WO2017144590A1

WO2017144590A1 - Biocatalyst for manufacturing ethanol from corn

Info

Publication number: WO2017144590A1
Application number: PCT/EP2017/054173
Authority: WO
Inventors: Anaïs GUILLAUME; Yoann Arnaud CARRE; Antoine Serge Dominique Drevelle; Aurore THORIGNE
Original assignee: Etablissements J. Soufflet
Priority date: 2016-02-23
Filing date: 2017-02-23
Publication date: 2017-08-31
Also published as: FR3047999A1

Abstract

The invention relates to a biocatalyst for manufacturing ethanol from amylaceous raw material, said biocatalyst having at least a protease activity, a cellulase activity that is not a β-glucosidase activity, and a β-glucosidase activity, the biocatalyst being obtainable in a biocatalyst manufacturing process that involves a step in which a substrate is fermented in a solid medium using an Aspergillus tubingensis SCC027 strain registered on 1 February 2016 with the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, Netherlands) under number CBS 136961, or a natural variant of said strain.

Description

Biocatalyst for the production of ethanol from maize

The present invention relates to biocatalysts for accelerating and improving the production of ethanol by fermentation of starchy raw materials.

The rapid and rapid reduction of fossil energy resources and the pollution problems associated with their use are at the origin of the need to find alternative sources of energy for these fuels.

Biomass from agricultural products and / or co-products is now a significant source of carbon and interest in replacing fossil fuels. Ethanol produced from the fermentable sugars contained in the plants can thus be used in vehicles equipped with combustion engines. The increase in this use has resulted in a rapid increase in bioethanol production in recent years in both North America and Europe, particularly from wheat and / or maize crops or their agricultural byproducts.

The generalization of the use of bioethanol as fuel remains, however, still sought to fight for example against global warming.

Such generalization will, however, imply a need to increase wheat and / or maize crops for bioethanol production. However, the world's cultivable areas remain limited, generating tensions between the areas of cultures dedicated to the production of ethanol and those dedicated to food and feed.

Therefore, although processes for making ethanol from starchy feedstock, particularly from wheat or corn flour, are now commonly used by those skilled in the art, there is still a great need for increase in production yields of these processes. Moreover, given the quantities targeted, even a 1% increase in ethanol production yield from starchy raw material is a particularly beneficial increase for both producers and farmers.

The present inventors have surprisingly identified a strain of Aspergillus tubingensis for producing, by fermentation in a solid medium, a biocatalyst whose enzymatic composition allows, when it is added during the fermentation stage, and or yeast propagation, a process for the manufacture of ethanol from starchy material and in particular maize, to significantly accelerate the production of ethanol, to increase the final ethanol concentration of at least 1% and reduce the production of glycerol, a co-product of ethanol. This strain, called strain SCC027 was filed on ¹ February 2016 with the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the number CBS 136961.

By performing a proteomic study of the biocatalyst obtained using this strain, the inventors have shown that it has an original enzymatic composition, including activities mainly proteases, cellulases and hemicellulases.

They also demonstrated that nearly 90% of the effect observed with this biocatalyst on ethanol production was due to a combination of 5 families of enzymatic activities: the protease, cellulase, xylanase, β-glucosidase and pectinase activities, and more particularly to a combination of 7 specific fungal enzymes: aspergillopepsin A, endoglucanase, endoxylanase A, endoxylanase C, β-glucosidase A, cellobiohydrolase and polygalacturonase C.

Moreover, all these enzymatic activities are produced simultaneously by the strain of Aspergillus tubingensis SCC027 by fermentation. The production process of this biocatalyst is therefore practical to implement, since it does not require different fermentation conditions depending on the enzymatic activities produced.

The inventors have furthermore shown that the production of glycerol, a co-product of the ethanol production process, was significantly reduced in the presence of this biocatalyst. However, the level of glycerol production is an indicator of the performance of ethanol production by yeast. The reduction of glycerol can also have beneficial effects on the downstream ethanol production process under industrial conditions, in particular on the evaporation of vinasses.

The present invention therefore a strain of Aspergillus tubingensis SCC027 filed on ¹ February 2016 with the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the number CBS 136961.

The present invention also relates to a natural variant strain of Aspergillus tubingensis SCC027 filed on ¹ February 2016 at the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the number CBS 136961, allowing producing, by solid-state fermentation on a substrate comprising a mixture of wheat bran and rapeseed meal, a biocatalyst which, when added to the simultaneous saccharification-fermentation medium of an ethanol manufacturing process from corn flour, allows to obtain an Ethanol Gain at least equal to that obtained under the same conditions with a biocatalyst produced by solid-state fermentation of the Aspergillus tubingensis SCC027 strain on a substrate comprising a mixture of wheat and rapeseed meal. The present invention also relates to a process for producing a biocatalyst for producing ethanol from starchy raw material, comprising the following steps:

a) providing a substrate,

b) seeding the substrate provided in step a) with the strain of Aspergillus tubingensis SCC027 according to the invention or a natural variant thereof according to the invention, and c) fermentation by fermentation in a solid medium said substrate with the strain under appropriate conditions to allow simultaneous production by the strain of a protease activity, a cellulase activity that is not a β-glucosidase activity, and a β-glucosidase activity.

Another subject of the present invention relates to a biocatalyst for producing ethanol from starchy feedstock, having at least one protease activity, a cellulase activity which is not a β-glucosidase activity, and a β-glucosidase activity, obtainable by the biocatalyst manufacturing process according to the invention.

The present invention also relates to a biocatalyst for producing ethanol from starchy raw material, said biocatalyst comprising:

(i) at least one protease activity,

(ii) at least one cellulase activity that is not a β-glucosidase activity, (iii) at least one xylanase activity,

(iv) at least one β-glucosidase activity, and

(v) at least one pectinase activity,

said protease, cellulase, xylanase, β-glucosidase and pectinase activities being produced simultaneously by the same filamentous fungal strain.

The present invention also relates to the use of a biocatalyst according to the invention, for the manufacture, by fermentation of starchy feedstock, of ethanol.

It also relates to a process for producing ethanol from starchy raw material comprising a step of fermenting the starchy raw material with a yeast in the presence of the biocatalyst according to the invention.

Detailed description of the invention

Definitions

The nomenclature used for the classification of enzymes is the nomenclature of enzymes of the International Union of Biochemistry and Molecular Biology (IUBMB).

By "protease" is meant here an enzyme of class EC 3.4, which cleaves the peptide bonds. Preferably, the fungal protease is an endopeptidase. Particularly preferably, the fungal protease is one or a mixture of Aspergillus protease (s), in particular one or a mixture of protease (s) of Aspergillus genus nigri, more particularly one or a mixture of protease (s). Aspergillus tubingensis, more particularly aspergillopepsin. Most preferably, the fungal protease is Aspergillopepsin A.

By "aspergillopepsin A" is meant here an enzyme of the class EC 3.4.23.18, which is an aspartate endopeptidase, which cleaves the internal peptide bonds of proteins in acidic conditions and with a broad specificity. This enzyme typically has at least 60.4% sequence identity with Haspergillopepsin A-like aspartic endopeptidase "of sequence SEQ ID NO: 1, whose accession number Gl of the National Center for Biotechnology Information (NCBI) is 145249508, from sequencing of the genome of DSM Aspergillus niger strain CBS 513.88.

By "cellulase" is meant here an enzyme which cleaves glycoside bonds in β-D-1, 4 of cellulose, lichenine and β-glucans cereals.

Preferably, the cellulase is one or a mixture of cellulase (s) of Aspergillus, in particular of Aspergillus genus nigri, more particularly Aspergillus niger. Preferably, the cellulase is an endoglucanase, a cellobiohydrolase or a mixture thereof.

By "endoglucanase" is meant here an enzyme of the GH6 glycoside hydrolase family, class EC 3.2.1.4 cleaving the internal β-1, 4-glycosidic bonds of cellulose chains. This enzyme preferably has at least 43.6% Hendoglucanase sequence identity of sequence SEQ ID NO: 2, whose accession number Gl of the National Center for Biotechnology Information (NCBI) is 350633077, resulting from sequencing. of the genome of the Aspergillus niger strain ATCC 1015.

By "cellobiohydrolase" is meant here an enzyme of the GH7 glycoside hydrolase family, class EC 3.2.1 .91 which releases the cellobiose molecules non-reducing ends of cellulose chains. This enzyme typically has at least 25.6% sequence identity with the "Probable 1,4-beta-D-glucan cellobiohydrolase A" of sequence SEQ ID NO: 3, including the accession number Gl of the National Center for Biotechnology Information (NCBI) is 294956478, resulting from genome sequencing of the Aspergillus niger strain CBS 513.88 from DSM.

By "xylanase" is meant here an enzyme of class EC 3.2.1.8 of the nomenclature of enzymes of the International Union of Biochemistry and Biology Molecular (IUBMB) which degrades xylose polymers linked by β-1 -4 bonds, thus degrading hemicellulose in particular.

Preferably, the xylanase is one or a mixture of Aspergillus xylanase (s), in particular Aspergillus genus nigri, more particularly Aspergilus tubingensis. Preferably, the xylanase is an endoxylanase of class EC 3.2.1.8. Particularly preferably, the xylanase is an endoxylanase A, an endoxylanase C or a mixture thereof.

By "endoxylanase A" is meant here an enzyme of the GH1 1 glycoside hydrolase family, of the EC 3.2.1.8 class, which cleaves the internal β-1, 4-xylosidic bonds of the xylan chains. This enzyme typically has at least 73.7% sequence identity with "endo-1,4-beta-xylanase A" of sequence SEQ ID NO: 4, including the accession number Gl of the National Center for Biotechnology. Information (NCBI) is 145228427, resulting from genome sequencing of the Aspergillus niger strain CBS 513.88 from DSM.

By "endoxylanase C" is meant here an enzyme of the GH10 glycoside hydrolase family, of the EC 3.2.1.8 class, which cleaves the internal β-1, 4-xylosidic bonds of the xylan chains. This enzyme typically has at least 71.2% sequence identity with the "probable endo-1,4-beta-xylanase C" of sequence SEQ ID NO: 5, including the accession number Gl of the National Center for Biotechnology Information (NCBI) is 292495278, resulting from genome sequencing of the Aspergillus niger strain CBS 513.88 from DSM.

By "β-glucosidase" is meant here an enzyme of the GH3 glycoside hydrolase family, of the class EC 3.2.1 .21 cleaving the β-1, 4-glycosidic external bonds of cellulose chains with release of β-D -glucose.

Preferably, the β-glucosidase is one or a mixture of β-glucosidase (s) of Aspergillus, in particular Aspergillus genus nigri, more particularly Aspergillus tubingensis. More particularly, beta-glucosidase is beta-glucosidase A.

