Method for separating beta-xylosidase enzyme from an enzyme mixture

Abstract

The present invention relates to a method for separating beta-xylosidase enzymes from a mixture of enzymes comprising beta-xylosidase enzymes and other enzymes, such that the beta-xylosidase enzymes to be separated have no histidine moiety, and such that said beta-xylosidase enzymes are separated from the remainder of the enzyme mixture by immobilized metal affinity chromatography (IMAC).

Description

[0001] PROCESS FOR THE SEPARATION OF THE BETA-XYLOSIDASE ENZYME FROM A

[0002] ENZYME MIXTURE

[0003] Technical field

[0004] The present invention relates to the production of enzymes, of the cellulolytic and / or hemicellulolytic type, in particular in the context of the production of sugars from cellulosic or lignocellulosic materials involving enzymatic hydrolysis of these materials. The sugars can be used / recovered as they are, or continue their conversion into alcohol, in particular ethanol, by fermentation.

[0005] Prior art

[0006] 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. Examples include the landmark work of the National Renewable Energy Laboratory (Process Design and Economies for Biochemical Conversion of Lignocellulosic Biomass to Ethanol, Humbird et al., NREL / TP-5100-57764, May 2011).

[0007] Lignocellulosic materials are cellulosic materials, i.e., made up of cellulose, 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, in this text they can be grouped under the generic term biomass.

[0008] Wood, straw, and corn cobs are the most commonly used lignocellulosic materials, but other resources, dedicated forest crops, residues from alcohol-producing, sugar-producing, and cereal-producing plants, products and residues from the paper industry, and products from the processing of lignocellulosic materials can also be used. Most of them consist of approximately 35 to 50% cellulose, 20 to 30% hemicellulose, and 15 to 25% lignin.

[0009] The process for the biochemical transformation of lignocellulosic materials into sugars, and then possibly into ethanol-type alcohol, comprises a physicochemical pretreatment step, followed by an enzymatic hydrolysis step using an enzyme cocktail. It may continue with an ethanolic fermentation step of the released sugars, the ethanolic fermentation and enzymatic hydrolysis being able to be carried out simultaneously, followed 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.

[0010] The enzyme cocktail used for hydrolysis is a mixture of cellulolytic enzymes (also called cellulases) and / or hemicellulolytic enzymes. Cellulolytic enzymes exhibit three main types of activity: endoglucanases, exoglucanases, and cellobiases, the latter also known as p-glucosidases. Hemicellulolytic enzymes, in particular, exhibit xylanase activities.

[0011] The most widely used cellulolytic microorganism for the industrial production of enzyme cocktails is the fungus Trichoderma reesei. Wild strains have the ability to secrete, in the presence of an inducing carbon substrate, such as cellulose, the enzyme cocktail considered best suited to cellulose hydrolysis. Other proteins with 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 for xylose, which, when combined with an inducing carbon substrate such as cellulose or lactose, significantly improves the so-called xylanase activity.

[0012] More specifically, in the context of the production of so-called second-generation (2G) bioethanol from lignocellulosic biomass, one of the main challenges is to degrade cellulose and hemicellulose fibers through pretreatment of the biomass (treatment, for example, with an acidic or basic liquor, then cooking or steam explosion) and then through the action of cellulolytic and hemicellulolytic enzymes, which depolymerize the fibers. Cellobiohydrolases (CBH1 and CBH2) make it possible to produce sugar oligomers such as cellobiose, cellotriose and other glucose oligomers produced from cellulose. β-glucosidase makes it possible to degrade cellobiose (and also other oligomers) into glucose that can be directly assimilated by yeast for the production of bioethanol.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 p-xylosidase allows the degradation of these xylose oligomers to produce xylose. Xylanases are generally inhibited by xylobiose and short xyloligosaccharides, and the lack of p-xylosidases is then responsible for the rate-limiting step in xylan hydrolysis.

[0013] From patent application WO 2011 / 079048, we learn 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 allows the quantity of enzymes required to be reduced. It also allows the hydrolysis of alkyl-xylosides.

