Method for separating beta-xylosidase from enzyme mixture

Abstract

The present invention relates to a method for separating a beta-xylosidase from an enzyme mixture comprising a beta-xylosidase and other enzymes wherein the beta-xylosidase to be separated is free of histidine groups and said beta-xylosidase is separated from the remainder of the enzyme mixture by immobilized metal ion affinity chromatography (IMAC).

Description

TECHNICAL FIELD

[0001] 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 in the enzymatic hydrolysis of cellulosic or lignocellulosic materials. The sugars can be used as such / profitable utilized, or further converted into alcohols, in particular ethanol, by fermentation. PRIOR ART

[0002] Since the 1970s, the conversion of lignocellulosic materials into ethanol after hydrolysis of the constituent polysaccharides into fermentable sugars has been the subject of numerous studies. For example, reference can be made to the reference study of the National Renewable Energy Laboratory (Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol, Humbird et al., NREL / TP-5100-57764, May 2011).

[0003] Lignocellulosic materials are cellulosic materials, i.e., materials composed of cellulose, hemicellulose, and lignin, hemicellulose being a polysaccharide composed essentially of pentoses and hexoses, and lignin being a macromolecule with a complex structure and high molecular weight based on phenolic compounds. For the sake of brevity, they are grouped together in this text under the general term "biomass".

[0004] Wood, straw, and corn cobs are the most widely used lignocellulosic materials, but other resources, dedicated forestry crops, residues from alcohol-producing plants, sugar-producing plants, and cereal plants, products and residues from the paper industry, and products from the conversion of lignocellulosic materials are available. Most of them consist of approximately 35% to 50% cellulose, 20% to 30% hemicellulose, and 15% to 25% lignin.

[0005] The method of biochemically converting lignocellulosic materials into sugars and then optionally into alcohols of the ethanol type includes a physico-chemical pretreatment step, followed by an enzymatic hydrolysis step using an enzyme mixture. This can then be followed by an ethanol fermentation step of the released sugars, where ethanol fermentation and enzymatic hydrolysis can be carried out simultaneously, and then an ethanol purification step. An example of such a method for converting biomass into ethanol is described in patent EP 3484945, to which reference can be made for further details.

[0006] The enzyme mixture for hydrolysis is a mixture of cellulolytic enzymes (also known as cellulases) and / or hemicellulolytic enzymes. Cellulolytic enzymes have three main types of activity: endoglucanase, exoglucanase, and cellobiase, the latter also known as β-glucosidase. Hemicellulolytic enzymes particularly have xylanase activity.

[0007] The cellulolytic microorganism most commonly used in the industrial production of enzyme mixtures is the fungus Trichoderma reesei. The wild-type strain has the ability to secrete an enzyme mixture that is considered most suitable for cellulose hydrolysis in the presence of a carbon-based inducer substrate (such as cellulose). Other proteins with properties crucial for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, such as xylanase. The presence of a carbon-based inducer substrate is essential for the expression of cellulolytic enzymes and / or hemicellulolytic enzymes. The nature of the carbon substrate has a strong influence on the composition of the enzyme mixture. This is the case for xylose, which, when combined with a carbon-based inducer substrate such as cellulose or lactose, makes it possible to significantly improve the "xylanase" activity.

[0008] More specifically, in the context of the production of "second-generation" (2G) bioethanol from lignocellulosic biomass, one of the main challenges lies in the pretreatment of the biomass (such as treatment with acid or alkali solutions, followed by cooking or steam explosion), and then the degradation of cellulose and hemicellulose fibers through the action of cellulolytic enzymes and hemicellulolytic enzymes that 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 other oligomers) into glucose, which can be directly assimilated by yeast to produce bioethanol. The degradation of hemicellulose is carried out by xylanase or xylobiohydrolases and is capable of forming xylose oligomers (xylobiose, xylotriose, and other xylose oligomers). The action of β-xylosidase makes it possible to degrade these xylose oligomers to produce xylose. Xylanase is usually inhibited by xylobiose and short xylooligosaccharides, and the lack of β-xylosidase is the reason for the rate-limiting step in xylan hydrolysis.

