Method for separating the beta-xylosidase enzyme from an enzyme mixture

Abstract

The present invention relates to a method for separating beta-xylosidase enzymes from an enzyme mixture comprising beta-xylosidase enzymes and other enzymes, wherein the beta-xylosidase enzymes to be separated are devoid of a histidine group, and wherein said beta-xylosidase enzymes are separated from the remainder of the enzyme mixture by immobilized metal ion affinity chromatography (IMAC). Figure for the abstract: Fig. 1

Description

Title of the invention: Method for separating the beta-xylosidase enzyme from a mixture of enzymes technical field

[0001] The present invention relates to the production of cellulolytic and / or hemicellulolytic enzymes, particularly for the production of sugars from cellulosic or lignocellulosic materials involving enzymatic hydrolysis of these materials. The sugars can be used / valued as is, or further converted into alcohol, particularly ethanol, by fermentation. Prior art

[0002] Since the 1970s, the transformation of lignocellulosic materials into ethanol, after hydrolysis of the constituent polysaccharides into fermentable sugars, has been the subject of numerous studies. For example, one can cite the seminal work of the National Renewable Energy Laboratory (Process Design and Economies for Bio-chemical Conversion of Lignocellulosic Biomass to Ethanol, Humbird et al., NREL / TP-5100-57764, May 2011).

[0003] Lignocellulosic materials are cellulosic materials, that is, materials composed of cellulose and hemicellulose, which are polysaccharides essentially made up of pentoses and hexoses, as well as lignin, which is a macromolecule with a complex structure and high molecular weight based on phenolic compounds. For the sake of brevity, they may be grouped under the generic term biomass in this text.

[0004] Wood, straw, and corn cobs are the most commonly used lignocellulosic materials, but other resources, such as dedicated forest crops, residues from al-oil-producing, sugar, and cereal crops, products and residues from the paper industry, and by-products of lignocellulosic material processing, are also usable. These materials consist mostly of approximately 35 to 50% cellulose, 20 to 30% hemicellulose, and 15 to 25% lignin.

[0005] The process for the biochemical transformation of lignocellulosic materials into sugars, and then optionally into ethanol, comprises a physicochemical pretreatment step, followed by an enzymatic hydrolysis step using an enzyme cocktail. This can be followed by an ethanolic fermentation step of the released sugars, with ethanolic fermentation and enzymatic hydrolysis being able to be carried out simultaneously, and then by an ethanol purification step. An example of such a process converting biomass into ethanol is described in patent EP 3,484,945, to which reference may be made for further details.

[0006] The enzyme cocktail used for hydrolysis is a mixture of cellulolytic (also called cellulases) and / or hemicellulolytic enzymes. Cellulolytic enzymes exhibit three main types of activity: endoglucanases, exoglucanases, and cellobiases, the latter also being called [3]glucosidases. Hemicellulolytic enzymes notably exhibit xylanase activity.

[0007] The most widely used cellulolytic microorganism for the industrial production of the enzyme cocktail is the fungus Trichoderma reesei. Wild-type strains have the ability to secrete, in the presence of an inducing carbon substrate, such as cellulose, the enzyme cocktail considered best suited for cellulose hydrolysis. Other proteins possessing properties essential for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, such as xylanases. The presence of an inducing carbon substrate is essential for the expression of cellulolytic and / or hemicellulolytic enzymes. The nature of the carbon substrate has a strong influence on the composition of the enzyme cocktail. This is the case with xylose, which, when combined with an inducing carbon substrate such as cellulose or lactose, significantly improves xylanase activity.

[0008] More specifically, in the context of second-generation (2G) bioethanol production from lignocellulosic biomass, one of the main challenges is to degrade cellulose and hemicellulose fibers through biomass pretreatment (treatment, for example, with an acidic or basic solution, followed by cooking or steam explosion) and then by the action of cellulolytic and hemicellulolytic enzymes, which depolymerize the fibers. Cellobiohydrolases (CBH1 and CBH2) produce sugar oligomers such as cellobiose, cellotriose, and other glucose oligomers from cellulose. [3-Glucosidase] degrades cellobiose (and other oligomers) into glucose that can be directly assimilated by yeast for bioethanol production.The degradation of hemicelluloses is carried out by xylanases or xylobiohydrolases, and allows the formation of xylose oligomers (xylobiose, xylotriose, and other xylose oligomers). The action of [3-xylosidase] allows the degradation of these xylose oligomers to produce xylose. Xylanases are generally inhibited by xylobiose and short xyloligosaccharides, and the lack of [3-xylosidases] is then responsible for the rate-limiting step of xylan hydrolysis.

[0009] From patent application WO 2011 / 079048, it is stated that, in a process for the simultaneous hydrolysis and fermentation of biomass (SSF for "Simultaneous Saccarification and Fermentation"), increasing the beta-xylosidase activity of the enzyme cocktail used for enzymatic hydrolysis has a beneficial effect on the enzymatic hydrolysis of certain biomasses, since it reduces the amount of enzymes required. It also allows for the hydrolysis of alkyl- xylosides.