[0014] It is therefore interesting to isolate the beta-xylosidases present in enzyme mixtures produced by microorganisms, for example to enrich a given enzyme cocktail with beta-xylosidases. And to do this, different techniques have already been proposed, in particular to first separate, in the culture medium, the fungus from the enzymes it has produced. Thus, patent US-3,398,055 teaches 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 flowing them through a column using cotton, and eluting them with a basic solution.

[0015] Patent WO 2018 / 015228 proposes separating the enzymes from the fungus by a succession of steps of treatment 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.

[0016] It is also known to separate beta-xylosidase from a mixture of enzymes by fractional precipitation with ethanol, as described in the publication by V. CORTEZ and AL "Xylanase and p-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, insofar as it requires the use of a solvent, and it imposes numerous steps, which makes it expensive and complex to implement.

[0017] The invention therefore 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 aims more particularly at a separation technique that is efficient and deployable on an industrial scale.

[0018] Summary of the invention

[0019] The invention firstly relates to a process for separating beta-xylosidase enzymes from a mixture of enzymes comprising beta-xylosidase enzymes and other enzymes, such that: - the beta-xylosidase enzymes to be separated are devoid of a histidine group,

[0020] - and such that said beta-xylosidase enzymes are separated from the rest of the enzyme mixture by affinity chromatography on immobilized metal ions (hereinafter also referred to by its acronym IMAC for the English term “Immobilized Metal Affinity Chromatography”).

[0021] IMAC chromatography is known to separate proteins that have histidine groups exposed on their surface, either naturally or through genetic modification, in the latter case we speak of a histidine "tag" or a histidine "cluster" added to the protein. We can refer in particular to the publication by V. GABERC-POREKAR et al "Perspectives of immobilized-metal affinity chromatography" J Biochem. Biophys. Methods. 2001 Oct. 30; 49 (1-3) 335-60.

[0022] 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 sought to separate in an enzymatic cocktail produced by microorganisms.

[0023] And this is very advantageous in several ways:

[0024] - beta-xylosidase enzymes can be isolated using this technique without having previously modified them to have these histidine "tags" or "clusters". By avoiding modifying them, we simplify their method of obtaining / separating them, of course, by eliminating a genetic modification step. But we also limit any risk of loss of performance by modifying their behavior / alteration of their activity due to the presence of these histidine groups (many cases of enzymes that have had their activity altered following the addition of a histidine "tag" have been described in the literature, particularly in the case of metalloenzymes and multimeric enzymes),

[0025] - the IMAC type chromatography separation technique is very efficient: it can be deployed on an industrial scale, the materials needed to carry out 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 the beta-xylosidases, are excellent.

[0026] - the separation can be carried out in a single step, resulting in a process that is easier to implement and faster. Generally, the other enzymes in said mixture may comprise at least one enzyme chosen from cellulases, hemicellulases, and / or from hemicellulases.

[0027] Generally, the other enzymes in said mixture may include beta-glucosidases, endoglucanases, and possibly cellobiohydrolases.

[0028] 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 in the same way to enzyme mixtures containing a higher proportion of beta-xylosidases.

[0029] Preferably, immobilized metal ion affinity chromatography IMAC uses:

[0030] - a solid immobile phase which comprises a matrix on which metal ions are fixed by chelating agents,

[0031] - and a liquid mobile phase called eluent.

[0032] The matrix of the immobile phase can advantageously be chosen from at least one of the following compounds: agarose gel, crosslinked dextran gel, silica.

[0033] The chelating agents may advantageously be chosen from at least one of the following compounds: iminodiacetic acid IDA, nitrolotriacetic acid NTA, tris [carboxymethyl] ethylene diamine TED.

[0034] The metal ions can advantageously be chosen from: metal ions of transition metals, in particular chosen from

[0035] - the divalent ions of Cu (II), Ni (II), Zn (II), Co (II),

[0036] - trivalent metal ions of metals, in particular chosen from trivalent ions of Fe (III), Al (III), Ga (III),

[0037] - or tetravalent metal ions, notably the Zr(IV) metal ion.

[0038] According to a preferred embodiment of the invention, the enzyme mixture is derived from the production of enzymes by a microorganism, in particular by a filamentous fungus, for example of the genus Trichoderma, preferably of the species Trichoderma reesei.