[0009] Patent application WO 2011 / 079048 teaches that, in the method of simultaneous hydrolysis and fermentation of biomass (SSF, i.e., Simultaneous Saccharification and Fermentation), increasing the β-xylosidase activity of the enzyme mixture for enzymatic hydrolysis has a beneficial effect on the enzymatic hydrolysis of certain biomass, as it makes it possible to reduce the amount of enzymes required. It also makes it possible to hydrolyze alkyl xylosides.

[0010] Thus, it is advantageous to separate the β-xylosidase present in an enzyme mixture produced by microorganisms, for example to enrich a given enzyme mixture in β-xylosidase. For this purpose, various techniques have been proposed, in particular first separating the fungus from the enzymes it produces in a culture medium. 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 filter under vacuum. The enzymes are then separated by passing them through a column using cotton and by elution with an alkaline solution.

[0011] Patent WO 2018 / 015228 proposes separating the enzymes from the fungus by a series of culture medium treatment steps, including a step of filtering the culture medium via a filter press, followed by a tangential microfiltration step of the resulting liquid phase.

[0012] It is also known to separate β-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 advantageous technique, but it is not without drawbacks, since it requires the use of solvents and many steps, making it expensive and complex to implement.

[0013] An object of the present invention is thus to develop an improved technique for separating enzymes from an enzyme mixture, and more particularly a technique for separating β-xylosidase from a mixture containing β-xylosidase and other types of enzymes. It more particularly relates to a separation technique that is very efficient and can be deployed on an industrial scale. Summary of the Invention

[0015] The first subject of the present invention is a method for separating β-xylosidase from an enzyme mixture containing β-xylosidase and other enzymes, wherein:

[0016] - the β-xylosidase to be separated does not contain histidine groups,

[0017] - and the β-xylosidase is separated from the rest of the enzyme mixture by immobilized metal ion affinity chromatography (hereinafter also denoted by its acronym IMAC, i.e., Immobilized Metal Affinity Chromatography).

[0018] IMAC chromatography is known for separating proteins with histidine groups exposed on their surface, whether naturally exposed or due to genetic modification; in the latter case, the histidine is referred to as a "tag" or "cluster" added to the protein. Reference can also be made to the publication by V. Gaberc-Porekar et al., "Perspectives of immobilized-metal affinity chromatography", J Biochem. Biophys. Methods., October 30, 2001; 49(1-3) 335-60.

[0019] Now, quite unexpectedly, it has been found in the present invention that this chromatographic technique is still capable of separating enzymes that do not contain histidine groups, and very particularly the β-xylosidase that the inventors have attempted to isolate in a mixture of enzymes produced by microorganisms.

[0020] This is very advantageous in many respects:

[0021] - The β-xylosidase can be separated by this technique without having to pre-modify them to have these histidine tags or clusters. By avoiding modifying them, of course, the way of obtaining / separating them is simplified by eliminating the genetic modification step. But it also limits any risk of performance loss due to changes in their behavior / their activity caused by the presence of these histidine groups (many cases of enzymes whose activity has changed after adding histidine tags have been described in the literature, especially in the case of metalloenzymes and multimeric enzymes).

[0022] - The technique of separation by IMAC chromatography is very efficient: it can be deployed on an industrial scale, the materials required for this type of chromatography are stable and thus can be stored without the risk of degradation, the elution conditions are generally not drastic, and the reactants used are usually reusable, which makes it economically advantageous, and its results in terms of the selectivity of the separated enzyme, in this case especially β-xylosidase, are excellent.

[0023] - The separation can be carried out in a single step, resulting in a method that is easier and faster to implement.

[0024] Generally, the other enzymes in the mixture may include at least one enzyme selected from cellulase, hemicellulase, and / or an enzyme selected from hemicellulase.

[0025] Generally, the other enzymes in the mixture may include β-glucosidase, endoglucanase, and possibly cellobiohydrolase.