[0010] It is therefore of interest to isolate the beta-xylosidases present in enzyme mixtures produced by microorganisms, for example, to enrich a given enzyme cocktail with beta-xylosidases. To this end, various techniques have already been proposed, notably for first separating the fungus from the enzymes it has produced in the culture medium. For example, US patent 3,398,055 describes the separation and purification of cellulases produced by the fungus Trichoderma reesei: The fungus is separated from the enzymes by filtration with a rotary vacuum filter. The enzymes are then separated by passing them through a column using cotton and eluting them with a basic solution. Patent WO 2018 / 015228 proposes to separate the enzymes from the fungus by a succession of treatment steps of a culture medium, including a step of filtration of the culture medium by a filter press, then a step of tangential microfiltration of the liquid phase obtained. It is also known to separate beta-xylosidase from an enzyme mixture by fractional precipitation with ethanol, as described in the publication by V. Cortez et al., "Xylanase and β-xylosidase separation by fractional precipitation," Process Biochemistry, Volume 35, Issues 3-4, 1999, Pages 277-283. This is an interesting technique, but it is not without drawbacks, as it requires the use of a solvent and involves numerous steps, making it costly and complex to implement.

[0011] The invention aims to develop an improved technique for separating enzymes from a mixture of enzymes, and more particularly, a technique for separating beta-xylosidases from a mixture containing beta-xylosidases and other types of enzymes. It is particularly aimed at a separation technique that is efficient and deployable on an industrial scale. Summary of the invention

[0012] The invention relates firstly to a method for separating beta-xylosidase enzymes from an enzyme mixture comprising beta-xylosidase enzymes and other enzymes, such as: - the beta-xylosidase enzymes to be separated are devoid of histidine group, - and such that the said beta-xylosidase enzymes are separated from the rest of the enzyme mixture by immobilized metal ion affinity chromatography (hereafter also referred to by its acronym IMAC for the Anglo-Saxon term "Immobilized Metal Affinity Chromatography").

[0013] IMAC-type chromatography is known to separate proteins that have histidine groups exposed on their surface, either naturally or by In the latter case, genetic modification is referred to as a histidine "tag" or "cluster" added to the protein. See, in particular, the publication by V. GABERC-POREKAR et al., "Perspectives of μ-mobilized-metal affinity chromatography," J Biochem. Biophys. Methods. 2001 Oct. 30; 49 (1-3) 335-60.

[0014] However, quite surprisingly, it was discovered in the context of the present invention that this chromatography technique was nevertheless capable of separating enzymes lacking a histidine group, and in particular the beta-xylosidases that the inventors were seeking to separate in an enzymatic cocktail produced by microorganisms.

[0015] And this is very advantageous in several respects: - Beta-xylosidase enzymes can be isolated using this technique without first modifying them to have these histidine "tags" or "clusters." By avoiding modification, their extraction / separation method is simplified, of course, by eliminating a genetic modification step. But it also limits any risk of performance loss due to changes in their behavior or altered activity caused by the presence of these histidine groups (numerous cases of enzymes whose activity has been altered following the addition of a histidine "tag" have been described in the literature, particularly in the case of metalloenzymes and multimeric enzymes). - The IMAC type chromatography separation technique is very efficient: it can be deployed on an industrial scale, the materials needed to operate this type of chromatography are stable and can therefore be stored without risk of degradation, the elution conditions are generally not severe, the reagents used are generally reusable, which makes it economically attractive, and its results in terms of selectivity in the separated enzymes, particularly here beta-xylosidases, are excellent. - the separation can be carried out in a single step, resulting in a simplified and faster implementation process.

[0016] Generally, the other enzymes of said mixture may include at least one enzyme selected from cellulases, hemicellulases, and / or from hemicellulases.

[0017] Generally, the other enzymes of said mixture may include beta-glucosidases, endoglucanases, and possibly cellobiohydrolases.

[0018] Beta-xylosidases may constitute at least 1% by weight, in particular between 2 and 15% by weight or between 3 and 8% by weight, of all the enzymes present in the mixture. This is the content generally found in enzyme cocktails produced by Trichoderma Reseei, but, naturally, the invention applies equally to enzyme mixtures containing a higher proportion of beta-xylosidases.

[0019] Preferably, immobilized metal ion affinity chromatography (IMAC) uses: - a stationary solid phase comprising a matrix to which metallic ions are fixed by chelating agents, - and a liquid mobile phase called eluent.

[0020] The matrix of the stationary phase can advantageously be chosen from at least one of the following compounds: agarose gel, cross-linked dextran gel, silica.

[0021] Chelating agents may advantageously be chosen from at least one of the following compounds: iminodiacetic acid IDA, nitrolotriacetic acid NTA, tris[carboxymethyl]ethylenediamine TED.

[0022] The metal ions may advantageously be chosen from: the metal ions of transition metals, in particular chosen from - the divalent ions of Cu (II), Ni (II), Zn (II), Co (II), - trivalent metallic ions of metals, in particular selected from the trivalent ions of Fe (III), Al (III), Ga (III), - or tetravalent metal ions, in particular the Zr(IV) metal ion.