[0039] The separation method according to the invention may comprise a preliminary step of separating a culture medium comprising the mixture of enzymes and a microorganism having produced said mixture, said preliminary step aiming to separate the microorganism from said mixture of enzymes 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 having 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 hand. This liquid phase may optionally be concentrated, then it can be treated according to the invention.

[0040] The separation method according to the invention may also comprise a step of treating the must, whether or not it has been separated from the rest of the culture medium, said treatment comprising 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.

[0041] 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 mixture of these two liquid phases.

[0042] 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.

[0043] According to a first variant of the separation method of the invention, the chromatography is carried out continuously in a chromatography column containing an immobile, solid phase capable of being continuously crossed by a liquid mobile phase called eluent.

[0044] According to another variant, the separation by chromatography according to the invention is carried out in batch, by bringing into contact a stationary chromatography phase with the mixture comprising beta-xylosidase enzymes and other enzymes, in a liquid medium, to constitute a reaction medium in a container for a given duration, then by eluting the solid part of said reaction medium in order to extract the beta-xylosidases therefrom.

[0045] In this variant, the separation may comprise a step of mixing the immobile phase with the mixture of enzymes 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 therefrom.

[0046] The separation by chromatography according to the invention fixes the beta-xylosidases on the immobile 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 makes it possible, in particular, to change the pH of the immobile phase.

[0047] The invention also relates to the enzyme beta-xylosidase, in particular 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 at least 20 or at least 30 or at least 35 pmoles of p-nitrophenol.min -1 . mg -1 enzyme. It is a high specific activity, which demonstrates an efficient separation resulting in a high purity of the beta-xylosidase enzyme thus separated.

[0048] The method for measuring the specific activity, known to those skilled in the art, consists of placing the purified enzyme in the presence of PNP-Xylose (p-nitrophenyl-pD-xylopyranoside). Under the action of beta-xylosidase, the released PNP is monitored by spectroscopy and the specific activity is calculated using a standard range of PNP.

[0049] An example of beta-xylosidase targeted by the present invention is a protein Xylan 1,4 beta-xylosidase obtained from Trichoderma reesei of reference XP 006964075.1 in NCBI (acronym for National Center for Biotechnology Information) and described in the Uniprot database under the reference Q92458_HYPJE (EC: 3.2.1.37; taxonomic identifier 51453 NCBI; sequence version 2 of 01 / 06 / 1998, : Gene: bxl1, -Organism: Hypocrea jecorina (Trichoderma reesei)).

[0050] Also covered 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.

[0051] The invention relates more generally to any beta-xylosidase, which can be obtained in particular 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, cia vatus, aculeatus, awamori, fia vus.

[0052] 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%. The purity was evaluated, in a known manner, by electrophoresis on SDS-PAGE gel (a polyacrylamide gel containing sodium dodecyl sulfate), then analyzed with the Image-Lab software, available from the company BIO-RAD.

[0053] We therefore obtain a very pure enzyme, which makes it highly recoverable. This is a result that is all the more remarkable since the separation according to the invention can be carried out on enzymatic cocktails that can contain dozens, or even a hundred, different enzymes, such as those produced by microorganisms such as Trichoderma for example.

[0054] The invention also relates to the use of beta-xylosidase enzymes, in particular obtained according to the process described above, for enriching a mixture of enzymes produced by a microorganism with beta-xylosidase enzymes.

[0055] 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 recalcitrance to sugars or alcohol (ethanol type).

[0056] Another use is to valorize these beta-xylosidase enzymes as such, for applications that specifically call for beta-xylosidase activity.

[0057] Beta-xylosidase can be purified and sold pure for biotechnological applications, either to degrade or to produce xylo-oligosaccharides.

[0058] Beta-xylosidase can be supplemented to an enzymatic cocktail low in beta-xylosidase for industrial applications in the field of degradation of lignocellulosic biomass in order to produce sugars that can be used in bioproducts or for the production of alcohols, including bioethanol.

[0059] List of figures

[0060] Figure 1 represents the FPLC profile (acronym for the English expression “Fast Protein Liquid Chromatography”, which is a known technique for rapid chromatography of proteins in the liquid phase) of the separation of beta-xylosidase from a mixture of enzymes according to an exemplary embodiment of the invention.

