, that is to say both for the growth of the microorganism and the induction of the expression system.
- This carbon source can be used more particularly by genetically improved strains and in particular recombinant strains.
- the operating conditions of pH and temperature, for the growth stage and the production stage are as follows:
- a pH of 4.4 is preferably chosen and a temperature of 27 ° C during the growth phase and a pH of 4 or 4.4 also, and a
- vvm (aeration rate expressed in volume of air per volume of reaction medium and per minute) applied during the process is between 0.3 and 1.5 min 1 and the rpm (speed of rotation) must make it possible to regulate the pressure in 0 2 between 20% and 60%.
- aeration of 0.3 to 0.5 vvm and a stirring enabling the pressure to be adjusted to 0 2 to 30% or 40% are chosen.
- the carbon substrate chosen for obtaining the biomass is introduced into the fermenter before sterilization, or is sterilized separately and introduced into the fermenter after sterilization.
- the concentration of carbon substrate is between 200 and 600 g / L depending on the degree of solubility of the carbon substrates used (in particular as regards the inducing substrate).
- a preculture of the strains is carried out in 2 Fernbach flasks with a useful volume of 500 ml, which are inoculated with each a tube of spores of T reesei TR3002 and CL847. They are placed in an INFORS HT Multitron commercial reference incubator at 30 ° C, with orbital shaking of 150 rpm for 72 hours. They are then dispatched to 8 vacuum sterilized flasks (80 mL per flask) which will be used to inoculate each fermenter / bioreactors.
- the operating conditions for the production of cellulases from the strains obtained at the end of preculture are as follows:
- the experiments have two phases.
- a sample is taken each day with monitoring of the dry weight, glucose, lactose, galactose, xylose concentrations.
- Culture supernatants of 5 mL are stored at 4 ° C. for the enzymatic assays and proteins produced at the end of the culture.
- Example 1 is carried out from the strain TR3002. This strain is described in the following publications: Ben Chaabane F, Jourdier E, Licht R, Cohen C and Monot F (2012)
- the production phase is carried out at pH 4 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass and per hour.
- Example 2 is carried out from the strain TR3002.
- the growth phase is carried out at pH 4.8 at 27 ° C and with a glucose concentration of 15 g / L
- the production phase is carried out at pH 4.8 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.
- Example 3 is made from the strain TR3002.
- the growth phase is carried out at pH 5.5 at 27 ° C and with a glucose concentration of 15 g / L
- the production phase is carried out at pH 5.5 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.
- Example 4 is made from the strain TR3002.
- the growth phase is carried out at pH 4.4 at 27 ° C and with a glucose concentration of 15 g / L
- the production phase is carried out at pH 4.4 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.
- Example 5 is carried out from the strain CL847.
- This strain is described in the following publications: - Jourdier E, Poughon L, Larroche C, Monot F and Ben Chaabane F (2012) "A new stoichiometric miniaturizationstrategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains” .
- Microbial Cell Factories 1 1, 70 Impact Factor: 3.60
- Jourdier E, Ben Chaabane F, Poughon L Jourdier E, Ben Chaabane F, Poughon L
- the growth phase is carried out at pH 4 at 27 ° C and with a glucose concentration of 15 g / L
- the production phase is carried out at pH 4 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass and per hour.
- Example 6 is made from the CL847 strain.
- the growth phase is carried out at pH 4.4 at 27 ° C and with a glucose concentration of 15g / L
- the production phase is carried out at pH 4.4 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.
- Example 7 is carried out from the CL847 strain.
- the growth phase is carried out at pH 4.8 at 27 ° C and with a glucose concentration of 15 g / L
- the production phase is carried out at pH 4.8 at 25 ° C, with a lactose concentration of 220 g / L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.
- Example 8 is made from the CL847 strain.
- the growth phase is carried out at pH 5.5 at 27 ° C and with a glucose concentration of 15 g / L
- FIGS. 1 and 2 represent photographs of the bioreactors of examples 1, 2, 3, 5, 6, 7, 8 after 24 hours: (the reference 1 corresponds to example 1, and so on).
- Figure 3 indicates the production in g / L of biomass (line with dots) and proteins (line with triangles) as a function of time expressed in hours for comparative example 2
- Figure 4 represents the same type of graph for example 1 according to the invention
- FIG. 5 for example 4 according to the invention: the comparison of these graphs confirms that the protein production remains at the same level, whether or not the growth pH is lowered .
- the enzyme activity levels of the enzymes produced were also evaluated by measuring the activity known as filter paper ("FPase").
- FPase filter paper
- This method allows to measure the overall activity of the enzyme pool (endoglucanases and exoglucanases).
- the FPase activity is measured on Whatman N ° 1 paper (procedure recommended by the IUPAC biotechnology commission): the test portion of the enzymatic solution is determined, which achieves 4% progress in the enzymatic reaction in 60 minutes.
- the principle of the filter paper activity is to determine by dosage at DNS (dinitrosalicylic acid) the amount of reduced sugars from Whatman N ° 1 paper.
- the FPase values obtained for the 8 examples, and therefore reflecting the overall activity of the enzymatic cocktail, and therefore its quality, have conventional values for the strains used, namely between 0.8 and 1 IU / mg.
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CN201980077783.4A CN113195703A (zh) | 2018-11-26 | 2019-11-04 | 通过属于丝状真菌的菌株生产酶的方法 |
CA3117434A CA3117434A1 (fr) | 2018-11-26 | 2019-11-04 | Procede de production d'enzymes par une souche appartenant a un champignon filamenteux |
EP19797284.7A EP3887503A1 (fr) | 2018-11-26 | 2019-11-04 | Procede de production d'enzymes par une souche appartenant a un champignon filamenteux |
CN202410179110.2A CN117987396A (zh) | 2018-11-26 | 2019-11-04 | 通过属于丝状真菌的菌株生产酶的方法 |
US17/295,198 US20220017933A1 (en) | 2018-11-26 | 2019-11-04 | Method for the production of enzymes by a strain belonging to a filamentous fungus |
BR112021008030-9A BR112021008030A2 (pt) | 2018-11-26 | 2019-11-04 | processo de produção de enzimas por uma capa pertencente a um fungo filamentoso |
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- 2019-11-04 BR BR112021008030-9A patent/BR112021008030A2/pt unknown
- 2019-11-04 US US17/295,198 patent/US20220017933A1/en active Pending
- 2019-11-04 CN CN201980077783.4A patent/CN113195703A/zh active Pending
- 2019-11-04 CN CN202410179110.2A patent/CN117987396A/zh active Pending
- 2019-11-04 WO PCT/EP2019/080130 patent/WO2020108923A1/fr unknown
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FR3088934B1 (fr) | 2024-07-26 |
BR112021008030A2 (pt) | 2021-07-27 |
CN113195703A (zh) | 2021-07-30 |
EP3887503A1 (fr) | 2021-10-06 |
CA3117434A1 (fr) | 2020-06-04 |
CN117987396A (zh) | 2024-05-07 |
FR3088934A1 (fr) | 2020-05-29 |
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