[0023] According to a preferred embodiment of the invention, the enzyme mixture is produced by a microorganism, in particular by a filamentous fungus, for example of the genus Trichoderma, preferably of the species Tri-choderma reesei.

[0024] The separation process according to the invention may include a preliminary step of separating a culture medium comprising the enzyme mixture and a microorganism that produced said mixture, said preliminary step being aimed at separating the microorganism from said enzyme mixture and comprising, in particular, one or more successive filtrations of the culture medium. This preliminary separation may, for example, be carried out as described in patent WO 2018 / 015228. Thus, after solid / liquid separation, the microorganism that produced the enzymes (also called must) is obtained in solid / semi-solid form on the one hand, and the soluble enzymes in liquid (aqueous) phase on the other. This liquid phase may optionally be concentrated, and then it can be processed according to the invention.

[0025] The separation process according to the invention may also include a step of treating the must, having been separated or not from the rest of the culture medium, said treatment including cooling the must and then separating the must from a so-called additional liquid phase containing an additional quantity of enzyme mixture, as taught in patent EP 3 174 979.

[0026] If this additional separation is carried out on the already separated must, the liquid phase obtained after the solid / liquid separation described above can then be mixed with this additional liquid phase, and the process according to the invention can be carried out on the a mixture of these two liquid phases.

[0027] The process according to the invention can naturally be carried out on a liquid phase containing the mixture of enzymes which has been previously concentrated.

[0028] According to a first variant of the separation process of the invention, chromatography is carried out continuously in a chromatography column containing an immobile, solid phase suitable for being continuously traversed by a liquid mobile phase called eluent.

[0029] According to another embodiment, the chromatographic separation according to the invention is carried out by batch, by bringing into contact a stationary chromatographic phase with the mixture comprising beta-xylosidase enzymes and other enzymes, in liquid medium, to constitute a reaction medium in a container for a given time, then eluting the solid part of said reaction medium in order to extract the beta-xylosidases.

[0030] In this variant, the separation may include a step of mixing the immobile phase with the enzyme mixture in solution, then an optional decantation step, then a step of isolating the solid phase from the reaction medium, then an optional washing step, then a step of eluting the isolated solid phase to extract the beta-xylosidases.

[0031] The chromatographic separation according to the invention fixes the beta-xylosidases on the stationary phase preferably at a pH between 6.5 and 9, and the beta-xylosidases are preferably eluted by changing the nature, composition or concentration of the eluent, which allows, in particular, changing the pH of the stationary phase.

[0032] The invention also relates to the beta-xylosidase enzyme, particularly produced by a fungus such as Aspergillus or Trichoderma, and in particular obtained by the separation process as described above, and which has a specific activity of at least 10 pmoles of p-nitrophenol.min⁻¹.mg⁻¹ of enzyme, in particular of at least 20 or at least 30 or at least 35 pmoles of p-nitrophenol.min⁻¹.mg⁻¹ of enzyme. This is a high specific activity, which demonstrates efficient separation resulting from a high purity of the beta-xylosidase enzyme thus separated. The method for measuring specific activity, known to those skilled in the art, consists of placing the purified enzyme in the presence of PNP-Xylose (p-nitrophenyl-[3-D-xylopyranoside]. Under the action of beta-xylosidase, the released PNP is monitored spectroscopically, and the specific activity is calculated using a PNP standard curve.

[0033] An example of a beta-xylosidase targeted by the present invention is a Xylan 1,4 beta-xylosidase protein obtained from Trichoderma reesei, reference XP_006964075.1 in NCBI (acronym for National Center for Biotechnology Information) and described in the Uniprot database under reference Q92458_HYPJE (EC: 3.2.1.37; taxonomic identifier 51453 NCBI; sequence version 2 of 01 / 06 / 1998, : Gene: bxll, -Organism: Hypocrea jecorina (Trichoderma reesei)). Also targeted are all beta-xylosidases with sequences having at least 50% identity with this beta-xylosidase, in particular at least 60% or at least 65% or at least 80% or at least 85% or at least 90% or at least 95% or at least 98% or 99% with this beta-xylosidase.

[0034] The invention relates more generally to any beta-xylosidase, which can in particular be obtained with a fungus of the genus Trichoderma, in particular the species Trichoderma reesei or citrinoviride or orientale or longibrachiatum or arundinaceum, or with a fungus of the genus Aspergillus, in particular the species Aspergillus niger, japonicus, oryzae, clavatus, aculeatus, awamori, flavus.

[0035] The invention also relates to the beta-xylosidase enzyme, in particular obtained by the separation process described above, and which has a purity greater than or equal to 90%, generally greater than or equal to 95% or 97%. Purity was assessed, in a known manner, by SDS-PAGE gel electrophoresis (a polyacrylamide gel containing sodium dodecyl sulfate), then analyzed with the hnage-Lab software, available from the company BIO-RAD.