[0061] Figure 2 represents an electrophoresis result with an SDS-PAGE gel of beta-xylosidase after FPLC purification (described later). Figure 3 represents a graph of the activities of the beta-xylosidases separated according to 2 exemplary embodiments 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 on the ordinate -1 . mg -1 of enzyme.

[0062] Description of the embodiments

[0063] The invention will be described in detail below, using figures and examples given for illustration purposes and which are therefore in no way limiting.

[0064] The invention provides a method for separating a particular enzyme from a mixture of enzymes: the enzyme beta-xylosidase.

[0065] She is particularly interested in separating this enzyme from an enzymatic cocktail produced by a microorganism, more particularly by the Trichoderma fungus, notably Trichoderma reesei, and which is the subject of the examples and detailed descriptions which follow.

[0066] But the invention applies in a similar manner to the separation of this enzyme from any mixture of enzymes containing it, and in particular from any enzymatic cocktail produced by microorganisms which contain this enzyme in variable proportions.

[0067] The method according to the invention makes it possible to purify the p-xylosidase of T. reesei simply and quickly, regardless of the type of T. reesei strain used.

[0068] This is a process for purifying an enzyme of interest (beta-xylosidase) from a complex enzyme mixture (around a hundred enzymes) carried out in a single step. This requires first producing the enzymes and separating the mycelium.

[0069] The description below details the variant of the invention using a chromatography column, operating continuously. But the invention can be implemented without a column, in a similar manner, by batch.

[0070] To implement the separation method according to the invention, a preliminary separation of the culture medium, comprising the enzymatic cocktail and the Trichoderma reesei fungus, is first carried out. For this, the culture medium is subjected, within a period of less than 24 hours, from the end of production, to a separation on a filter press packed with a cloth having a porosity of 3-20 pm, so as to obtain a filtrate having a corrected optical density OD 600 nm of less than 2.5. The liquid phase obtained is subjected to tangential microfiltration on a ceramic membrane having a cut-off threshold of between 0.5 and 1.4 pm, so that the corrected optical density OD 600 nm does not exceed 0.1. The separation on the filter press and the microfiltration are carried out at 20-30 ° C, preferably 22-27 ° C.

[0071] At the end of the separation on a filter press, we generally obtain 5-10% by weight of solid residue (called "cake"), and 90-95% by weight of filtrate. Advantageously, the microfiltration of the filtrate obtained at the end of the filter press is carried out within a maximum of 30 hours, and preferably 24 hours maximum.

[0072] Preferably, the tangential microfiltration is carried out on a ceramic membrane having a cut-off threshold between 0.8 and 1.4 pm.

[0073] 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 of between 5 and 15 kDa.

[0074] The filtration method described here follows the teaching of patent WO 2018 / 015228, which should be referred to for further details.

[0075] The resulting retentate is then passed through an IMAC affinity column to separate enzymes with 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-terminus of the protein. It is sometimes referred to as a hexahistidine tag or 6xHis-tag.

[0076] It is worth noting, however, that the beta-xylosidase purified from the cocktail does not include a histidine tag (nor any of the other enzymes in the enzyme mixture here).

[0077] Purification on IMAC column

[0078] 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.

[0079] The resulting supernatant is loaded onto a column using immobilized metal ion affinity chromatography (IMAC). This type of affinity chromatography is based on the mechanism of chelation of immobilized metal cations. It generally allows the purification of proteins with a histidine tag from the supernatant containing a complex mixture of different proteins of biological origin. Chelation of the metal ion (usually divalent) is a process that allows the formation of a complex between a metal cation and a ligand fixed on a solid phase. Thanks to this chelation, the metal ions remain immobilized in a column, into which the enzyme mixture to be fractionated or purified is passed. The bonds between the metal ion and the ligand are generally formed in a pH range between 7 and 8.In order to maintain this pH, the column is first equilibrated using a buffer solution.

[0080] The solution in which the sample can be solvated ideally has a high ionic strength to reduce nonspecific electrostatic interactions, but these ions should not themselves bind with metals. The solution is also preferably neutral or slightly alkaline, since the interactions between histidine groups and metals are deactivated in the presence of protons that occupy the binding sites on the amino acid. Examples of such solutions are 50 mM Tris-acetate (tris(hydroxymethyl)aminomethane acetate) (CH3COO ), or 20 to 50 mM sodium phosphate. Tris-HCl (tris(hydroxymethyl)aminomethane HCl) is used to purify enzymes whose protein-metal interactions are quite strong.