[0026] The β-xylosidase can constitute at least 1% by weight, in particular from 2% to 15% by weight, or from 3% to 8% by weight, of all the enzymes present in the mixture. This is the content typically encountered in enzyme mixtures produced by Trichoderma reesei, but of course, the present invention is equally applicable to enzyme mixtures containing a higher proportion of β-xylosidase.

[0027] Preferably, immobilized metal ion affinity chromatography (IMAC) uses:

[0028] - A solid stationary phase comprising a matrix on which metal ions are immobilized via a chelating agent,

[0029] - And a liquid mobile phase called an eluent.

[0030] The matrix of the stationary phase can advantageously be selected from at least one of the following compounds: agarose gel, cross-linked dextran gel, silica.

[0031] The chelating agent can advantageously be selected from at least one of the following compounds: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris[carboxymethyl]ethylenediamine (TED).

[0032] The metal ions can advantageously be selected from: metal ions of transition metals, in particular selected from:

[0033] - Divalent ions Cu(II), Ni(II), Zn(II), Co(II),

[0034] - Trivalent metal ions, in particular selected from the trivalent ions Fe(III), Al(III), Ga(III),

[0035] - Or tetravalent metal ions, in particular the metal ion Zr(IV).

[0036] According to a preferred embodiment of the present invention, the enzyme mixture is obtained by producing enzymes through a microorganism, in particular through a filamentous fungus, such as a fungus of the genus Trichoderma, in particular the species Trichoderma reesei.

[0037] The separation method according to the present invention may include a preliminary step of separating a culture medium comprising the enzyme mixture and the microorganism producing the mixture, the preliminary step being intended to separate the microorganism from the enzyme mixture, particularly including filtration of the culture medium or several successive filtrations. This preliminary separation can be carried out, for example, as described in patent WO 2018 / 015228. Thus, in the solid / liquid separation, first, the microorganism producing the enzyme in solid / semi-solid form (also called must) is obtained, and second, the soluble enzyme in the (aqueous) liquid phase is obtained. This liquid phase can be optionally concentrated and then can be processed according to the present invention.

[0038] The separation method according to the invention may further comprise the step of treating the mash which may have been separated from the rest of the culture medium or may not have been separated from the rest of the culture medium, said treatment comprising cooling the mash and then separating the mash and an "extra" liquid phase containing an additional amount of the enzyme mixture, as taught in patent EP 3174979.

[0039] If this additional separation is carried out on the already separated mash, the liquid phase obtained after the above solid / liquid separation can subsequently be mixed with this extra liquid phase, and the method according to the invention can be carried out on the mixture of these two liquid phases.

[0040] Of course, the method according to the invention can be carried out on the liquid phase containing the enzyme mixture which has been pre-concentrated beforehand.

[0041] According to a first variant of the separation method according to the invention, chromatography is carried out continuously in a chromatographic column containing a solid stationary phase through which a liquid mobile phase called an eluent can pass continuously.

[0042] According to another variant, the chromatographic separation according to the invention is carried out batchwise - by bringing the stationary chromatographic phase into contact with a mixture containing β-xylosidase and other enzymes in a liquid medium for a given time to form a reaction medium in a container, and then by eluting the solid part of the reaction medium to extract β-xylosidase therefrom.

[0043] In this variant, the separation may include the steps of mixing the stationary phase with the dissolved enzyme mixture, then an optional decantation step, then the step of separating the solid phase from the reaction medium, then an optional washing step, and then the step of eluting the separated solid phase to extract β-xylosidase therefrom.

[0044] The chromatographic separation according to the invention preferably immobilizes β-xylosidase on the stationary phase at a pH between 6.5 and 9, and preferably elutes β-xylosidase by changing the nature, composition or concentration of the eluent (which makes it possible in particular to change the pH of the stationary phase).

[0045] One subject of the invention also lies in β-xylosidase, which is particularly produced by fungi such as Aspergillus or Trichoderma, and is particularly obtained by the separation method as described above, and which has a specific activity of at least 10 μmol p-nitrophenol·min -1 ·mg -1 enzyme, in particular at least 20 or at least 30 or at least 35 μmol p-nitrophenol·min -1 ·mg -1 enzyme. This high specific activity resulting from the high purity of the β-xylosidase separated therefrom demonstrates an efficient separation.