[0036] A very pure enzyme is thus obtained, making it highly valuable. This result is all the more remarkable as the separation according to the invention can be carried out on enzyme cocktails that can contain dozens, or even a hundred, of different enzymes, such as those produced by microorganisms like Trichoderma for example.

[0037] The invention also relates to the use of beta-xylosidase enzymes, in particular obtained according to the process described above, to enrich in beta-xylosidase enzymes a mixture of enzymes produced by a microorganism.

[0038] They can thus be added, in a controlled manner, in a process for converting different types of lignocellulosic biomass into sugar(s) (saccharification including enzymatic hydrolysis of the biomass) or into alcohol (saccharification and fermentation) having different recalcitrances into sugars or into alcohol (ethanol type).

[0039] Another use is to valorize these beta-xylosidase enzymes as such, for applications which specifically call upon beta-xylosidase activity.

[0040] Beta-xylosidase can be purified and sold pure for biotechnological applications, whether for degrading or for producing xyloligosaccharides.

[0041] Beta-xylosidase can be supplemented with an enzyme cocktail low in beta-xylosidase for industrial applications in the field of lignocellulosic biomass degradation in order to produce sugars that can be used in bioproducts or for the production of alcohols, including bioethanol. List of figures

[0042] [Fig.1] Fig. 1 represents the FPLC profile (acronym for the English expression "Fast Protein Liquid Chromatography", which is a known technique for rapid liquid-phase protein chromatography) of the separation of beta-xylosidase from an enzyme mixture according to one embodiment of the invention. [Fig.2] Figure [Fig.2] represents an electrophoresis result with an SDS-PAGE gel of beta-xylosidase after FPLC purification (described later). [Fig.3] Figure 3 represents a graph of the activities of beta-xylosidases separated according to 2 examples of embodiment of the invention, in the form of histograms, with the identification of examples 1 and 2 on the abscissa, and their activities expressed in pmoles of p-nitrophenol.min'.mg of enzyme on the ordinate. Description of the implementation methods

[0043] The invention will be described in detail below, with the aid of figures and examples given by way of illustration and which are therefore in no way limiting.

[0044] The invention proposes a method for separating a particular enzyme in a mixture of enzymes: the beta-xylosidase enzyme. She is particularly interested in separating this enzyme from an enzymatic cocktail produced by a microorganism, more specifically by the fungus Trichoderma, notably Trichoderma reesei, and which is the subject of the examples and detailed descriptions that follow.

[0045] But the invention applies similarly to the separation of this enzyme from any mixture of enzymes containing it, and in particular from any enzyme cocktail produced by microorganisms which contain this enzyme in varying proportions.

[0046] The process according to the invention allows for the simple and rapid purification of [3-xylosidase from T. reesei regardless of the type of T. reesei strain used.

[0047] This is a process for purifying an enzyme of interest (beta-xylosidase) from a complex enzyme mixture (about one hundred enzymes) in a single step. This requires prior production of the enzymes and separation of the mycelium.

[0048] The description below details the variant of the invention using a chromatography column, operating continuously. However, the invention can be implemented without a column, in a similar manner, by batch. Preliminary steps

[0049] To implement the separation process according to the invention, a preliminary separation of the culture medium, comprising the enzyme cocktail and the Trichoderma reesei fungus, is first carried out. For this purpose, the culture medium is subjected, within 24 hours of the production shutdown, to separation using a filter press lined with a cloth having a porosity of 3-20 µm, so as to obtain a filtrate having a corrected optical density OD 600nm of less than 2.5. The resulting liquid phase is subjected to tangential microfiltration using a ceramic membrane having a cutoff threshold between 0.5 and 1.4 µm, so that the corrected optical density OD 600nm does not exceed 0.1. The filter press separation and microfiltration are carried out at 20-30°C, preferably 22-27°C.

[0050] After separation using a filter press, 5-10% by weight of solid residue (called "cake") is generally obtained, and 90-95% by weight of filtrate. Advantageously, the microfiltrate obtained after filter pressing is carried out within a maximum of 30 hours, and preferably within a maximum of 24 hours.

[0051] Preferably, tangential microfiltration is carried out on a ceramic membrane having a cut-off threshold between 0.8 and 1.4 pm.

[0052] The liquid phase obtained after microfiltration can be subjected to ultrafiltration, preferably on a ceramic membrane, and even more preferably on a ceramic membrane having a cut-off threshold between 5 and 15kDa. The filtration method described here is based on the teachings of patent WO 2018 / 015228, to which reference should be made for further details.

[0053] The resulting retentate is then passed through an IMAC affinity column to separate enzymes bearing a polyhistidine tag (also called a "His-tag"). This is an amino acid motif in a protein consisting of at least six histidine residues, often inserted at the N- or C-terminal end of the protein. It is sometimes referred to as a "hexahistidine tag" or "6xHis-tag". It is worth noting, however, that the beta-xylosidase purified from the cocktail does not include a histidine tag (nor do any of the other enzymes in the enzyme mixture here). IMAC column purification

[0054] The supernatant containing the enzymes (microfiltration permeate or ultrafiltration retentate), i.e. the cellulases produced by Trichoderma reesei, is stored between 4°C and 30°C, but preferably below 10°C.