[0081] For the eluent, an acidic solution with a pH gradient of 7 to 4 can be chosen, in order to protonate the amino acids interacting with the IMAC matrix, which induces a drastic decrease in the affinity of the enzyme for the resin. Alternatively, an imidazole solution can also 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, (often used to regenerate the column).

[0082] 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 an enzyme cocktail using this fungus are described in patents FR 3 024463, FR 3 049 957, FR 3 085 961, FR 3 088 934. An improvement to the process for increasing the level 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.

[0083] The process of producing the enzyme cocktail begins with a propagation phase, generally carried out in small reactors of increasing size, with the aim of multiplying the filamentous fungus, and limiting the duration of the latency phase and the risks of contamination.

[0084] 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 into the final large volume reactor.

[0085] The enzyme production process comprises two phases, detailed as follows according to a preferred embodiment:

[0086] - 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 of between 10 and 90 g / L,

[0087] - 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, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, and / or a crude extract of water-soluble pentoses originating from the pretreatment of a cellulosic biomass, said production phase being carried out with a concentration of production carbon substrate of between 150 and 400 g / L.

[0088] The microorganisms used in the process for producing an enzymatic cocktail according to the invention are strains of fungi belonging to the species Trichoderma reesei.

[0089] The most efficient industrial strains are those belonging to the species Trichoderma reesei, modified to improve the enzymatic cocktail by mutation-selection processes.

[0090] Strains improved by recombinant DNA techniques can also be used. These strains are grown in stirred and aerated reactors under conditions compatible with their growth and enzyme production.

[0091] As examples of strains and methods of obtaining them, we can recall that classical genetic techniques by mutation have allowed the selection of strains of Trichoderma reesei hyperproducing cellulases such as the MCG77 strains (Gallo

[0092] - 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 hyperproductive 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 allowing Trichoderma reesei to use sucrose as a carbon source. These strains have retained their hyperproductivity and their ability to be cultivated in fermenters.

[0093] The carbon substrate for growth 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 of enzymatic hydrolysates of lignocellulosic materials and extracts of the hemicellulosic fraction in the form of monomers originating from pretreated lignocellulosic substrate, used alone or in a mixture.

[0094] 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.

[0095] Said carbon growth substrate 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.

[0096] Preferably, said growth phase a) is carried out over a period of between 30 and 70 hours, preferably between 30 and 40 hours.

[0097] 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 of the order of 27°C.

[0098] Said inducing carbon substrate used in said production phase b) is advantageously fed in fed-batch phase with a limiting flow of between 30 and 80 mg per gram of cells per hour. The temperature is generally the same as in step a).

[0099] At the end of the enzyme production step, a medium is generally obtained containing a dry matter concentration of between 10 and 45 g / L (corresponding to the dry mushroom), the enzymes are all soluble in water. The pellet measured after centrifugation (4000 rpm, 5 minutes) is greater than 15% and often of the order of 30%, and even up to 60%. It corresponds to the percentage of the volume occupied by the solid compared to the total volume of the sample. The aim of the invention is to separate the enzymes from the fungus, then to purify the beta-xylosidase, to market it or to add it specifically to a mixture of enzymes, in a biochemical process involving enzymes, if they are limiting.

[0100] The invention can be applied to any mixture of enzymes produced by a microorganism, called an enzyme cocktail, including enzyme cocktails resulting from a reaction medium containing the microorganism which has been treated, in particular by cooling, as described in patent EP3174979. solid / liquid separation in which the fungus is separated from the liquid.

[0101] The liquid contains the enzymes and residual salts.

[0102] - Once the enzymes have been separated from the mycelium, the pH of the supernatant (containing the enzymes) must be adjusted to a pH range of 6.5-9, preferably to pH 8 (buffer change by desalting column, filtration, ultrafiltration, pressure (stirred cell commercially available under the name Amicon from Merck, or ultrafiltration cell, commercially available under the name Pellicon with Ultracel 10kD membrane also from Merck)

[0103] - Once the cellulase cocktail is buffered, an IMAC type column described previously is used.