[0046] Methods for measuring specific activity known to those skilled in the art consist in placing the purified enzyme in the presence of PNP-xylose (p-nitrophenyl-β-D-xylopyranoside). Under the action of β-xylosidase, the released PNP is monitored spectroscopically and the specific activity is calculated using a PNP standard range.

[0047] An example of the β-xylosidase according to the invention is the xylan 1,4-β-xylosidase protein obtained from Trichoderma reesei with the reference number XP_006964075.1 in NCBI (the acronym for the National Center for Biotechnology Information in the United States), and is described under the reference number Q92458_HYPJE in the Uniprot database (E.C. 3.2.1.37; taxonomic identifier 51453 NCBI; sequence version 2 of 01 / 06 / 1998, Gene: bxl1, -organism: Hypocrea jecorina (Trichoderma reesei)).

[0048] It also relates to all β-xylosidases having at least 50% sequence identity with such β-xylosidase, in particular having 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% sequence identity with such β-xylosidase.

[0049] The invention more generally relates to any β-xylosidase that can be obtained in particular with fungi of the genus Trichoderma, in particular the species Trichoderma reesei or Trichoderma citrinoviride or Trichoderma orientale or Trichoderma longibrachiatum or Trichoderma arundinaceum, or with fungi of the genus Aspergillus, in particular the species Aspergillus niger, Aspergillus japonicus, Aspergillus oryzae, Aspergillus clavatus, Aspergillus aculeatus, Aspergillus awamori or Aspergillus flavus.

[0050] A subject of the invention also lies in the β-xylosidase obtained in particular by the above separation method, which has a purity greater than or equal to 90%, usually greater than or equal to 95% or 97%.

[0051] In a known manner, the purity is evaluated by electrophoresis on an SDS-PAGE gel (a polyacrylamide gel containing sodium dodecyl sulfate), and then analyzed using the Image-Lab software available from Bio-Rad.

[0052] High-purity enzymes are thus obtained, making it possible to use them profitably. This result is all the more remarkable since it is possible to carry out the separation according to the invention on enzyme mixtures which may contain several tens or even approximately a hundred different enzymes, such as those produced by microorganisms such as Trichoderma.

[0053] Another subject of the invention lies in the use of β-xylosidases obtained in particular according to the above-described method for enriching enzyme mixtures produced by microorganisms with β-xylosidase.

[0054] It is thus possible to add them in a controlled manner to processes for converting different types of lignocellulosic biomass into sugars (saccharification, including enzymatic hydrolysis of biomass) or into alcohols (saccharification and fermentation), which have various recalcitrances to sugars or alcohols (type of ethanol).

[0055] Another use lies in the fact that these β-xylosidases can be used profitably as such in applications which particularly require β-xylosidase activity.

[0056] β-xylosidases can be purified and sold in pure form for biotechnological applications, whether for degradation or for the production of xylo-oligosaccharides.

[0057] β-xylosidases can be added to enzyme mixtures poor in β-xylosidase for industrial applications in the field of degradation of lignocellulosic biomass in order to produce sugars which can be used profitably in biological products, or for the production of pure, especially including bioethanol.

[0058] List of figures

[0059] Figure 1 Figure representing an FPLC profile (acronym for Fast Protein Liquid Chromatography, a known technique for fast chromatography of proteins in the liquid phase) for the separation of β-xylosidase from an enzyme mixture according to an exemplary embodiment of the invention.

[0060] Figure 2 Figure representing the results of SDS-PAGE gel electrophoresis of β-xylosidase after (hereinafter) FPLC purification.

[0061] Figure 3 Graph showing in the form of a histogram the activities of β-xylosidases separated according to two exemplary embodiments of the invention, where Examples 1 and 2 are marked on the x-axis, in μmol p-nitrophenol·min -1 ·mg -1 and their activities expressed in enzyme are on the y-axis.