[0055] The resulting supernatant is loaded onto a column by immobilized metal ion affinity chromatography, also known as IMAC (an acronym for the corresponding English term "Immobilized Metal Affinity Chromatography"). This type of affinity chromatography is based on the mechanism of metal cation chelation. immobilized. It generally allows the purification of proteins possessing a histidine "tag" from the supernatant containing a complex mixture of different proteins of biological origin.

[0056] Metal ion chelation (generally divalent) is a process that allows the formation of a complex between a metal cation and a ligand attached to a solid phase. Through this chelation, the metal ions remain immobilized in a column, through which the enzyme mixture to be fractionated or purified is flowed. The bonds between the metal ion and the ligand generally form within a pH range of 7 to 8. To maintain this pH, the column is first equilibrated using a buffer solution.

[0057] The solution in which the sample can be solvated ideally exhibits high ionic strength to reduce nonspecific electrostatic interactions, but these ions must not themselves bind with the metals. The solution is also preferably neutral or slightly alkaline, since the interactions between the histidine groups and the metals are deactivated in the presence of protons that occupy the binding sites on the amino acid. Examples of this type of solution are Tris-acetate (tris(hydroxymethyl)aminomethane acetate (CH3COO), 50 mM) or sodium phosphate 20 to 50 mM. Tris-HCl (tris(hydroxymethyl)aminomethane HCl) can be used to purify enzymes in which the interactions between the protein and the metals are quite strong.

[0058] For the eluent, an acidic solution with a pH gradient of 7 to 4 can be chosen to protonate the amino acids interacting with the IMAC matrix, thereby drastically reducing the enzyme's affinity for the resin. Alternatively, an imidazole solution can be used to replace the proteins at the binding sites (to exchange the ligands). Finally, the metal ion can also be extracted with a strong chelating agent such as ethylenediaminetetraacetic acid (EDTA), which is often used to regenerate the column. Production stage

[0059] The enzyme cocktail on which the separation process according to the invention is carried out is produced by Trichoderma reesei in a conventional production line, by aerated fermentation. Examples of processes for producing enzyme cocktails using this fungus are described in patents FR 3 024 463, FR 3 049 957, FR 3 085 961, and FR 3 088 934. An improvement to the process for increasing the levels of beta-glucosidase and / or beta-xylosidase by cooling the must obtained at the end of production is described in patent EP 3 174 979.

[0060] The process for producing the enzyme cocktail begins with a propagation phase, generally implemented in small reactors of increasing size, in order to multiply the filamentous fungus and limit the duration of the phase of latency and the risks of contamination. When this production is deemed sufficient (mushroom concentration greater than or equal to 10 g / L, preferably greater than or equal to 15 g / L), the culture medium is transferred to the final large-volume reactor. The enzyme production process comprises two phases, detailed as follows according to a preferred embodiment: - a phase a) of growth of said microorganism in the presence of at least one carbon growth substrate in a closed aerated reactor, said growth phase being carried out with a concentration of carbon growth substrate between 10 and 90 g / L, - a phase b) of production of the enzymatic cocktail, in which at least one inducing carbon substrate is introduced, said inducing carbon substrate being chosen from the group formed by lactose, cellobiose, sophorose, residues obtained after ethanolic fermentation of monomeric sugars of enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses from the pretreatment of cellulosic biomass, said production phase being carried out with a concentration of production carbon substrate between 150 and 400 g / L.

[0061] The microorganisms used in the process of producing an enzyme cocktail according to the invention are strains of fungi belonging to the species Trichoderma reesei.

[0062] The most efficient industrial strains are strains belonging to the species Trichoderma reesei, modified to improve the enzymatic cocktail by mutation-selection processes.

[0063] Strains improved by genetic recombination techniques can also be used. These strains are cultured in stirred and aerated reactors under conditions compatible with their growth and enzyme production.

[0064] As examples of strains and methods of obtaining them, it can be recalled that classical genetic techniques by mutation have allowed the selection of strains of Trichoderma reesei hyperproducing cellulases such as strains MCG77 (Gallo - US patent 4275 167), MCG 80 (Allen, AL and Andreotti, RE, Biotechnol-Bioengi 1982, 12, 451-459 1982), RUT C30 (Montenecourt, BS and Eveleigh, DE, Appl. Environ. Microbiol. 1977, 34, 777-782) and CL847 (Durand et al, 1984, Proc. Colloque SFM "Génétique des microorganismes industriels". Paris. H. HESLOT Ed, pp 39-50). The improvements have made it possible to obtain hyper-producing strains, less sensitive to catabolic repression on monomeric sugars in particular, glucose for example, compared to wild strains. Recombinant strains have also been obtained from Trichoderma reesei strains such as Qm9414, RutC30, CL847, by cloning heterologous genes, For example, the invertase of Aspergillus niger allows Trichoderma reesei to use sucrose as a carbon source. These strains have retained their hyperproductivity and their ability to be cultured in fermenters.