[0104] It should be noted that the tested enzyme mixtures have a composition of the following type (the contents indicated are expressed in abundance and are approximate data but which give an idea of ​​the distribution of the enzymes in the mixture):

[0105] - CEL7A = CBH1 content approximately 35%,

[0106] - CEL6A = CBH2 content: approximately 30%,

[0107] - BGL1 content = Beta-glucosidase 1: approximately 5%

[0108] - Endoglucanase I content = Cel7B: approximately 10%

[0109] - Endoglucanase II content = Cel5A: approximately 10%

[0110] - content of Others (enzymes and / or other compounds): approximately 10%

[0111] For examples of secretome analyses of modified Trichoderma reesei strains, RUT-C30 and CL847, see 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 its table I.

[0112] The HisTrap Crude column (Cytiva, 5mL), preloaded with nickel ion Ni 2+, 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".

[0113] The equilibration buffer can be Tris, Bis-Tris, Phosphate, 4(2-hydroxyethyl)-1-piperazine ethanesulfonic acid also called HEPES, or any other buffer solution 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.

[0114] - Once the column is equilibrated, the clarified supernatant can be filtered and then loaded onto the column by a peristaltic pump, by a system called "Fast Protein Liquid Chromatography System", a system which generally includes a pump, a UV detector, a conductivity measuring device, a fraction collector and valves allowing in particular to pass from one column to another. Such a system is notably commercially available from the company BIO-RAD. Another chromatographic system is also available from the company Cytiva under the name "AKTA pure protein purification system".

[0115] Gravity separation techniques can also be used.

[0116] Column washing is performed in the presence of 0 to 40 mM imidazole, typically with 20 mM imidazole.

[0117] Surprisingly, and this had never been observed before, the beta-xylosidase of T. reesei binds to the solid phase of IMAC, and can be easily purified on this type of resin, while it does not present any histidine "tag".

[0118] To elute beta-xylosidase from T. reesei, a gradient or elution in the presence of 500 mM imidazole must be performed, or the pH must be decreased to reduce the enzyme's affinity for the solid phase.

[0119] 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-pD-xylopyranoside (4-NPX) and releases para-nitrophenol pNP). The specific activity of beta-Xylosidase is evaluated with 4-Nitrophenyl pD-Xylopyranoside (4-NPX) as substrate with activities at 50°C between 10 and 100 pmoles p-nitrophenol.min-1 .mg enzymes-1 and generally at least 20 and about 35 pmoles p-nitrophenol.min -1 .mg enzymes-1 (or more). The same protocol is used as for measuring beta glucosidase activity. We just change the substrate (para-nitrophenyl-b-D-Xylopyranoside (pNP)) and use beta-xylosidase. The principle of measuring specific activity with this type of reagent is well known in the literature.

[0120] Examples

[0121] Example 1

[0122] The experiments were carried out in the laboratory, on enzymatic cocktails produced by Trichoderma reesei according to the procedure described above, from the CL847 strain, already cited and also described in the publication Jourdier E. and 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;11:70. doi: 10.1186 / 1475-2859-11-70. PMID:22646695; PMCID: PMC3434075.).

[0123] Example 2

[0124] 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 the reference 130G9.

[0125] The following description concerns 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 on a Pellicon membrane (10 kDa) and then diluted three times in 50 mM Tris-Cl buffer A pH 8, 50 mM NaCl.

[0126] 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 pm (PES filter, VWR, 514-2073) with a syringe.

[0127] 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 crosslinked sepharose and coupled to Nickel ions via chelating groups.

[0128] The column is pre-equilibrated with 7 column volumes in 50 mM Tris-Cl pH8, 50 mM NaCl buffer A. A linear gradient is then applied for 10 column volumes to 50 mM Tris-Cl pH8, 50 mM NaCl, 500 mM Imidazole buffer B. The protein is eluted in a homogeneous and symmetrical peak. It is then concentrated and washed three times in buffer A by centrifugation at 5000 rpm with ultrafiltration units commercially available under the name Vivaspin (10 kDa) from Sartorius, in order to remove imidazole. The pure protein was then analyzed on SDS-PAGE gel and by mass spectrometry. These analyses made it possible to unambiguously demonstrate that the protein purified / separated from the rest of the enzymes in the starting cocktail is beta-xylosidase. Note that, on the other hand, we did not separate the CBH2 which, however, has a histidine “tag”, which is doubly surprising.