Preferably, "β-glucosidase A" has at least 30.2% sequence identity with "beta-glucosidase A" of sequence SEQ ID NO: 6, including the accession number Gl of the National Center for Biotechnology Information. (NCBI) is 145254958, from genome sequencing of DSM Aspergillus niger strain CBS 513.88.

By "pectinase" is meant here an enzyme capable of degrading pectin, a polysaccharide present in the cell walls of plants.

Preferably, the pectinase is one or a mixture of Aspergillus pectinase (s), in particular Aspergillus genus nigri, more particularly Aspergillus tubingensis. Preferably, the pectinase is a polygalacturonase of the class EC 3.2.1.15 of the nomenclature of enzymes of the International Union of Biochemistry and Molecular Biology (IUBMB), which cleaves the glycosidic linkages a-1, 4 between the galacturonic acid residues. More particularly, the pectinase is a polygalacturonase C.

By "polygalacturonase C" is meant here an enzyme of the GH28 glycoside hydrolase family, class EC 3.2.1 .15 cleaving the internal α-1,4-galacturonic linkages of galacturonic acid chains. This enzyme typically has at least 28.7% sequence identity with Hendopolygalacturonase C- of sequence SEQ ID NO: 7, whose accession number Gl of the National Center for Biotechnology Information (NCBI) is 145251 107, resulting from sequencing. genome of the Aspergillus niger strain CBS 513.88 from DSM.

In the context of the invention, the name of an enzymatic class or family (eg protease, cellulase, xylanase, β-glucosidase and pectinase) preferably refers to the enzymatic activity produced by this family, while the name of a specific enzyme (eg Aspergillopepsin A, Endoxylanase A, Endoxylanase C, Polygalacturonase C) refers to the protein as such.

Thus, a composition comprising an enzymatic family X comprises the enzyme activity X and optionally the enzyme X, as such.

The enzymatic activities of the enzymes mentioned above can be determined by any technique well known to those skilled in the art. Examples of methods for determining each of the enzymatic classes mentioned above are described in the section "Enzyme Activities" below.

The percentage identity as defined herein can be calculated by many methods well known to those skilled in the art. Preferably, the percent identity corresponds to the number of identical amino acids obtained for optimal matched alignment (i.e., alignment maximizing the number of identical amino acids) of a sequence of a protein with a reference protein, over their entire length, divided by the total number of amino acids of the larger of the two aligned sequences. Alignment can be done manually or using computer programs such as the EMBOSS-Needle program (Needleman & Wunsch (1970) J. Mol Biol., 48: 443-453). The identity percentage is preferably calculated using the program EMBOSS: needle (global) with the matrix Blosum62 and the following parameters: "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5.

In the context of the invention, the references NCBI G1 are those that were available on December 21, 2015.

Aspergillus tubingensis strain The present invention relates to a strain of Aspergillus tubingensis SCC027 filed on ¹ February 2016 with the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the number CBS 136961.

The present invention also relates to a natural variant strain of Aspergillus tubingensis SCC027 filed on ¹ February 2016 at the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under the number CBS 136961, allowing to produce, by solid state fermentation on a substrate comprising a mixture of wheat bran and rapeseed meal, in particular a mixture of wheat bran and rapeseed meal in a 30/70 ratio, a biocatalyst which, when it is added in the simultaneous saccharification-fermentation medium of a process for producing ethanol from corn flour, makes it possible to obtain an Ethanol Gain at least equal to that obtained under the same conditions with a biocatalyst produced by fermentation in a solid medium of the strain Aspergillus tubingensis SCC027 on a substrate comprising a mixture of wheat bran and rapeseed cake, in particular a mixture of wheat bran and wheat bran. rapeseed in a 30/70 ratio.

By "natural variant" is meant here a strain obtained without genetic manipulation, from a natural reference strain, the strain thus obtained making it possible to produce, by fermentation in a solid medium, on a substrate comprising a mixture of wheat bran and rapeseed meal, in particular a mixture of wheat bran and rapeseed meal in a 30/70 ratio, a biocatalyst which, when added to the simultaneous saccharification-fermentation medium of a process for the manufacture of rapeseed ethanol from maize flour, makes it possible to obtain an Ethanol Gain as described in the section "Manufacture of ethanol" below, at least equal to that obtained under the same conditions with a biocatalyst produced by fermentation in a solid medium of the reference strain on a substrate comprising a mixture of wheat bran and rapeseed cake, in particular a mixture of wheat bran and rapeseed meal in a 30/70 ratio.

The natural variant can thus be obtained by cross-breeding and / or hybridization of Aspergillus strains and / or by spontaneous mutation and / or by random mutagenesis, for example following exposure to stress conditions, to a UV treatment or to a treatment with other mutagenic agents.

The genetic inheritance of a natural variant has not been modified by genetic engineering. The natural variant is therefore not a genetically modified organism.

"Ethanol Gain" is defined in the "Ethanol Manufacturing" section below.

Preferably, the natural variant according to the invention makes it possible to produce, under the conditions described in Example 1, a biocatalyst which, when it is added in the simultaneous saccharification-fermentation medium of an ethanol manufacturing process. as described in Example 2, makes it possible to obtain an Ethanol Gain as described in the "Manufacture of ethanol" section at least equal to that obtained under the same conditions with a biocatalyst produced under the conditions described in the example 1, by strain SCC027.

Biocatalyst manufacturing process

The present invention also relates to a process for manufacturing a biocatalyst for producing ethanol from starchy raw material, comprising the following steps:

a) providing a substrate,

b) seeding the substrate provided in step a) with the strain of Aspergillus tubingensis SCC027 or a natural variant thereof according to the invention, and

c) fermentation by fermentation in a solid medium said substrate with the strain under appropriate conditions to allow simultaneous production by the strain of a protease activity, a cellulase activity which is not a β-glucosidase activity and a β-glucosidase activity.

substratum

The substrate used for the production of the biocatalyst is preferably an agricultural or agro-industrial co-product.

By "agricultural or agro-industrial co-product" is meant a product created in the same process of transformation and at the same time as a major agricultural or agri-industrial product.

The substrate used for the production of the biocatalyst may in particular be a cereal co-product, an oleaginous crop co-product, an oilseed crop co-product, a sacchariferous co-product, a co-product of the processes for the transformation of cereal, oleaginous, oléoprotéagineuse, sacchariferous crops. , or a mixture of these.

Examples of substrates that can be used to produce the biocatalyst according to the invention include wheat bran, rapeseed meal, soybean meal, beet pulp, barley rootrush, corn ethanol seed, corn wheat ethanol and mixtures thereof.

Preferably, the substrate used for the production of the biocatalyst is selected from the group consisting of wheat bran, rapeseed meal, barley rootlet, ethanol cornseed, and mixtures thereof. More preferably, the substrate used for producing the biocatalyst comprises wheat bran and / or rapeseed cake. Most preferably, the substrate used for the production biocatalyst comprises or consists of a mixture of wheat bran and rapeseed cake.

Wheat bran and rapeseed meal may be present in this mixture in mass proportions ranging from 10/90 (ie 10% wheat bran to 90% rapeseed meal) at 50/50 (ie 50% wheat bran for 50% rapeseed meal), preferably 15/85 (ie 15% wheat bran for 85% rapeseed cake) at 45/55 (ie 45% wheat bran for 55% rapeseed cake), preferably 20/80 (ie 20% wheat bran for 80% rapeseed cake) at 40/60 (ie 40% wheat bran for 60% oilcake). rapeseed), preferably from 25/75 (ie 25% wheat bran to 75% rapeseed meal) to 35/65 (ie 35% wheat bran to 35% rapeseed cake), so more preferred 30/70 (ie 30% wheat bran for 70% rapeseed cake).

The substrate used for the production of the biocatalyst can be ground beforehand.

The substrate used for the production of the biocatalyst is preferably pre-moistened and heat-treated to reduce the endogenous flora of the substrate, to pasteurize or sterilize it.

In a particularly preferred manner, the substrate used for the production of the biocatalyst is pre-moistened so as to reach a solids content of 35 to 90%, preferably a solids content of 40 to 80%, preferably a solids content of 45 to 70%. %, preferably a solids content of 50 to 65%, preferably 55% of dry matter.

The heat treatment may consist of heating, for example in an autoclave, typically at 105 ° C., for example for 35 minutes, or pasteurization, typically at 105 ° C., for example for 15 minutes. It is also possible to heat treat the substrate used for the production of the biocatalyst by injecting water vapor.

Preferably, the pH is adjusted during the humidification in a range from 4 to 5.5, preferably to 4.9, for example with nitric acid or sulfuric acid, in order to promote the implantation of the microorganism cultivated and starting the fermentation in solid medium.

The humidification is preferably determined so that the water content of the substrate used for the production of the biocatalyst is between 40 and 70% of the total mass of the substrate and water. Thus, the substrate used for the production of the biocatalyst preferably has an initial dry matter of between 30 and 60%, preferably 45%. seeding

The seeding of the substrate used for the production of the biocatalyst can be carried out with any suitable inoculum. The strain of Aspergillus tubingensis used during the fermentation in solid medium can thus be previously subjected to a sporulation step before being inoculated in the form of spores. The inoculum dose will then advantageously be at least 5.10 ⁶ viable spores / g of initial dry matter, preferably at least 10 ⁷ viable spores / g of initial dry matter. Alternatively, the strain of Aspergillus tubingensis used during fermentation in solid medium can be inoculated as mycelium. The dose of inoculum will then advantageously be between 100 and 500 g of liquid culture medium comprising the mycelium per kg of initial dry matter.

Fermentation in a solid medium

By "solid state fermentation" is meant here the cultivation of microorganisms on wet solid supports, on inert carriers or on insoluble substrates that can be used as a source of carbon and energy. The fermentation process takes place in the absence or near-absence of free water in the space between the substrate particles.

Fermentation in solid medium can be carried out in any suitable reactor. Fermentation in a solid medium is preferably conducted for controlled arrest prior to sporulation of the Aspergillus tubingensis strain used. The fermentation in solid medium is thus preferably carried out for a period of 1 to 4 days, preferably between 20 and 80 hours, between 35 and 60 hours, between 40 and 55 hours, particularly preferably between 45 hours and 45 hours. 50 h.

The temperature of the medium is preferably maintained between 30 ° C and 36 ° C ± 0.5 ° C, more preferably at 33 ° C ± 0.5 ° C, which corresponds to the optimum range of activity of the strains. Aspergillus tubingensis used to produce the biocatalyst.