[0065] The carbonaceous growth substrate of said microorganism used in said growth phase a) of the process according to the invention is advantageously chosen from industrial soluble sugars, and preferably from glucose, lactose, xylose, liquid residues obtained after ethanolic fermentation of monomeric sugars from enzymatic hydrolysates of lignocellulosic materials and extracts of the hemicellulosic fraction in the form of monomers from pretreated lignocellulosic substrate, used alone or in mixture.

[0066] Depending on its nature, said carbon growth substrate is introduced into the closed reactor before sterilization, or is sterilized separately and introduced into the closed reactor after sterilization of the latter.

[0067] Said carbon substrate for growth is used in said growth phase a) at an initial concentration most often between 20 and 90 g of carbon substrate per liter of reaction volume.

[0068] Preferably, said phase a) of growth is carried out over a period of between 30 and 70 h, preferably between 30 and 40 h.

[0069] Preferably, said growth phase a) operates at a pH of 4.8 and at a temperature of 20-30°C, generally 22-27°C, preferably in the order of 27°C.

[0070] Said carbon inducing substrate used in said production phase b) is advantageously fed in a fed-batch phase with a limiting flux of between 30 and 80 mg per gram of cells per hour. The temperature is generally the same as in step a).

[0071] At the end of the enzyme production step, a medium is generally obtained with a dry matter concentration of between 10 and 45 g / L (corresponding to the dry mushroom). The enzymes are all water-soluble. The pellet measured after centrifugation (4000 rpm, 5 minutes) is greater than 15% and often around 30%, and even up to 60%. It corresponds to the percentage of the volume occupied by the solid relative to the total volume of the sample.

[0072] The object of the invention is to separate the enzymes from the fungus, then to purify the beta-xylosidase, for commercialization or for specifically adding it to a mixture of enzymes, in a biochemical process involving enzymes, if they are limiting.

[0073] The invention can be applied to any mixture of enzymes produced by a microorganism, called an enzyme cocktail, including enzyme cocktails derived from a reaction medium containing the microorganism that has been treated, in particular by cooling, as described in patent EP3174979.

[0074] Solid / liquid separation steps in which the fungus is separated from the liquid,

[0075] The liquid contains the enzymes and residual salts.

[0076] - Once the enzymes have been separated from the mycelium, the pH of the supernatant (containing the enzymes) must be adjusted within a pH range of 6.5-9, preferably to pH 8 (buffer change by desalination column, filtration, ultrafiltration, under pressure (stirring cell commercially available under the name Amicon from Merck, or ultrafiltration cell, commercially available under the name Pellicon with Ultracel lOkD membrane also from Merck) - Once the cellulase cocktail has been buffered, an IMAC type column described previously is used. It should be noted that the tested enzyme mixtures have a composition of the type (the indicated contents are expressed in abundance and are approximate data but which give an idea of ​​the distribution of enzymes in the mixture): - CEL7A content = CBH1 approximately 35%, - CEL6A = CBH2 content: approximately 30% - BGL1 (Beta-glucosidase 1) content: approximately 5% - Endoglucanase I (Cel7B) content: approximately 10% - Endoglucanase II (Cel5A) content: approximately 10% - Content of Other (enzymes and / or other compounds): approximately 10%

[0077] For examples of secretome analyses of modified Trichoderma reesei strains, RUT-C30 and CL847, reference can be made to the publication "Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretrory strains", by I; HERPOEL-GIMBERT et al. Biotechnology for biofuels, article number 18(2008) published on December 23, 2008, and in particular to its table I.

[0078] The HisTrap Crude column (Cytiva, 5 mL), pre-charged with nickel ion Ni2+, is equilibrated at a pH between 6.5 and 9, preferably at pH 8. This type of column is available from Cytiva under the full name* HisTrap FF crude histidine-tagged protein purification column. The equilibration buffer can be Tris, Bis-Tris, Phosphate, 4(2-hydroxyethyl)-l-piperazine ethanesulfonic acid also called HEPES, or any other buffer solution being in the pH range between 6.5 and 9. The buffer solution can contain salts (NaCl, KCl) between 0-500 mM but preferably at 50 mM. - Once the column is equilibrated, the clarified supernatant can be filtered and then loaded onto the column by a peristaltic pump, using a system called a "Fast Protein Liquid Chromatography System," a system that generally includes a pump, a UV detector, a conductivity measurement device, a fraction collector, and... Valves allow, in particular, switching between columns. Such a system is commercially available from BIO-RAD. Another chromatographic system is also available from Cytiva under the name "AKTA Pure Protein Purification System". Gravity separation techniques can also be used.

[0079] The column washing is carried out in the presence of 0 to 40 mM imidazole, generally with 20 mM imidazole.

[0080] Surprisingly, and this had never been observed before, the beta- T. reesei xylosidase binds to the solid phase of IMAC, and can be easily purified on this type of resin, while it does not have any histidine "tag". To elute beta-xylosidase from T. reesei, a gradient or elution must be performed in the presence of 500 mM imidazole, or the pH must be decreased to reduce the enzyme's affinity with the solid phase.