[0062] Description of the embodiments

[0063] The present invention will be described in detail below with reference to the accompanying drawings and examples, which are given by way of example and are in no way limiting.

[0064] The present invention provides a method for separating a specific enzyme: β-xylosidase from an enzyme mixture.

[0065] The present invention more particularly relates to the separation of this enzyme from an enzyme mixture produced by microorganisms, more particularly by fungi of the genus Trichoderma, especially Trichoderma reesei, which is the subject of the following examples and detailed description.

[0066] However, the present invention is similarly applicable to the separation of this enzyme from any enzyme mixture containing this enzyme, especially any enzyme mixture produced by microorganisms containing different proportions of this enzyme.

[0067] The method according to the present invention makes it possible to simply and rapidly purify β-xylosidase from Trichoderma reesei, regardless of the type of Trichoderma reesei strain used.

[0068] The method is a method for purifying the enzyme of interest (β-xylosidase) from a complex mixture of enzymes (about 100 enzymes) in a single step. This requires the prior production of the enzyme and the separation of the mycelium.

[0069] The following description details a variant of the present invention using a continuously operating chromatographic column. However, the present invention can also be carried out batchwise in a similar manner without a column.

[0070] Prior steps

[0071] To implement the separation method according to the present invention, a preliminary separation of the medium containing the enzyme mixture and the fungus Trichoderma reesei is first carried out. For this purpose, within less than 24 hours after the production is stopped, the medium is separated on a filter press lined with a fabric having a porosity of 3 - 20 μm to obtain a filtrate with a corrected optical density OD 600nm of less than 2.5. The resulting liquid phase is subjected to tangential microfiltration on a ceramic membrane with a cut-off threshold between 0.5 and 1.4 μm so that the corrected optical density OD 600nm 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.

[0072] At the end of the separation on the filter press, a solid residue ("filter cake") of 5 - 10% by weight and a filtrate of 90 - 95% by weight are generally obtained. Advantageously, the microfiltration of the filtrate obtained at the end of the filter press is carried out within at most 30 hours, preferably at most 24 hours.

[0073] Preferably, the tangential microfiltration is carried out on a ceramic membrane with a cut-off threshold between 0.8 and 1.4 μm.

[0074] The liquid phase obtained after microfiltration can be subjected to ultrafiltration, preferably on a ceramic membrane, more preferably on a ceramic membrane with a retention threshold between 5 and 15 kDa.

[0075] The filtration method described herein adopts the teachings of patent WO 2018 / 015228, and further details can be obtained by referring to it.

[0076] Then the obtained retentate is passed through an IMAC affinity column capable of separating enzymes with a polyhistidine tag (also known as "His-tag"). This is an amino acid motif consisting of at least six histidine residues in a protein, usually inserted at the N- or C-terminus of the protein. It is sometimes denoted by the name "hexahistidine tag" or "6xHis-tag".

[0077] However, it is clearly pointed out that the β-xylosidase purified from this cocktail does not contain a histidine tag (nor does any other enzyme in the enzyme mixture here).

[0078] Purification on an IMAC column

[0079] The enzyme-containing supernatant (the permeate from microfiltration or the retentate from ultrafiltration), namely the cellulase produced by Trichoderma reesei, is stored between 4 °C and 30 °C, but preferably below 10 °C.

[0080] The obtained supernatant is loaded onto an immobilized metal ion affinity chromatography column (also known as IMAC, the acronym for Immobilized Metal Affinity Chromatography). This type of affinity chromatography is based on the chelation mechanism of immobilized metal cations. This generally enables the purification of proteins with a histidine tag from the supernatant of a complex mixture containing proteins from various biological sources.

[0081] The chelation of (usually divalent) metal ions is a process capable of forming a complex between the metal cation and the ligand immobilized on the solid phase. Due to this chelation, the metal ions remain fixed in the column into which the enzyme mixture to be fractionated or purified is poured. The bond between the metal ion and the ligand is usually formed in the pH range between 7 and 8. To maintain this pH, the column is pre-equilibrated with a buffer solution.