[0129] Figure 1 represents the FPLC (Fast Protein Liquid Chromatography) profile of the purification of beta-xylosidase, correlated with the polyacrylamide electrophoresis gel containing sodium dodecyl sulfate (SDS PAGE, BIO-RAD, Mini Protean TGX Strain-Free Precast gel 10%- 456-8035), according to example 1. It shows band 1 corresponding to the enzyme purified by this chromatographic technique, which was cut out of the gel and then analyzed by mass spectrometry. The protein identified by mass spectroscopy 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.

[0130] Figure 2 is an image of the SDS-PAGE gel, for example 1. which allows the separation of proteins according to their molecular masses after denaturing the proteins. In order to have an indication of the molecular mass, the left band shows 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 band, the purified beta-xylosidase was loaded and then analyzed. The result of electrophoresis with an SDS-PAGE gel of beta-xylosidase shows that the molecular mass 1 corresponds to that predicted by the DNA sequence, i.e. between 75 kDa and 100 kDa. In addition, it is noted that the protein is pure. The same type of result is obtained with example 2.

[0131] Following the various purifications, the activity was measured on different batches of purified enzymes, and the activity results are shown in the histogram in Figure 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.

[0132] In conclusion, this IMAC purification technique of beta-xylosidase allows to obtain this enzyme in a fast, simple and very efficient way, and without using a histidine tag. The affinity of beta-xylosidase for the IMAC type column is a discovery that could not be expected. The beta-xylosidase separated according to the invention was identified by mass spectrometry, and the specific activity thereof, evaluated with para-nitrophenyl-bD-Xylopyranoside (pNPX) as substrate, varies between at least 15 or 20 and 35 pmoles of p-nitrophenol.min-1 . mg -1of enzyme or more.

[0133] This enzyme has industrial interest to stimulate the degradation of xylan or xylose oligomers with different degrees of Polymerization (DP) such as xylobiose, xylotriose or with higher xylose DP.

[0134] It can be used alone, or combined with other enzymatic mixtures / cocktails depending on needs and applications.

Claims

Claims 1. 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 remainder of the enzyme mixture by affinity chromatography on immobilized metal ions IMAC.

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

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

4. Separation process according to 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 one of the preceding claims, characterized in that the affinity chromatography on immobilized metal ions IMAC uses: - a solid immobile 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, crosslinked dextran gel, silica.

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

8. Separation method according to one of claims 5 to 7, characterized in that the metal ions are chosen from: metal ions of transition metals, in particular chosen from divalent ions of Cu (II), Ni (II), Zn (II), Co (II), trivalent metal ions of metals, in particular chosen 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 one of the preceding claims, characterized in that the mixture of enzymes is derived from the production of enzymes by a microorganism, in particular by a filamentous fungus, for example of the genus Trichoderma, in particular the species Trichoderma 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. Separation method according to 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 having produced said mixture, said preliminary step aiming to separate the must from said liquid mixture of enzymes and comprising in particular one or several successive filtrations of the culture medium.

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

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

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

14. Separation method according to the preceding claim, characterized in that the separation comprises a step of mixing the immobile phase with the mixture of enzymes 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 therefrom.

15. Separation method according to one of the preceding claims, 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.

16. Beta-xylosidase enzymes (1) obtained by the separation process according to one of the preceding claims, characterized in that they have a specific activity of at least 10 pmoles of p-nitrophenol.min -1 . mg -1 of enzyme, in particular at least 20 or at least 30 pmoles of p-nitrophenol.min -1 . mg -1 of enzyme.

17. Beta-xylosidase enzymes (1) obtained by the separation process according to one of claims 1 to 15, characterized in that they have a purity greater than or equal to 90%, in particular greater than or equal to 95% or 97%.

18. Use of the beta-xylosidase enzymes (1) obtained according to the process according to one of claims 1 to 15 for enriching an enzymatic cocktail produced by a microorganism with beta-xylosidase enzymes.

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