The water content of the substrate is preferably substantially maintained between 45 and 60% during the fermentation, more preferably 55%, for example by periodically carrying water to compensate for the loss of water in the medium. The term "substantially maintained" means that it is tolerable for the moisture content to be a value deviating by 2% units of the 45-60% range for a relatively short period of time between two successive adjustments of the rate of moisture or at the end of fermentation. The moisture content of the substrate may indeed have a tendency to decrease during fermentation by evaporation under the effect of the temperature increase generated by the fungal growth. Aeration, preferably continuously, is preferably carried out during fermentation in solid medium, in order to provide the oxygen necessary for the fermentation and to avoid the excessive accumulation of carbon dioxide produced by the fermentation and / or avoid excessive accumulation of heat.

Stirring may be conducted to avoid the formation of impermeable masses. The agitation can be implemented by means of stirring arms, blades or spatulas or worm. The agitation, however, preferably remains moderate. Preferably, the stirring is discontinuous, typically stirring is conducted every 3 h to 13 h.

The fermentation step c) is carried out under appropriate conditions to allow the simultaneous production by the strain of a protease activity as defined in the "Definitions" section above, of a cellulase activity which is not not β-glucosidase and β-glucosidase activity, as defined in the "Definitions" section above.

By "simultaneous production" of several enzymatic activities is meant here that the enzymatic activities are produced by the Aspergillus tubingensis strain in a single fermentation step during which the fermentation conditions (temperature, pH, composition of the medium, aeration) make it possible to obtain all the enzymatic activities sought.

The fermentation step c) is preferably carried out under appropriate conditions to allow the simultaneous production by the strain of at least 60 U of PAC protease activity per gram of initial dry matter of substrate, preferably from minus 100 U of PAC protease activity per gram of initial substrate dry matter, preferably at least 130 U of PAC protease activity per gram of initial substrate dry matter, of at least 60 U of CMCell cellulase activity per gram of initial substrate dry matter, preferably at least 1 10 U of CMCell cellulase activity per gram of initial substrate dry matter, and at least 100 U of β-Glu β-glucosidase activity per gram initial substrate dry matter, preferably at least 200 U β-Glu β-glucosidase activity per gram of initial substrate dry matter.

Preferably, the fermentation step c) is carried out under appropriate conditions to allow further simultaneous production by the strain.

at least 75 U of xylanase activity AXCn per gram of initial dry matter of substrate, preferably at least 130 U of xylanase activity AXCn per gram of initial dry matter of substrate, preferably of at least 180 U of xylanase activity AXCn per gram of initial dry matter of substrate, and / or

at least 2000 U of PG pectinase activity per gram of initial dry matter of substrate, preferably at least 2900 U of PG pectinase activity per gram of initial dry matter of substrate, preferably at least

3800 U of PG pectinase activity per gram of initial substrate dry matter, preferably at least 4500 U of pectinase PG activity per gram of initial substrate dry matter.

The fermentation step c) is preferably carried out under conditions to allow the simultaneous production by the strain of at least one aspergillopepsin A, as defined in the "Definitions" section above, as a protease, of at least one endoglucanase, as defined in the "Definitions" section above, as cellulase, and at least one β-glucosidase, as defined in the "Definition" section above.

The fermentation product thus obtained is a moist solid product.

By "initial substrate dry matter" or "MSi" is meant here the mass of substrate dry matter used to produce the biocatalyst by fermentation in solid medium, at the beginning of this fermentation. During the fermentation process in a solid medium, the growth of microorganisms such as Aspergillus consumes a portion of the substrate, and thus generates a loss of substrate dry matter which typically represents between 15 and 30%, for example 20% of the material. initial dry.

The amounts of biocatalysts, when used as defined below, are related to the amount of initial dry matter of the corresponding substrate.

The levels of enzymatic activities are related to the amount of initial dry matter of the corresponding substrate, so that the extracted forms can be compared with the raw forms of the biocatalyst, or biocatalysts from fermentation or strain can be compared with mass losses. different.

Packaging of the fermented product

The biocatalyst obtained after fermentation in solid medium above can be in raw form or extracted.

By "raw form" is meant here that the biocatalyst comprises at least protease, cellulase and β-glucosidase, as well as the strain of Aspergillus tubingensis used for the fermentation in solid medium and the substrate used during the preparation. fermentation in solid medium, possibly crushed. This raw form contains typically particles having a diameter of between 50 μηι and 5 mm depending on the type of ground material generated.

The biocatalyst obtained after the fermentation in solid medium can also be dried or frozen for preservation.

The drying is preferably carried out at a moderate temperature so as not to affect the enzymatic activities, for example in an oven preferably at 40 ° C., until a humidity of for example 8% or, for example, 10% is obtained.

Freezing is preferably carried out on the moist fermented product, for example at -20 ° C.

The biocatalyst obtained after the fermentation in solid medium can also undergo a new grinding before being conditioned.

"Extracted form" here means that the combination of enzymes of the biocatalyst, in particular at least protease, cellulase and β-glucosidase, more particularly at least protease, cellulase, xylanase, β-glucosidase and pectinase, more particularly at least aspergillopepsin A, endoglucanase, endoxylanase A, endoxylanase C, β-glucosidase A, cellobiohydrolase and polygalacturonase C, are isolated from the Aspergillus tubingensis strain and the substrate used during fermentation in solid medium with the strain. of Aspergillus tubingensis by extraction processes that can implement any technique well known to those skilled in the art, in particular by extraction in aqueous solution, extraction in alcoholic solution, extraction by solvents, high pressure homogenization, supercritical extraction, extraction by fluidized beds, grinding, cryogenic grinding, decompression, cavitation, bubbling, ultrasonic extraction, adsorption on r esins or zeolites. The combination of enzymes in liquid form can then be purified by any technique well known to those skilled in the art, in particular by centrifugation, filtration, ultrafiltration, chromatography, use of membranes or precipitation.

The biocatalyst in extracted form may further be stabilized, dried or frozen for preservation.

The stabilization of the biocatalyst can be carried out by any means known to those skilled in the art, such as the addition of glycerol, sorbate, potassium salt, etc.

The drying of the biocatalyst in extracted form can be carried out by any means known to those skilled in the art, such as lyophilization, atomization, etc.

biocatalyst

The present invention relates to a biocatalyst for producing ethanol from starchy raw material, comprising at least one protease activity, an activity cellulase which is not a β-glucosidase activity and β-glucosidase activity, as defined in the "Definitions" section above, obtainable by the biocatalyst manufacturing method according to the invention.

In a particular embodiment, the biocatalyst comprises as protease at least one aspergillopepsin A. In a particular embodiment, the biocatalyst comprises as cellulase at least one endoglucanase and / or at least one cellobiohydrolase. In a particular embodiment, the biocatalyst comprises at least one β-glucosidase A as β-glucosidase. In a particularly preferred embodiment, the biocatalyst comprises at least one aspergillopepsin A, at least one endoglucanase and at least one β-glucosidase. AT.

In a preferred embodiment, said protease activity is present in the biocatalyst at a dose of at least 60 UAP / g of initial dry matter of the substrate, preferably at a dose of at least 100 UAP / g of material. initial dry substrate, preferably at a dose of at least 130 UAP / g of initial dry matter substrate. In another preferred embodiment, said cellulase activity which is not a β-glucosidase activity is present in the biocatalyst at a dose of at least 60 U CMCell / g of initial dry matter of substrate, preferably at a dose of at least 1 10 U CMCell / g of initial substrate dry matter. In another preferred embodiment, said β-glucosidase activity is present in the biocatalyst at a dose of at least 100 U β-Glu / g of initial dry matter of substrate, preferably at a dose of at least 200 U β-Glu / g of initial dry matter of substrate.

The term "initial dry substrate material" is defined in the "Biocatalyst Manufacturing Process" section above.

Preferably, the biocatalyst obtainable by the manufacturing method according to the invention further comprises at least one xylanase activity and / or at least one pectinase activity, said activities being produced simultaneously by the Aspergillus tubingensis strain.

In a particularly preferred manner, the biocatalyst obtainable by the process according to the invention also comprises:

at least 75 U of xylanase activity AXCn per gram of initial dry matter of substrate, preferably at least 130 U of AXCn xylanase activity per gram of initial dry matter of substrate, preferably at least 180 U of xylanase activity AXCn per gram of initial dry matter of substrate, and / or

at least 2000 U of PG pectinase activity per gram of initial substrate dry matter, preferably at least 2900 U of PG pectinase activity per gram of initial substrate dry matter, preferably at least 3800 U of PG pectinase activity per gram of initial substrate dry matter, preferably at least 4500 U of PG pectinase activity per gram of initial substrate dry matter.

Preferably, the biocatalyst according to the invention comprises, as xylanase, an endoxylanase A, an endoxylanase C or a mixture thereof, and / or as a pectinase a polygalacturonase C.

In a particularly preferred embodiment, all the enzymes present in the biocatalyst according to the invention are simultaneously produced by a single strain of filamentous fungus, in particular a single Aspergillus strain, in particular a single strain of Aspergillus genus. nigri, more particularly Aspergillus tubingensis, more preferably the strain Aspergillus tubingensis SCC027 or a natural variant thereof as defined in the section "Aspergillus tubingensis strain" above.

(i) at least one protease activity,

(iv) at least one β-glucosidase activity, and

(v) at least one pectinase activity,

said protease, cellulase, xylanase, β-glucosidase and pectinase activities being produced simultaneously the same strain of filamentous fungus.

Preferably, the biocatalyst comprises as protease (i) at least one acid protease, preferably at least one aspergillopepsin, most preferably at least one aspergillopepsin A.

Preferably, the biocatalyst comprises as cellulase (ii) at least one endoglucanase, a cellobiohydrolase or a mixture thereof.

Preferably, the biocatalyst comprises as xylanase (iii) at least one endoxylanase or a mixture of endoxylanases, preferably at least one endoxylanase A, an endoxylanase C or a mixture thereof.

Preferably, the biocatalyst comprises, as β-glucosidase (iv), at least one β-glucosidase A.

Preferably, the biocatalyst comprises as pectinase (v) at least one polygalacturonase, preferably at least one polygalacturonase C. In a preferred embodiment, the biocatalyst according to the invention comprises:

(i) at least one aspergillopepsin A,

(ii) at least one endoglucanase and one cellobiohydrolase,

(iii) at least one endoxylanase A and one endoxylanase C,

(iv) at least one β-glucosidase A, and

(v) at least one polygalacturonase C,

said aspergillopepsin A, endoglucanase, cellobiohydrolase, endoxylanase A, endoxylanase C, β-glucosidase A and polygalacturonase C, being produced simultaneously by the same filamentous fungal strain.

Preferably, said filamentous fungus is Aspergillus, preferably an Aspergillus section nigri, more particularly Aspergillus tubingensis, most preferably Aspergillus tubingensis strain SCC027 or a natural variant thereof as defined in the section. "Aspergillus tubingensis strain" above.