[0082] The solution in which the sample can be solvated ideally has a high ionic strength to reduce non-specific electrostatic interactions, but these ions themselves do not need to bind to the metal. This solution is also preferably neutral or slightly alkaline because the interaction between the histidine group and the metal is inactivated in the presence of protons occupying the binding site on the amino acid. An example of this type of solution is 50 mM Tris-acetate (tris(hydroxymethyl)aminomethane acetate (CH3COO- )) or 20 to 50 mM sodium phosphate. Tris-HCl (tris(hydroxymethyl)aminomethane HCl) makes it possible to purify enzymes that have a fairly strong interaction between the protein and the metal.

[0083] For the eluent, it is possible to select an acidic solution with a pH gradient from 7 to 4 to protonate the amino acids interacting with the IMAC matrix, which causes a sharp decrease in the affinity of the enzyme for the residues. Alternatively, it is also possible to use an imidazole solution to displace the protein at the binding site (to exchange the ligand). Finally, it is also possible to extract metal ions with a strong chelating agent such as ethylenediaminetetraacetic acid EDTA (usually used for column regeneration).

[0084] Production steps

[0085] The enzyme mixture to which the separation method according to the invention is applied is produced by Trichoderma reesei by aerated fermentation in a conventional production line. Examples of methods for producing enzyme mixtures with the aid of this fungus are described in patents FR 3024463, FR 3049957, FR 3085961 and FR 3088934. An improvement in the method for increasing the content of β-glucosidase and / or β-xylosidase by cooling the mash obtained at the end of production is described in patent EP 3174979.

[0086] The method for producing the enzyme mixture begins with a propagation phase, usually carried out in small reactors of increasing size, with the aim of multiplying the filamentous fungus and limiting the duration of the lag phase and the risk of contamination.

[0087] When the production is considered sufficient (fungus concentration greater than or equal to 10 g / l, preferably greater than or equal to 15 g / l), the culture medium is transferred to a large-volume final reactor.

[0088] The enzyme production method comprises two phases, described in detail according to a preferred embodiment:

[0089] - a growth phase a) of the microorganism in an aerated closed reactor in the presence of at least one carbon-based growth substrate, the growth phase being carried out at a carbon-based growth substrate concentration of 10 to 90 g / l,

[0090] - a phase b) of producing the enzyme mixture, in which at least one carbon-based inducer substrate is introduced, the carbon-based inducer substrate being selected from lactose, cellobiose, sophorose, the residue obtained after ethanol fermentation of the monomeric sugars of the enzymatic hydrolysis product of cellulose biomass and / or the crude extract of water-soluble pentoses derived from the pretreatment of cellulose biomass, the production phase being carried out at a carbon-based production substrate concentration of 150 to 400 g / l.

[0091] The microorganism used in the process for producing the enzyme mixture according to the invention is a fungal strain belonging to the Trichoderma reesei species.

[0092] The most efficient industrial strains are strains belonging to the Trichoderma reesei species that have been modified by mutation-selection methods to improve the enzyme mixture.

[0093] Strains improved by genetic recombination techniques can also be used. These strains are cultured in a stirred and aerated reactor under conditions compatible with their growth and the production of enzymes.

[0094] As an example of the strains and the methods for obtaining them, it can be reiterated that conventional genetic mutation techniques have made it possible to select Trichoderma reesei strains with high cellulase productivity, such as strain MCG77 (Gallo – US Patent 4275167), MCG 80 (Allen, A.L. and Andreotti, R.E., Biotechnol.-Bioeng. 1982, 12, 451-459, 1982), RUT C30 (Montenecourt, B.S. and Eveleigh, D.E., Appl. Environ. Microbiol. 1977, 34, 777-782) and CL847 (Durand et al., 1984, Proc. Colloque SFM "Génétique des microorganismes industriels" [Genetics of industrial microorganisms]. Paris. H. Heslot Ed., pp. 39-50). These improvements have made it possible to obtain high-yield strains that are less sensitive to catabolic repression by monomeric sugars, especially glucose for example, compared to wild-type strains.