[0081] The beta-xylosidase enzyme from T. reesei purified under these conditions is of high purity (verification was done by mass spectrometry), and it is active (the activity test shows that the enzyme is active on 4-nitrophenyl-[3-D-xylopyranoside (4-NPX) and releases para-nitrophenol pNP). The specific activity of beta-xylosidase is evaluated using 4-Nitrophenyl[3-D-Xylopyranoside (4-NPX)] as a substrate, with activities at 50°C ranging from 10 to 100 pmol p-nitrophenol.min⁻¹.mg enzymes⁻¹ and generally at least 20 and approximately 35 pmol p-nitrophenol.min⁻¹.mg enzymes⁻¹ (or higher). The same protocol is used as for measuring beta-glucosidase activity. The only difference is the substrate (para-nitrophenyl-β-D-Xylopyranoside (pNP)) and the use of beta-xylosidase. The principle of measuring specific activity with this type of reagent is well-established in the literature. Examples

[0082] Example 1 The experiments were carried out in the laboratory, on enzymatic cocktails produced by Trichoderma reesei according to the operating procedure described above, from the strain CL847, already cited and also described in the publication Jourdier E. et Al., “A newstoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains” (Microb Cell Fact. 2012 May 30;ll:70. doi: 10.1186 / 1475-2859-11-70. PMID:22646695; PMCID: PMC3434075.).

[0083] Example 2 The experiments were carried out in the laboratory, on enzymatic cocktails produced by Trichoderma reesei according to the procedure described above, from the strain, described in Table 1 of patent EP 3 174 979 (SEQ Id nO: 7 in nucleic acid, SEQ Id nO: 8 in polypeptide) under reference 130G9.

[0084] The following description relates to the treatment of the culture medium obtained in each of the two examples: The extracellular medium was separated from the mycelium by filtration. The extracellular medium containing the enzymes secreted by Trichoderma reesei was removed by filtration through a Pellicon membrane (10 kDa) and then diluted three times in buffer A 50 mM Tris-Cl pH8, 50 mM NaCl.

[0085] This step has the double advantage of removing low molecular weight molecules present in the culture medium and bringing the pH back to 8. The protein extract is then centrifuged for 10 min at 5000 rpm and filtered at 0.2 µm (PES filter, VWR, 514-2073) with a syringe. The protein solution is then loaded onto the HisTrap column (Cytiva, HisTrap™ FF and HisTrap Crude, 5 mL) identified above, which has an immobile phase based on cross-linked sepharose coupled to Nickel ions via chelating groups.

[0086] The column is pre-equilibrated with 7 column volumes in buffer A 50 mM Tris-Cl pH 8, 50 mM NaCl. A linear gradient is then applied for 10 column volumes to buffer B 50 mM Tris-Cl pH 8, 50 mM NaCl, 500 mM imidazole. The protein is eluted with a homogeneous and symmetrical peak. It is then concentrated and washed three times in buffer A by centrifugation at 5000 rpm using commercially available ultrafiltration units under the name Vivaspin (1000 kDa) from Sartorius to remove the imidazole. The pure protein was then analyzed by SDS-PAGE and mass spectrometry. These analyses unambiguously demonstrated that the purified protein, separated from the remaining enzymes in the starting cocktail, is beta-xylosidase. Note that, on the other hand, we did not separate the CBH2s which, however, have a histidine "tag", which is doubly surprising.

[0087] Figure 1 shows the FPLC (Fast Protein Liquid Chromatography) profile of the beta-xylosidase purification, correlated with the sodium dodecysulfate-containing polyacrylamide electrophoresis gel (SDS-PAGE, BIO-RAD, Mini Protean TGX Strain-Free Precast gel 10%-456-8035), according to Example 1. Band 1, corresponding to the enzyme purified by this chromatographic technique, is shown. This enzyme was cut from the gel and then analyzed by mass spectrometry. The protein identified by mass spectrometry corresponds to the protein XP_006964075.1 in NCBI and UniProtKB Reference: Q92458_HYPJE, already cited above. The same type of result is obtained with Example 2.

[0088] Figure 2 is an image of the SDS-PAGE gel, for example 1, which allows proteins to be separated according to their molecular weights after denaturing them. To provide an indication of molecular weight, the left-hand band It features markers of different molecular sizes: 15 kDa, 20 kDa, 25 kDa, 37 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa, and 250 kDa. On the right-hand side, purified beta-xylosidase was loaded and then analyzed. The SDS-PAGE gel electrophoresis results for beta-xylosidase show that molecular mass 1 corresponds to that predicted by the DNA sequence, i.e., between 75 kDa and 100 kDa. Furthermore, the protein is found to be pure. The same type of result is obtained with Example 2.