Preferably, said at least one protease activity is present in the biocatalyst at a dose of at least 60 UAP / g of initial dry matter of substrate, preferably at a dose of at least 100 UAP / g dry matter. initial substrate, preferably at a dose of at least 130 UAP / g of initial substrate dry matter. Preferably, said at least one cellulase activity which is not a β-glucosidase activity, is present in the biocatalyst at a dose of at least 60 U CMCell / g of initial dry matter of substrate, preferably at least 1 10 U CMCell / g of initial dry matter of substrate. Preferably, said at least one xylanase activity is present in the biocatalyst at a dose of at least 75 U AXCn per gram of initial dry matter of substrate, preferably at least 130 U AXCn per gram of initial dry matter of the substrate, preferably at least 180 U AXCn per gram of initial dry matter of substrate. Preferably, said at least one β-glucosidase activity is present in the biocatalyst at a dose of at least 100 U β-Glu per gram of initial dry matter of substrate, preferably at least 200 U β-Glu per gram of material initial dry substrate. Preferably, said at least one pectinase activity is present in the biocatalyst at a dose of at least 2000 U PG per gram of initial dry matter of substrate, preferably at least 2900 U PG per gram of substrate initial dry matter, of preferably at least 3800 U PG per gram of initial substrate solids, preferably at least 4500 U PG per gram of initial substrate solids.

In a particularly preferred manner, the biocatalyst according to the invention comprises: (i) at least one protease activity at a dose of at least 60 UAP / g of initial substrate dry matter, preferably at a dose of at least 100 UAP / g of initial substrate dry matter, preferably at a dose of at least 130 UAP / g of initial substrate dry matter,

(ii) at least one cellulase activity at a dose of at least 60 U CMCell / g initial substrate dry matter, preferably at least 1 U CMCell / g initial substrate dry matter,

(iii) at least one xylanase activity at a dose of at least 75 U AXCn per gram of substrate initial dry matter, preferably at least 130 U AXCn per gram of substrate initial dry matter, preferably at least 180 U AXCn per gram of initial dry matter of substrate,

(iv) at least one β-glucosidase activity at a dose of at least 100 U β-Glu / g of initial substrate dry matter, preferably at least 200 U β-Glu / g of initial substrate dry matter; and

(v) at least one pectinase activity at a dose of at least 2000 U PG per gram of substrate initial dry matter, preferably at least 2900 U PG per gram of initial substrate dry matter, preferably at least 3800 U PG per gram of initial dry matter of substrate, preferably at least 4500 U PG per gram of initial dry matter of substrate,

said protease, cellulase, xylanase, β-glucosidase and pectinase activities being produced simultaneously by the same filamentous fungal strain, preferably the same Aspergillus strain, in particular the same Aspergillus section nigri strain, more particularly the same strain of Aspergillus tubingensis, more particularly the same strain of Aspergillus tubingensis SCC027 or a natural variant thereof as defined in the section "Aspergillus tubingensis strain" above.

Ethanol manufacturing

The subject of the present invention is also the use of the biocatalyst according to the invention for the manufacture, by fermentation of starchy raw material, of ethanol.

The present invention also relates to a process for producing ethanol from starchy raw material comprising a step of fermenting the starchy raw material with a yeast in the presence of the biocatalyst according to the invention.

The fermentation step of the ethanol production process according to the invention is preferably a step of saccharification-simultaneous fermentation, in the presence of the biocatalyst according to the invention. By "starchy raw material" is meant here a cereal or tuber, which contains starch as the major source of carbon. In particular, "starchy raw material" here means maize, wheat, barley, sorghum, cassava, potato, alone or as a mixture. Preferably, the starchy raw material is corn or wheat. In a particular embodiment, the starchy raw material is maize. In another particular embodiment, the starchy raw material is wheat.

The method of manufacturing ethanol according to the invention preferably comprises the following steps:

A) a step of mixing a mill of the starchy raw material with water, and optionally clarified vinasses,

B) optionally a step of cooking the mixture prepared in step A),

C) optionally a step of pre-liquefaction of the starch of said mixture,

D) a step of liquefaction of the starch, in the presence of starch liquefying enzymes, for hydrolyzing the starch to dextrins,

E) optionally a yeast propagation step, and

F) a step of simultaneous saccharification-fermentation of dextrins and / or fermentable sugars, in the presence of the biocatalyst according to the invention, saccharification enzymes of dextrins and yeasts, optionally propagated in step E), so as to produce ethanol.

Step A) mixing

The mixture typically contains 20 to 40% by mass of dry matter, preferably between 25 and 35%, preferably 30% by mass of dry matter.

Step B) cooking

The mixture prepared in stage A), typically containing from 20 to 40% by mass of dry matter, preferably between 25 and 35%, preferably 30% by mass of dry matter, is preferably sent in a jet-cooker. typically between 100 and 130 ° C, preferably between 105 and 120 ° C, preferably with a steam injection, preferably for a period of 5 to 20 min, preferably for 15 min, so as to gelatinize and destructure the granular starch of the raw material mixed.

This cooking step B) is preferably carried out when the starchy raw material is maize.

Step C) pre-liquefaction

The mixture, typically containing from 20 to 40% by mass of dry matter, preferably between 25 and 35%, preferably 30% by mass of dry matter, is preferably placed in the presence of a dezosising enzyme complex. The pre-liquefaction enzymes that can be used during this step are well known to those skilled in the art. These are typically viscosity reducing enzymes such as hemicellulases and / or beta-glucanases.

Depending on the enzymes used, the pH may be adjusted with an acid or base solution, for example sulfuric acid or ammonium hydroxide. The pH can typically be adjusted to 4.5 to 6.5, preferably 5.5.

The pre-liquefaction is preferably carried out by applying a temperature increase phase of preferably 15 min, making it possible to reach a temperature typically of 55 ° C maintained for 15 to 45 min, preferably 30 min.

Vats in which the pre-liquefaction step is carried out can further be stirred, preferably continuously.

This pre-liquefaction stage C) is preferably carried out when the starchy raw material is wheat.

Step D) Liquefaction

The mixture prepared in step A) and having optionally undergone steps B) or C), typically containing from 20 to 40% by mass of dry matter, preferably between 25 and 35%, preferably 30% by mass of material dry, is preferably brought into the presence of liquefying enzymes. The liquefaction enzymes that can be used during this step are well known to those skilled in the art. These are typically α-amylases, in particular bacterial α-amylases, preferably thermostable bacterial α-amylases. Other enzymes may be further used in this step.

Depending on the enzymes used, the pH may be adjusted beforehand with a solution of acid or base, for example sulfuric acid or ammonium hydroxide. The pH can typically be adjusted to 4.5 to 6.5, preferably 5.5. Depending on the enzyme used, a calcium salt may also be added to enhance the stability of the enzyme.

The liquefaction is preferably carried out at a temperature of 80 to 95 ° C, preferably between 85 and 90 ° C, for 60 to 150 min, preferably 90 min.

The tanks in which the liquefaction stage is carried out can also be stirred, preferably continuously.

Step E) of yeast propagation

The yeasts that can be used for the manufacture of ethanol are well known to those skilled in the art and may in particular be yeasts of the Saccharomyces genus. Preferably, the yeasts used for the manufacture of ethanol are not genetically modified organisms. Preferably, the yeasts are introduced into the simultaneous fermentation or saccharification-fermentation medium, in step F) defined below, after having undergone a propagation step. This propagation step allows yeast development sufficient to inoculate the fermentation medium or simultaneous saccharification-fermentation at the dose mentioned above. The propagation step is typically carried out by culturing active dry yeasts in a liquefied wort, typically containing from 15 to 30% by mass of dry matter, preferably between 20 and 25%, preferably 20% by weight of dry matter. Typically, the propagation step according to the invention has the following characteristics:

• The temperature is between 30 ° C and 37 ° C, preferably 35 ° C.

The pH can be adjusted at the beginning of the propagation stage with an acid or a base, for example sulfuric acid or ammonium hydroxide, preferably between 4.5 and 5.8, preferably between 5 and 5.5, preferably at 5.3. Preferably, there is no pH adjustment during the continuation of the propagation step.

The assembly is preferably stirred throughout the duration of the propagation step, so as to aerate the medium.

• A nitrogen addition may be carried out, in mineral or organic form, for example urea, so as to provide the yeast a complementary source of nitrogen. For example a supply of urea of 2 to 4 g / l of liquefied must can be carried out, preferably 3 g / liter of liquefied must.

• The propagation time can be between 4 h and 24 h, preferably between 8h and 12h.

The biocatalyst according to the invention can be added during the propagation step, instead of the simultaneous saccharification-fermentation step defined below, so as to guarantee a contribution during the saccharification-fermentation step. simultaneously, of the order of 0.01 to 5 kg of initial biocatalyst substrate dry matter per tonne of starchy raw material, preferably between 0.4 and 1.2 kg of initial biocatalyst substrate dry matter per tonne of starchy raw material.

Step F) simultaneous saccharification-fermentation

During this step, the wort liquefied according to stage D), typically containing from 20 to 40% by mass of dry matter, preferably between 25 and 35%, preferably 30% by mass of dry matter, is preferably used. in the presence of saccharification enzymes. The saccharification enzymes that can be used during this step are well known to those skilled in the art. It is typically glucoamylase. Other enzymes may be further used in this step.

In the simultaneous saccharification-fermentation step of the ethanol production process according to the invention, the saccharification-fermentation must is placed in the presence of yeasts, possibly propagated by step E) above. Typically, the simultaneous saccharification-fermentation step according to the invention has the following characteristics:

• The temperature is between 30 ° C and 35 ° C, preferably 32 ° C.

The pH can be adjusted at the beginning of the simultaneous saccharification-fermentation stage with an acid or a base, for example sulfuric acid or ammonium hydroxide, preferably between 4.5 and 5.8. preferably between 5 and 5.5, preferably at 5.3. Preferably, there is no pH adjustment during the subsequent simultaneous saccharification-fermentation step.

The whole can be stirred throughout the simultaneous saccharification-fermentation step, preferably continuously.

• A nitrogen addition may be carried out, in mineral or organic form, for example urea, so as to provide the yeast a complementary source of nitrogen. For example, a urea supply of 1 g / l of liquefied must can be carried out.

· The fermentation time may be between 20 hours and 80 hours, preferably between 40 hours and 60 hours.

Preferably, the biocatalyst according to the invention is present during the simultaneous fermentation or saccharification-fermentation stage at a concentration of 0.01 to 5 kg of initial biocatalyst substrate dry matter per ton of starchy raw material. , preferably between

0.4 and 1.2 kg of initial biocatalyst substrate dry matter per tonne of starchy feedstock.