[0089] Following the various purifications, the activity was measured on different batches of purified enzymes, and the activity results are shown in the histogram in [Fig. 3]. It can be seen that the beta-xylosidase enzyme purified from Examples 1 and 2 has a specific activity of approximately 35 to 38 pmoles of p-nitrophenol.min-1.mg 1 of enzyme.

[0090] In conclusion, this IMAC purification technique for beta-xylosidase allows for the rapid, simple, and highly efficient preparation of this enzyme, without the use of a histidine tag. The affinity of beta-xylosidase for the IMAC-type column is an unexpected finding. The beta-xylosidase separated according to the invention was identified by mass spectrometry, and its specific activity, evaluated with para-nitrophenyl-bD-Xylopyranoside (pNPX) as a substrate, varies between at least 15 or 20 and 35 pmoles of p-nitrophenol.min⁻¹.mg⁻¹ of enzyme or more. This enzyme has industrial interest for stimulating the degradation of xylan or xylose oligomers with different degrees of polymerization (DP) such as xylobiose, xylotriose or with higher DP of xylose. It can be used alone, or combined with other enzyme mixtures / cocktails depending on the needs and applications.

Claims

Demands

1. A method for separating beta-xylosidase enzymes (1) from an enzyme mixture comprising beta-xylosidase enzymes and other enzymes, characterized in that the beta-xylosidase enzymes to be separated are devoid of a histidine group, and in that said beta-xylosidase enzymes are separated from the rest of the enzyme mixture by immobilized metal ion affinity chromatography (IMAC).

2. Separation method according to the preceding claim, characterized in that the other enzymes of said mixture comprise at least one enzyme selected from cellulases and / or from hemicellulases.

3. Separation process according to any one of the preceding claims, characterized in that the other enzymes of said mixture include beta-glucosidases, endoglucanases, hemicellulases and optionally cellobiohydrolases.

4. Separation process according to any one of the preceding claims, characterized in that the beta-xylosidases (1) constitute at least 1% by weight, in particular between 2 and 15% by weight or between 3 and 8% by weight, of all the enzymes present in the mixture.

5. Separation method according to any one of the preceding claims, characterized in that the immobilized metal ion affinity chromatography IMAC uses: - a solid stationary phase which comprises a matrix on which metal ions are fixed by chelating agents, - and a liquid mobile phase called eluent.

6. Separation method according to the preceding claim, characterized in that the matrix of the immobile phase is chosen from at least one of the following compounds: agarose gel, cross-linked dextran gel, silica.

7. Separation process according to any one of claims 5 or 6, characterized in that the chelating agents are selected from at least one of the following compounds: iminodiacetic acid IDA, nitrolotriacetic acid NTA, tris[carboxymethyl]ethylenediamine TED.

8. Separation method according to any one of claims 5 to 7, characterized in that the metal ions are selected from: transition metal metal ions, in particular selected from divalent ions of Cu(II), Ni(II), Zn(II), Co(II), trivalent metal ions of metals, in particular selected from trivalent ions of Fe(III), Al(III), Ga(III) or tetravalent metal ions, in particular the metal ion of Zr(IV).

9. Separation method according to any one of the preceding claims, characterized in that the enzyme mixture is produced by a microorganism, in particular by a filamentous fungus, for example of the genus Trichoderma, in particular the species Tri-choderma reesei or citrinoviride or orientale or longibrachiatum or arundinaceum, or of the genus Aspergillus, in particular the species Aspergillus niger, japonicus, oryzae, clavatus, aculeatus, awamori, flavus.

10. A separation method according to any one of the preceding claims, characterized in that it comprises a preliminary step of separating a culture medium comprising the mixture of enzymes and a microorganism called must which produced said mixture, said preliminary step being aimed at separating the must from said liquid enzyme mixture and comprising in particular one or more successive filtrations of the culture medium.

11. Separation process according to the preceding claim, characterized in that it also includes a step of treating the must, having been separated or not from the rest of the culture medium, said treatment comprising cooling the must and then separating the must from a liquid containing an additional amount of enzyme mixture.

12. Separation method according to any one of the preceding claims, characterized in that the chromatography is carried out continuously in a chromatography column containing a stationary solid phase and capable of being continuously traversed by a liquid mobile phase called eluent.

13. Separation method according to any one of claims 1 to 11, characterized in that the chromatographic separation is carried out by batch, by bringing into contact a stationary chromatography phase with the mixture comprising beta-xylosidase enzymes and other enzymes, in liquid medium, to constitute a reaction medium in a container for a given time, and then eluting the solid part of said reaction medium in order to extract the beta-xylosidases.

14. Separation process according to the preceding claim, characterized in that the separation comprises a step of mixing the immobile phase with the enzyme mixture in solution, then an optional decantation step, then a step of isolating the solid phase from the reaction medium, then an optional washing step, then a step of eluting the isolated solid phase to extract the beta-xylosidases.

15. Separation method according to any one of the preceding claims, ca- characterized in that the separation by chromatography fixes the beta-xylosidases (1) on the immobile phase at a pH between 6.5 and 9, and in that the beta-xylosidases (1) are eluted by changing the nature, composition or concentration of the eluent.

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