The yeasts that can be used for the manufacture of ethanol are well known to those skilled in the art and may in particular be yeasts of the Saccharomyces genus. Preferably, the yeasts used for the manufacture of ethanol are not genetically modified organisms.

Typically, the simultaneous fermentation or saccharification-fermentation medium is inoculated with about 10 ⁶ to about 5 × 10 ⁸ CFU (colony-forming units) of yeast per ml of saccharification-fermentation medium, preferably about 10 ⁷ CFU of yeast per ml of yeast. simultaneous fermentation or saccharification-fermentation medium. Preferably, the yeasts are introduced into the simultaneous fermentation or saccharification-fermentation medium after having undergone a propagation step E) as defined above.

The biocatalyst according to the invention can be added partly during the propagation step E) defined above and partly during the simultaneous saccharification-fermentation step F), so as to guarantee a total supply, during the simultaneous saccharification-fermentation step, of the order of 0.01 to 5 kg of initial biocatalyst substrate dry matter per ton of starchy feedstock, preferably between 0.4 and 1.2 kg of material initial dry substrate of the biocatalyst per tonne of starchy raw material.

The performance of the ethanol production process is measured by the concentration of ethanol in the must at the end of the simultaneous saccharification-fermentation stage, relative to the dry matter content of the starchy raw material.

By "Ethanol Gain" is meant here the improvement of the production of ethanol, in particular after a fermentation, preferably simultaneous saccharification-fermentation, preferably after 51 hours of fermentation, preferably after 51 hours of simultaneous saccharification-fermentation, conducted in the presence of a biocatalyst obtained with the strain in question, compared to the production of ethanol under the same conditions in the absence of a biocatalyst (control), typically determined by the following equation:

Gain ethanol (%) = ([Ethanol] biocatalyst - [Ethanol] control) / [Ethanol] control

The inventors have shown that, surprisingly, the use of the biocatalyst according to the invention makes it possible to obtain a higher ethanol yield, a faster ethanol production kinetics, and a lower glycerol production than the absence of biocatalyst or by using an enzyme composition obtained by fermentation in solid medium with another strain of Aspergillus tubingensis with different enzymatic activities.

Measurement of enzymatic activities

Xylanase activity

Measurement of the xylanase activity can typically be carried out on birch xylan bound to a chromophore, Rémazol Brillant Blue R. The hydrolysis of this substrate by the xylanase activities indeed releases non-precipitable chromophore-bound oligomers after addition. an ethanol solution in the reaction medium. The concentration of non-precipitable oligomers is evaluated by measuring the absorbance at 590 nm. The reaction medium preferably used in this method is composed of 130 μl of a commercial solution of Birchwood azo-xylan (MEGAZYME) at 10 g / L, 50 μl of enzymatic solution diluted in 0 acetate buffer, 4 M, pH 4.70. The reaction is preferably carried out at 30 ° C for 20 minutes. The reaction is typically stopped by adding 500 μl of 96% ethanol. The optical density of the supernatant obtained after centrifugation (10 minutes at 3000 rpm at 20 ° C.) is read at 590 nm.

A unit of xylanase activity (AXCn) can then be defined as the amount of enzyme which, diluted at a rate of 1 unit / mL, at pH 4.70 and at 30 ° C., liberates, from a solution of Remazol Brillant Blue R xylane, oligomers not precipitable in ethanol such as the optical density of the supernatant is 0.93 to 590 nm.

Acid protease activity

Measurement of acid protease activity can typically be performed on casein. The casein can thus be hydrolysed into peptide fragments soluble in trichloroacetic acid (TCA).

A PAC unit is typically defined as the amount of enzyme which liberates soluble peptides at 40 ° C and pH 3.0 such that the 275 nm optical density of the hydrolyzate obtained after TCA precipitation is equivalent to that of a solution of tyrosine at 120 μg / mL.

An appropriate measurement technique of acidic protease activity is described below.

The reaction medium preferably used in this method is composed of 500 μl of a clear casein solution and filtered at 12 g / L (0.06 M lactate buffer, pH = 3), 100 μl of diluted enzyme solution. in 0.05 M lactate buffer, pH = 3. The reaction is preferably carried out at 40 ° C for 120 minutes. The reaction is typically stopped by adding 300 μl of a 10% trichloroacetic acid solution. The optical density of the supernatant obtained after centrifugation for 15 minutes at 3000 rpm at 20 ° C. is read at 275 nm.

Polygalacturonase activity

Measurement of polygalacturonase activity can typically be performed on pectins. Polygalacturonases hydrolyze the pectin chains of low degree of methylation and thus generate at the ends of the chain reducing groups on the galacturonic acid residues constituting the pectin. These reducing groups can be assayed with cyanoacetamide. The assay is preferably kinetic in 40 minutes at 31 ° C.

A polygalacturonase (PG) unit is typically defined as the amount of enzyme which liberates 1 μηιοΐβ from reducing groups in 1 min at 31 ° C and at pH = 4.7, from sodium polygalacturonate.

An appropriate measurement technique of polygalacturonase activity is described below.

The reaction medium preferably used in this method is composed of 750 μl of 0.1 M succinate buffer and pH = 4.7, 750 μl of an aqueous solution of commercial orange polygalacturonic acid (Sigma). at 6.5 g / L (adjusted to pH = 4.7 with sodium hydroxide), 15 μl of enzymatic solution diluted in succinate buffer at pH = 4.7 supplemented with bovine serum albumin (0.1 g / L). Enzyme kinetics are preferably carried out at 31 ° C by stopping the reaction at 10, 20, 30 and 40 minutes. For each time, the reaction is typically stopped by adding 70 μl of the reaction medium in a stop solution composed of 140 μl of borate buffer (0.2 M, pH = 10) and 70 μl of a solution of 2 cyanoacetamide at 20 g / l. A revelation step is performed by heating the sample for 12 minutes at 120 ° C and then cooling it to -20 ° C for 10 minutes. The optical density of the supernatant is read at 280 nm against water. A calibration range constructed with a monogalacturonic acid concentration range makes it possible to convert the obtained optical densities into moles of equivalent reducing groups.

Beta-glucosidase activity

Measurement of β-Glucosidase (β-Glu) activity can typically be performed on cellobiose. The enzyme indeed acts on this compound by releasing glucose.

A β-Glu unit is defined as the amount of enzyme that catalyzes the release of one micromole of glucose per minute under the conditions of the test (50 ° C, pH = 4.8).

An appropriate measurement technique of β-glucosidase activity is described below.

The reaction medium preferably used in this method is composed of 150 μl of a solution of cellobiose at 5.13 g / L (acetate buffer at pH = 4.8), 150 μl of enzymatic solution diluted in buffer 0.1 M acetate at pH = 4.8. The reaction is preferably carried out at 50 ° C for 30 minutes. The reaction is typically stopped by heating the reaction medium at 100 ° C for 10 minutes. The reaction medium is then cooled for 10 minutes in cold water. After centrifugation at 3000 rpm at 20 ° C for 10 minutes, the glucose concentration released into the supernatant is then assayed using a commercial enzyme glucose assay kit.

Cellulase activity

Measurement of the cellulase activity, in particular endoglucanase, can typically be carried out on partially depolymerized carboxymethyl cellulose linked to a chromophore: the Rémazol Brilliant Blue R. The hydrolysis of the substrate in fact releases bound low molecular weight oligomers to the chromophore. These are found in the supernatant after precipitation with ethanol and centrifugation. A measurement of the absorbance at 590 nm makes it possible to determine the cellulase activity.

A cellulase unit on carboxymethylcellulose (CMCell) can be defined as the amount of enzyme which, diluted at a rate of 1 U / ml and used under the conditions of the assay leads to a release of non-precipitable oligomers by the precipitation solution, such that the optical density of the supernatant is 1.

An appropriate measurement technique of cellulase activity is described below.

The reaction medium preferably used in this method is composed of 100 μl of commercial azo-CM-cellulose solution (MEGAZYME), 100 μl of enzymatic solution diluted in 0.1 M acetate buffer, pH 4.60. . The reaction is preferably carried out at 41 ° C for 10 minutes. The reaction is typically stopped by adding 500 μl of precipitation solution (40 g of sodium acetate trihydrate and 4 g of zinc acetate in 1 liter of 96% ethanol). The optical density of the supernatant obtained after centrifugation (10 minutes at 3000 rpm at 20 ° C.) is read at 590 nm.

The present invention will be further illustrated by the figures and examples below.

Brief description of the figures

Figure 1: Relative abundance of 7 enzymes selected from all the enzymes identified in the biocatalyst of Example 1 by proteomic analysis. FIG. 2: Monitoring of the production of C0 ₂ in reactors during the SSF reaction of an industrial liquid liquefied must, in the presence or absence of the biocatalyst according to the invention, produced with strain SCC027 incorporated at 1.2 kg MSi / t of maize, as described in Example 4.

3: Effect of the specific enzymes of the biocatalyst according to the invention, used alone or as a mixture, with respect to the overall effect of the biocatalyst according to the invention, produced with strain SCC027, on the ethanol gain obtained at the end of 51 hours of SSF. The value 100% corresponds to an ethanol gain of 1.5% calculated between the control condition and the biocatalyst condition.

Examples

Example 1 Production and Characterization of the Biocatalyst According to the Invention

This example describes a production process of the biocatalyst according to the invention obtained by fermentation in solid medium with the strain of Aspergillus tubingensis SCC027.

10 kg of initial dry matter of substrate consisting of a mixture of rapeseed meal and wheat bran, in a 70/30 ratio are used.

The substrate is pretreated by humidification at 60% dry matter and then pretreated in an autoclave at 105 ° C. for 35 min.

The substrate is then inoculated with 1 × 10 ⁷ viable spores of Aspergillus tubingensis SCC027 / g of initial dry matter.

The substrate has an initial dry matter before fermentation of 45%.

Fermentation in solid medium is then carried out for 51 h under the following conditions:

- Temperature: 33 ° C ± 0.5 ° C

- Dry matter: 45% ± 2%

Continuous 35 HZ ventilation

- Discontinuous agitation

After 51 hours of fermentation, the culture is stopped by adding propionic acid, then the medium is dried at 45 ° C for 18 h, until a solids content <10%.

The biocatalyst is finely ground using a Retsch ZM300 centrifugal grinder mounted with a 0.85 mm (18,000 rpm) grid. It is stored away from light and cold (-20 ° C). For its simultaneous saccharification-fermentation use of starchy raw material (see examples 2 to 6), the biocatalyst can be added either directly to the must in the form of a powder or extracted in water (1/10 or 1/20 p / w). v, stirring at room temperature for 10 to 15 minutes).

For enzymatic characterizations, the aqueous extract of the biocatalyst is centrifuged (10 min, 3000 rpm, 20 ° C) to separate the insoluble particles.

The determination of the primary structure of the proteins, in order to identify them, is carried out by a proteomic analysis according to the so-called "bottom-up" strategy which consists in analyzing the constituent peptides of the proteins to be identified by mass spectrometry. Soluble proteins underwent, after a reduction and alkylation step, digestion with trypsin.

The peptides thus obtained, after purification on ZipTip C ₁₈ microcolumn, are separated by nanoLC-type reverse-phase liquid chromatography. The chromatographic separations are carried out on a C18 75 μηι column of 50 cm with a particle size of 5 μηι (Acclaim PepMapl OO C18 column from Thermoscientific). The nanoLC is an Ultimate 3000 Rapid Separation LC Systems from Thermoscientific. The gradient used is a binary gradient. Solution A is a mixture of acetonitrile / 0.1% formic acid (2/98, v / v). Solution B is a mixture of acetonitrile / formic acid 0.1% (90/10, v / v). The flow rate is 0.220 μΙ_Ληίη at the nano pump and 15 μίΛηίη at the charge pump. The column is thermostated at 35 ° C.

The peptides thus separated are sequenced by high resolution mass spectrometry in tandem type LTQ-FT-ICR to obtain precise information on the amino acid sequence. The ionization of the peptides is done by a source of the electrospray type. The applied voltage is +1.70 kV. The method of acquisition in mass spectrometry is a method called data dependent acquisition in top 7, with dynamic exclusion, saved in LTQ centroid mode. Mass measurement is carried out with a high precision (of the order of the ppm) between 470 and 2000 m / z.

All experimental masses are compared to theoretical masses from protein databases where in silico digestion was performed. The identification of the proteins in the sample is done by comparing the two mass lists. Relative quantitation is performed by summing the intensity or area of the 3 major peptides of the identified protein. The ratio of the area obtained of each protein to the sum of the areas of all the proteins identified makes it possible to estimate the level of abundance of the protein in the sample. More than 70 enzymes were identified by this analysis.

The different proportions of proteins identified by proteomic analysis and selected for the invention are shown in FIG.

This figure shows that the main enzyme of the biocatalyst thus obtained is aspergillopepsin A (33.1%). Also present are endopolygalacturonase C (8.8%), 1,4 -3-D-glucan cellobiohydrolase (6.6%), endo-1, 4-3-xylanase A (3.6%), endo-1, 4-3-xylanase C (2.9%), endoglucanase (2.5%) and β-glucosidase (1.1%).

The inventors have also determined the level of the different enzymatic activities of the biocatalyst obtained by this production protocol. They compared these different activities with those obtained using other Aspergillus tubingensis strains (A tubingensis A and B) and a natural variant according to the invention of strain SCC027 in a similar production protocol. The enzymatic activities were determined by implementing the methods described above.

Table 1 summarizes the different enzymatic activities measured. Table 1: Comparison of the enzymatic profile of biocatalysts obtained with strain SCC027 and a natural variant of SCC027 compared to other strains of A. tubingensis

TC: rapeseed cake

SB: wheat bran

ND: not determined

These results show that the biocatalyst obtained with the strain of Aspergillus tubingensis SCC027 has enzymatic activities different from a composition obtained with other strains of Aspergillus tubingensis.

Example 2 Use of the Biocatalyst According to the Invention in the Production of Ethanol from Corn Flour, in a Vial This example shows the beneficial effect of the biocatalyst according to the invention on the production of ethanol from corn flour, in particular on the amount of ethanol produced.

Material and methods

General description of the ethanol production process

The ethanol production process implemented comprises the following steps: the raw material, here maize, is packaged, pasted then liquefied before being subjected to a simultaneous saccharification-fermentation step. In parallel, the yeast is rehydrated before being incorporated into the must for the simultaneous saccharification-fermentation step.

Conditioning of the corn raw material

The whole maize has been conditioned to obtain a mill, that is, a flour. For this, the corn was milled using a hammer mill (Electra). The size of the grid used was set at 0.8 mm. The grind obtained has a moisture content of 10% (+/- 2%). In the form of flour, the raw material is stored away from light in a dry place at room temperature.

Production of liquefied corn must

The liquefaction stage of the corn mill makes it possible to hydrolyze the starch into dextrins. The principle is to form a mixture of corn flour, water and clear vinasse industrial grade and heat it in the presence of liquefaction enzymes, so as to obtain a liquefied must. Thus, the must consists of 34% corn flour, 10% clear vinasse and 56% water. This must was prepared in a 4L bioreactor in double-jacketed glass (Cloup), at ambient temperature, with stirring between 250 and 350 rpm. After homogenization, the pH of the must was adjusted to 5.5 (by adding H ₂ SO ₄ ) in order to optimize the action of the liquefying enzymes. Alpha-amylase (Liquozyme SC DS, Novozymes) was added at 0.15 kg / t of corn. The wort was brought to 90 ° C and liquefied at this same temperature for 2h in the stirred reactor between 200 and 300 rpm. At the end of the liquefaction, the must was cooled to ambient temperature and adjusted to pH 5.3 (by addition of H ₂ SO ₄ ).

Ethanol production in vials

The simultaneous saccharification-fermentation step (SSF) was performed in baffled flasks. These are 250 ml baffle vials, closed with rubber stoppers hermetic, having a C0 ₂ output by needle of 1, 2 mm in diameter. The SSF consisted in bringing together:

• 140 g of liquefied corn must, 30% dry matter, pH 5.3;

• the active dry yeast Ethanol Red (LEAF Technologies), incorporated in 0.25 mg / ml of liquefied corn must, after being rehydrated for 30 min at 32 ° C. in a Tryptone Sel broth, to ensure an initial seeding of the order of 1 .10 ⁷ CFU yeasts / ml of must;

• urea at 1 g / l of liquefied corn must;

• glucoamylase (Spirizyme Excel XHS, Novozymes) at 0.42 kg / t of maize, so as to saccharify liquefied corn must.

The SSF must thus constituted was stirred at 120 rpm. The temperature was kept constant throughout this step at 32 ° C. No pH maintenance was performed. The fermentation was conducted for a period of 51 hours.

Finally, the biocatalyst obtained in Example 1, produced from strain SCC027 or one of its variants, was added at the beginning of SSF at different concentrations. In the so-called "control" condition, no biocatalyst addition has been made.

Each condition was applied in two separate vials (n = 2).

Results

By following the evolution of the weight of each of the flasks, it is possible to determine the amount of CO ₂ produced during the fermentation. Considered in the stoichiometric equation of ethanol production from glucose, the amount of CO ₂ produced makes it possible to estimate the production of ethanol for each of the fermentations conducted in flasks.

1 mole glucose -> 2 moles of CO ₂ + 2 moles of ethanol

Thus, by comparing the ethanol production after 51 hours of the so-called "control" fermentation compared to that of fermentations conducted in the presence of biocatalysts ("condition"), it is possible to calculate the ethanol gain:

Gain ethanol (%) = ([Ethanol] condition [Ethanol] control) / [Ethanol] control

Table 2: Ethanol Gains Obtained in Vials After 51 Hours of SSF of a Liquefied Mash of Corn, in the Presence of the SCC027 Biocatalyst or a Biocatalyst Obtained with a Natural Variant (SCC027-2 Variant), different doses of incorporation.

Addition rate of biocatalyst Gain Ethanol 51 h

Strain

(kg MSi / T corn) vs. Witness

variant SCC027-2 0.03 0.4%

variant SCC027-2 0.1 0.7% variant SCC027-2 0.3 0.8% variant SCC027-2 0.45 1, 2%

variant SCC027-2 0.6 1, 4%

SCC027 1, 2 1, 5%

As shown in Table 2, the production of ethanol after 51 hours of fermentation in a flask is improved with the use of biocatalysts obtained with strain SCC027 or its variants. The gain is variable depending on the strain and the incorporation dose. An ethanol gain of 1.5% is obtained with strain SCC027 at the incorporation dose of 1.2 kg MSi / t of corn. A variant of strain SCC027 gives rise to a similar ethanol gain (1.4%) at the dose of 0.6 kg MSi / t of maize, ie a dose half as much as with strain SCC027.

Example 3 Advantage of the Biocatalyst According to the Invention in the Production of Ethanol from Corn Flour, in a Vial, Compared with the Use of a Commercial Protease

This example shows the beneficial and advantageous effect of the biocatalyst according to the invention on the production of ethanol from corn flour, in particular on the amount of ethanol produced, compared to the implementation of a protease.

Material and methods

General description of the ethanol production process

The ethanol production process implemented comprises the following steps: the raw material, here industrial maize must, already liquefied, is directly subjected to a simultaneous saccharification-fermentation step. The yeast is rehydrated before being incorporated into the must for the simultaneous saccharification-fermentation step. Ethanol production in vials

The simultaneous saccharification-fermentation step (SSF) is done in baffled flasks. These are 250 ml baffled flasks, sealed with hermetic rubber stoppers, with a C0 ₂ output per needle of 1.2 mm diameter. The SSF consisted in bringing together:

· 140 g of industrially liquefied corn must, 32% dry matter, pH 5.2;

• the active dry yeast Ethanol Red (LEAF Technologies), incorporated in 0.25 mg / ml of liquefied corn must, after being rehydrated for 30 minutes at 32 ° C. in a Tryptone Sel broth, to ensure an initial seeding of the order of 1 .10 ⁷ CFU yeasts / ml of must;

• urea at 1 g / l of liquefied corn must;

The SSF must thus constituted was stirred at 120 rpm. The temperature was kept constant throughout this step at 32 ° C. No pH maintenance was performed. The fermentation was conducted for a period of 52 hours. Finally, the biocatalyst obtained in Example 1, produced from strain SCC027, was added at the beginning of fermentation at different concentrations. In comparison, the Lyvanol Acid® commercial protease (Lyven-Souletlet Biotechnologies) was added at the beginning of fermentation, at different concentrations, in order to guarantee the same contributions in protease activity (PAC) as with the biocatalyst produced with strain SCC027. . In the so-called "control" condition, no biocatalyst addition has been made. Each condition was applied in two separate vials (n = 2).

Results

1 mole glucose -> 2 moles of CO ₂ + 2 moles of ethanol Thus, by comparing the ethanol production at the end of 52 hours of the so-called "control" fermentation compared to that of fermentations conducted in the presence of biocatalysts (" condition "), it is possible to calculate the ethanol gain:

Gain ethanol (%) = ([Ethanol] status - [Ethanol] _control) / [Ethanol] _TEMOI n Table 3: Earnings ethanol obtained flask after 52 hours of SSF a liquefied mash industrial corn in the presence of the biocatalyst according the invention produced with the strain SCC027 or the commercial Lyvanol Acid® protease (Lyven - Soufflet Biotechnologies) at different incorporation doses.

Ethanol gains with Ethanol Gains with the

Dose of incorporation

Biocatalyst commercial proteinase

(U PAC / kg corn)

(Lyvanol Acid - Lyven) (SCC027)

10 0.1% 0.7% 30 0.4% 1, 2%

50 0.9% 1, 8%

100 0.7% 1, 6%

This example demonstrates the advantage of using the biocatalyst compared to that of a protease alone. As shown in Table 3, the ethanol gain potential follows a bell curve in both cases. It exceeds 1.5% with the use of the biocatalyst according to the invention while it is kept below the threshold of 1% with the use of the protease, even at high doses. An ethanol gain of 0.7% is achieved with the biocatalyst according to the invention for a contribution of protease activity of three to five times less than the necessary contribution of commercial protease for the same gain ethanol.

EXAMPLE 4 Use of the Biocatalyst According to the Invention in the Production of Ethanol from Corn Flour, in a Laboratory Fermenter

This example shows the beneficial effect of the biocatalyst according to the invention on the production of ethanol from corn flour, in particular on the final amount of ethanol produced and on the rate of ethanol production.

Material and methods

General description of the ethanol production process

The simultaneous saccharification-fermentation step (SSF) was performed in fermenters, model Biostat A + (Sartorius). They are reactors with a capacity of 2.5 I, equipped at the output of a condenser and a gas meter, model Milli GasCounter MGC-10 (Ritter), for online monitoring of C0 production. ₂ . The SSF consisted in bringing together:

· 2220 g of industrially liquefied corn must, 32% dry matter, pH 5.3;

• the active dry yeast Ethanol Red (LEAF Technologies), incorporated in 0.25 g / l of liquefied corn must, after being rehydrated for 30 minutes at 32 ° C. in a Tryptone Sel broth, to ensure an initial seeding of the order of 1 .10 ⁷ CFU yeasts / ml of must; • urea at 1 g / l of liquefied corn must;

The SSF must thus constituted was stirred at 350 rpm. The temperature was kept constant throughout this step at 32 ° C. No pH maintenance was performed. The fermentation was conducted for a period of 51 hours.

Finally, the biocatalyst obtained in Example 1, produced from strain SCC027, was added at the beginning of the SSF to 1.2 kg MSi / t of corn. In the so-called "control" condition, no biocatalyst addition has been made.

Each condition was applied in two separate bioreactors (n = 2).

At the end of the SSF reaction samples are taken in duplicate and analyzed by HPLC-RI (Modular Waters Isocratic HPLC e1515, Ion Exclusion Wat010295 column) to measure the concentrations of ethanol, glycerol and residual sugars present in the medium. .

Results

Figure 2 illustrates the significant effect of the implementation of the biocatalyst according to the invention on the rate of production of ethanol. Indeed, for the two fermenters in which the biocatalyst according to the invention was added at the beginning of fermentation, the production rate of CO ₂ is significantly increased compared to control fermenters. The maximum production rate of C0 ₂ in the presence of the biocatalyst according to the invention is 7.7 l / h whereas it is 6 l / h in the control condition. The maximum speed is measured after 12 h of SSF in the condition of addition of biocatalyst, and after 13 h in control condition.

The plateau of C0 ₂ production is reached after 45 h of SSF in the condition of addition of biocatalyst, while it has not yet been reached after 51 h of SSF in control condition.

Table 4: Ethanol and glycerol concentrations after 51 h of SSF of a liquefied corn must, in the presence or in the absence of the SCC027 biocatalyst according to the invention incorporated at 1.2 kg MSi / t of maize.

Bioreactor 1 Bioreactor 2 Difference

Mean standard deviation

Plvtl Plvt2 Plvtl Plvt2 SCC027 vs control

Ethanol (g / 1)

Witness 124.9 | 125.8 126.5 127.4 126.2 1, 08 SCC027 134.6 135.3 134.0 135.0 134.7 0.57 + 6.8%

Glycerol (g / i)

Witness 11, 8 12.3 12.0 12.1 12.0 0.21

SCC027 9.8 10.3 9.8 10.2 10.0 0.26 -16.5%

Table 4 above confirms the significant effect of the implementation of the biocatalyst according to the invention on the production of ethanol after 51 hours of SSF. In fact, the ethanol assayed in the fermenters containing the biocatalyst is 6 to 8% higher relative to the ethanol assayed in the control fermenters after 51 hours of SSF, specifying that at this stage the reaction of SSF the witness is not completed and has not reached his asymptote. In addition, the production of glycerol is significantly decreased in the presence of biocatalyst compared to the control. Indeed, after 51 hours of fermentation, the glycerol assay is lower by 16%. Glycerol is a by-product of ethanol. A lower production of glycerol indicates a modification of the utilization of glucose by the yeast and confirms the increase of the efficiency of conversion of glucose into ethanol by the yeast.

Example 5 Use of the Biocatalyst According to the Invention in the Production of Ethanol from Wheat Flour in a Vial

This example shows the beneficial effect of the biocatalyst according to the invention on the production of ethanol from starchy raw materials other than corn, in particular wheat flour, in particular on the quantity of ethanol produced and on the speed ethanol production.

Material and methods

General description of the ethanol production process

The ethanol production process implemented comprises the following steps: the raw material, here an industrial wheat flour, is liquefied before being subjected to a simultaneous saccharification-fermentation step. In parallel, the yeast is rehydrated before being incorporated in the liquefied must for the simultaneous saccharification-fermentation stage.

Production of liquefied must of wheat

The liquefaction stage of the wheat mill makes it possible to hydrolyze the starch into dextrins. The principle is to mix the wheat flour with water and clear vinasse, and to heat the mixture in the presence of liquefying enzymes, so as to obtain a liquefied must. Thus, the must consists of 28% wheat flour, 6% clear vinasse and 66% of water. This must was prepared in a 4 liter glass bioreactor (Cloup) at room temperature, with stirring at 250 rpm. The pH of the must was adjusted to 5.5 (by addition of NaOH) in order to optimize the action of the liquefying enzymes. Two liquefaction enzymes were used, namely alpha-amylase (Liquozyme SC DS, Novozymes) added to 0.15 kg / t of wheat and the hemicellulolytic complex unscrewing (Lyvanol Devisco Plus, Lyven-Soufflet Biotechnologies) added to 0 15 kg / t of wheat. The wort was first preredicated at 55 ° C for 30 min in the presence of the enzymes and then liquefied at 85 ° C for 1.5 h in the stirred reactor at 250 rpm. At the end of the liquefaction, the must was cooled to room temperature and adjusted to pH 4.5 (by addition of H ₂ SO ₄ ).

Ethanol production in vials

The simultaneous saccharification-fermentation step (SSF) was performed in baffled flasks. These are 250 ml baffle vials, closed with carded cotton stoppers. The SSF consisted in bringing together:

· 140 g of liquefied wheat must with 27.5% dry matter, pH 4.5;

The active dry yeast Ethanol Red (LEAF Technologies), incorporated in 0.25 mg / ml of liquefied wheat must, after being rehydrated for 30 minutes at 32 ° C. in a Tryptone Sel broth, to ensure an initial seeding of the order of 1 .10 ⁷ CFU yeasts / ml of must;

· Urea at 1 g / l of liquefied wheat must;

• glucoamylase (Distillase CS, Genencor) at 0.29 kg / t of wheat, so as to saccharify the liquefied must of wheat.

The wort thus formed was stirred at 105 rpm. The temperature was kept constant throughout this step at 32 ° C. No pH maintenance was performed. The SSF was conducted for 48 hours.

Finally, the biocatalyst obtained in Example 1, produced from strain SCC027, was added at the beginning of fermentation to 3 kg MSi / t of wheat. In the so-called "control" condition, no biocatalyst addition has been made.

Each condition was applied in two separate vials (n = 2).

Results

During the SSF reaction samples were taken and analyzed by HPLC-R1 (Modular Waters Isocratic E1515 HPLC, Ion Exclusion Wat010295 column) to measure the concentrations of ethanol, glycerol and sugars present in the medium. Table 5: Concentrations of ethanol and glucose after 24 h, 40 h and 48 h of SSF of a liquefied wort of wheat, in the presence or absence of the biocatalyst SCC027 incorporated at 3 kg MSi / t of wheat.

Table 5 demonstrates the beneficial effect of the implementation of the biocatalyst on ethanol production from wheat. Indeed, the production of ethanol after 48 hours of fermentation is significantly increased by 3.8%. The rate of ethanol production is also increased with significant differences in ethanol concentration at 24 and 40 hours of SSF, respectively 31% and 9%.

The kinetic effect of the implementation of the biocatalyst in fermentation is also visible on the yeast glucose consumption rate. After 40 h of SSF, the concentration of glucose in the medium containing the biocatalyst is similar to the concentration measured in the control medium after 48 h of SSF, indicating an acceleration of the end of the reaction of about 8 h.

Example 6 Comparison of the Effect of the Biocatalyst According to the Invention Relative to Enzymatic Compositions Obtained with Other Aspergillus Strains

This example shows that the beneficial effect of the biocatalyst according to the invention on the production of ethanol from corn flour, in particular on the amount of ethanol produced, is not observed with other enzyme compositions obtained with other strains of Aspergillus tubingensis than strain SCC027 and its variants.

Material and methods

General description of the ethanol production process

The ethanol production process implemented comprises the following steps: the raw material, here maize, is packaged, pasted then liquefied before being subjected to a simultaneous saccharification-fermentation step. In parallel, yeast is rehydrated before being incorporated into the must for the simultaneous saccharification-fermentation step.

Conditioning of the corn raw material

Production of liquefied corn must

The liquefaction stage of the corn mill makes it possible to hydrolyze the starch into dextrins. The principle is to constitute a mixture of corn flour, water and clear vinasse and to heat it in the presence of liquefying enzymes, so as to obtain a liquefied must. Thus, the must consists of 34% corn flour, 10% clear vinasse and 56% water. This must was prepared in a 4L bioreactor in double-jacketed glass (Cloup), at ambient temperature, with stirring between 250 and 350 rpm. After homogenization, the pH of the must was adjusted to 5.5 (by adding H ₂ SO ₄ ) in order to optimize the action of the liquefying enzymes. Alpha-amylase (Liquozyme SC DS, Novozymes) was added at 0.15 kg / t of corn. The wort was brought to 90 ° C and liquefied at this same temperature for 2h in the stirred reactor between 200 and 300 rpm. At the end of the liquefaction, the must was cooled to ambient temperature and adjusted to pH 5.3 (by addition of H ₂ SO ₄ ). Ethanol production in vials

The step of saccharification-simultaneous fermentation (SSF) was made in baffled flasks. These are 250 ml baffled flasks, sealed with hermetic rubber stoppers, with a C0 ₂ output per needle of 1.2 mm diameter. The SSF consisted in bringing together:

· 140 g liquefied corn must, 30% dry matter, pH adjusted to 5.3;

• dry yeast Ethanol Red (LEAF Technologies), incorporated in 0.25 mg / ml of liquefied corn must, after being rehydrated for 30 min at 32 ° C. in a Tryptone Sel broth, to ensure an initial seeding of the order of 1 .10 ⁷ CFU yeasts / ml of must;

· Urea at 1 g / l of liquefied corn must; • glucoamylase (Spirizyme Excel XHS, Novozymes) at 0.42 kg / t of maize, so as to saccharify liquefied corn must.

Finally, the biocatalyst obtained in Example 1, produced from strain SCC027, was added at the beginning of SSF at different concentrations. In comparison, other biocatalysts produced from the strains Aspergillus tubingensis B or Aspergillus tubingensis A were added at the beginning of SSF at different concentrations. In the so-called "control" condition, no biocatalyst addition has been made.

Each condition was applied in two separate vials (n = 2).

Results

1 mole glucose -> 2 moles of CO ₂ + 2 moles of ethanol

Thus, by comparing the production of ethanol after 51 hours of the so-called "control" fermentation compared with that conducted in the presence of a biocatalyst, it is possible to determine the ethanol gain.

Gain ethanol (%) = ([Ethanol] _con dition - [Ethanol] oin _CON) / [Ethanol] oin _CON

TABLE 6 Ethanol Gains Obtained in a Vial after 51 Hours of SSF from a Liquefied Corn Mash, in the Presence of the Biocatalyst According to the Invention Obtained with the SCC027 Strain or Other Biocatalysts Obtained with <ϋ Aspergillus tubingensis A Strains or B (those analyzed in Example 1), at different incorporation doses.

Strain Intake rate (g MSi / T corn) Gain Ethanol 51 h (%)

SCC027 690 0.9

SCC027 1,150 U

SCC027 2300 1, 5

A tubingensis strain B 690 -0.5

A tubingensis strain B 1 150 -1, 5

A tubingensis strain B 2300 -2.4

A tubingensis strain A 4600 0.3 The beneficial effect of using the SCC027 biocatalyst on ethanol production is not found with the use of biocatalysts obtained by fermentations of other strains of Aspergillus tubingensis, as shown in Table 6. At the same doses of incorporation and at higher doses than SCC027, no biocatalyst produced with the other strains tested allowed significant ethanol gains to be obtained after 51 h of SSF.

EXAMPLE 7 Comparison of the Effect of the Biocatalyst According to the Invention Compared to Enzymes Purified from the SCC027 Strain

This example shows that the beneficial effect of the biocatalyst according to the invention on the production of ethanol from corn flour, in particular on the total amount of ethanol produced, results from the combination of numerous enzymes produced during the production process. fermentation in solid medium of the strain.

Material and methods

Process for purifying the enzymes of the SCC027 biocatalyst

7 enzymes were purified from the biocatalyst obtained in Example 1: aspergillopepsin A, polygalacturonase C, endoxylanase A, endoglucanase, β-glucosidase, endoxylanase C and cellobiohydrolase (the latter two were obtained in a mixture).

Purification of these enzymes was performed after extraction of the biocatalyst in 0.02 M Histidine buffer pH 6.0 (1:10), centrifugation and filtration. Several separation techniques were combined: anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography and ammonium sulfate precipitation were used during the purification process.

The anion exchange chromatography is performed on a manually mounted column of 50 ml of Sephadex Q FF gel (GE Healthcare).

Chromatography is done on an ATKTA prime plus at 4 ° C. The flow rate is 10 ml / min. The gradient used is a binary gradient. Buffer A is 0.02 M Histidine solution pH 6.0. Buffer B is a solution of 0.02 M Histidine pH 6.0 and 1.0 M sodium chloride. The collected fractions are 6 ml.

Hydrophobic interaction chromatography was performed on a Hiprep phenyl HP 16/10 column (GE Healthcare). Chromatography is done on an ATKTA prime plus at 20 ° C. The flow rate is 3 ml / min. The gradient used is a binary gradient. The buffer A is a solution of 0.02 M Histidine pH 6.0 and 1.0 M ammonium sulfate. Buffer B is 0.02 M Histidine solution pH 6.0. The collected fractions are 6 mL.

Size exclusion chromatography was performed on a 75 μg Hiload Superdex column 16 mm in diameter and 600 mm long from GE Healthcare (120 ml gel). Chromatography is carried out on an ATKA purify at 20 ° C. The mobile phase is a solution of 0.1 M ammonium acetate pH 5.0. The flow rate is 1 ml / min. The sample volume injected is 2 ml. The fractions collected are 2 ml.

The protein mixture is fractionated by ammonium sulfate precipitation in a cascade of 40%, 55%, 65% and 100% salt saturation. Ammonium sulphate is added gradually in the form of rain. The precipitation is carried out for 30 min with stirring in ice. The precipitated mixture is centrifuged at 5000 g for 15 min at 4 ° C. The supernatant volume is measured and a new addition of ammonium sulfate is made to reach the second salt saturation plateau. This mixture is again precipitated 30 min in the ice with stirring. These steps are repeated for the different levels.

General description of the ethanol production process

The ethanol production process implemented comprises the following steps: the raw material, here maize, is packaged, pasted then liquefied before being subjected to a simultaneous saccharification-fermentation step. In parallel, the yeast is rehydrated before being incorporated into the simultaneous saccharification-fermentation must. Conditioning of the corn raw material

Production of liquefied corn must

The simultaneous saccharification-fermentation step (SSF) was performed in baffled flasks. These are 250 ml baffled flasks, sealed with hermetic rubber stoppers, with a C0 ₂ output per needle of 1.2 mm diameter. The SSF consisted in bringing together:

· 140 g liquefied corn must, 30% dry matter, pH 5.3;

· Urea at 1 g / l of liquefied corn must;

Finally, the biocatalyst obtained in Example 1, produced from strain SCC027, was added at the beginning of the SSF to 1.2 kg MSi / t of corn. In comparison, one or more fractions of purified enzymes of strain SCC027 was or were added at the beginning of SSF, so as to guarantee the same levels of contributions in enzymatic activity (s) that with the biocatalyst obtained with strain SCC027. In the so-called "control" condition, no biocatalyst addition has been made.

Results

By following the evolution of the weight of each of the flasks, it is possible to determine the amount of CO ₂ produced during the fermentation. Considered in the equation stoichiometric ethanol production from glucose, the amount of C0 ₂ produced is used to estimate the production of ethanol for each of the fermentations vial.

1 mole glucose -> 2 moles of CO ₂ + 2 moles of ethanol Thus, by comparing the ethanol production at the end of the 51 h of the so-called "control" fermentation compared with that conducted in the presence of a biocatalyst, it is possible to to determine the ethanol gain.

Gain ethanol (%) = ([Ethanol] _con dition - [Ethanol] _TEMOI n) / [Ethanol] _TEMOI n As shown in Figure 3, the inventors have determined that with a mixture of aspergillopepsin A, endoglucanase and β-glucosidase A, it was possible to increase the production of ethanol by 1.2% or 80% of the ethanol gain obtained with the crude biocatalyst. It should be noted that a mixture of aspergillopepsin A and β-glucosidase A makes it possible to reach 70% of the ethanol gain obtained with the biocatalyst, which represents the best combination of 2 enzymes among the tested enzymes produced by strain SCC027. , whereas this combination does not correspond to the combination of the two enzymes mainly produced by this strain.

The mixture of 7 enzymes among the 70 identified biocatalyst enzymes (aspergillopepsin A, polygalacturonase C, endoxylanase A, endoglucanase, β-glucosidase A, endoxylanase C and cellobiohydrolase) makes it possible to increase the production of ethanol at 51 hrs SSF. to achieve nearly 90% of the gain in ethanol obtained with the crude biocatalyst.

The optimal effect measured with the biocatalyst obtained according to the process of the invention with strain SCC027 is due to the combination of the multiple enzymes produced, including the 7 enzymes purified from the biocatalyst according to the invention.

Claims

1. Strain Aspergillus tubingensis SCC027 filed on ¹ February 2016 with the Centraalbureau voor Schimmelcultures (CBS, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands) under number CBS 136961.

2. Natural variant obtained from the strain of Aspergillus tubingensis as defined in claim 1, characterized in that it produces, by fermentation in a solid medium on a substrate comprising a mixture of wheat bran and rapeseed cake a biocatalyst having at least one fungal protease activity, a cellulase activity which is not a β-glucosidase activity, and a β-glucosidase activity, and which, when added to the simultaneous saccharification-fermentation medium of a process for producing ethanol from maize meal, induces an Ethanol Gain at least equal to that obtained under the same conditions with a biocatalyst produced by solid-state fermentation of the Aspergillus tubingensis strain SCC027 on a substrate comprising a mixture of wheat bran and rapeseed meal.

3. A process for producing a biocatalyst for producing ethanol from starchy raw material, comprising the following steps:

a) providing a substrate,

b) seeding the substrate provided in step a) with the Aspergillus tubingensis strain SCC027 according to claim 1 or a natural variant thereof according to claim 2, and

c) fermenting said substrate with the strain under solid conditions by solid-state fermentation to permit simultaneous production by the strain of protease activity, cellulase activity and β-glucosidase activity.

The manufacturing method according to claim 3, wherein the substrate is selected from the group consisting of wheat bran, rapeseed meal, soybean meal, beet pulp, barley rootlet, ethanol beans. corn, ethanol grains and mixtures thereof.

5. A biocatalyst for producing ethanol from starchy feedstock, having at least one fungal protease activity, a cellulase activity which is not a β-glucosidase activity, and a β-glucosidase activity obtained by the process according to claim 3 or 4.

6. Biocatalyst according to claim 5, comprising at least one aspergillopepsin A endoglucanase and β-glucosidase A.

7. Use of the biocatalyst according to claim 5 or 6, for the manufacture, by fermentation of starchy feedstock, of ethanol.

8. Use according to claim 7, wherein the starchy raw material is maize.

9. A process for producing ethanol from starchy raw material comprising a step of fermenting the starchy raw material with a yeast in the presence of the biocatalyst according to claim 5 or 6.

The method of manufacturing ethanol according to claim 9, comprising the steps of:

B) optionally a step of cooking the mixture prepared in step A),

C) optionally a step of pre-liquefaction of the starch of said mixture,

E) optionally a yeast propagation step, and

F) a simultaneous saccharification-fermentation step of the dextrins and / or fermentable sugars, in the presence of the biocatalyst according to claim 5 or 6, saccharification enzymes of dextrins and yeasts, optionally propagated in step E), way to produce ethanol.