[0095] Recombinant strains have also been obtained from strains of Trichoderma reesei, such as Qm9414, RutC30, CL847, by cloning heterologous genes, such as invertase from Aspergillus niger, to enable Trichoderma reesei to use sucrose as a carbon source. These strains maintain their high productivity and their ability to be cultured in a fermenter.

[0096] The carbon-based growth substrate for the microorganism used in the growth stage a) of the process according to the invention is advantageously selected from industrial soluble sugars, preferably selected from glucose, lactose, xylose, the liquid residue obtained after ethanol fermentation of the monomeric sugars of the enzymatic hydrolysate of lignocellulosic material, and the extract of the hemicellulose fraction in monomeric form derived from a pretreated lignocellulosic substrate, used alone or as a mixture.

[0097] Depending on its nature, the carbon-based growth substrate is introduced into the closed reactor before sterilization, or sterilized separately and introduced into the closed reactor after the closed reactor is sterilized.

[0098] The carbon-based growth substrate is used in the growth stage a) at an initial concentration usually between 20 and 90 g carbon substrate / liter of reaction volume.

[0099] Preferably, the growth stage a) is carried out for a time of 30 to 70 hours, preferably 30 to 40 hours.

[0100] Preferably, the growth stage a) is carried out at a pH of 4.8 and a temperature of 20 - 30°C, usually 22 - 27°C, preferably approximately 27°C.

[0101] The carbon-based inducer substrate used in the production stage b) is advantageously fed in a fed-batch phase mode at a limiting flow rate of 30 to 80 mg / g cell / hour. The temperature is usually the same as in step a).

[0102] At the end of the enzyme production step, a medium containing a solid concentration between 10 and 45 g / l (corresponding to dry fungi) is usually obtained; the enzyme is completely soluble in water. The pellet measured after centrifugation (4000 rpm, 5 minutes) is greater than 15%, usually approximately 30%, and even up to 60%. This corresponds to the percentage of the volume occupied by the solid relative to the total volume of the sample.

[0103] The object of the present invention is to isolate fungal enzymes and then purify β-xylosidase in order to sell it or specifically add it to a mixture of enzymes (if they are limiting) in biochemical processes involving enzymes.

[0104] The present invention is also applicable to any enzyme mixture produced by microorganisms, called an enzyme cocktail, including enzyme cocktails obtained from a reaction medium containing microorganisms that have been specifically treated by cooling as described in patent EP3174979.

[0105] The solid / liquid separation step where the fungus is separated from the liquid

[0106] The liquid contains enzymes and residual salts.

[0107] - Once the enzyme has been separated from the mycelium, the pH of the supernatant (containing the enzyme) should be adjusted to a pH range of 6.5 - 9, preferably to pH 8 (by desalting column, by filtration, by ultrafiltration, by buffer change under pressure) (a stirred cell available from Merck under the name Amicon, or an ultrafiltration cell with an Ultracel 10kD membrane also available from Merck under the name Pellicon)

[0108] - Once the cellulase mixture has been buffered, use the IMAC column described above.

[0109] It should be noted that the enzyme mixture tested had the following type of composition (the contents shown are expressed as abundances and are approximate data, but they give the idea of the enzyme distribution in the mixture):

[0110] - Content of CEL7A = CBH1: approximately 35%,

[0111] - Content of CEL6A = CBH2: approximately 30%,

[0112] - Content of BGL1 = β - glucosidase 1: approximately 5%,

[0113] - Content of endoglucanase I = Cel7B: approximately 10%,

[0114] - Content of endoglucanase II = Cel5A: approximately 10%,

[0115] - Content of others (enzymes and / or other compounds): approximately 10%.

[0116] For examples of the analysis of the secretomes of the modified Trichoderma reesei strains RUT - C30 and CL847, reference can be made to the following publication: "Comparative secretome analyses of two Trichoderma reesei RUT - C30 and CL847 hypersecretory strains", by I. et al., Biotechnology for biofuels, article number 18 (2008), published on December 23, 2008, especially Table I therein.

[0117] Pre - load nickel ions Ni 2+The HisTrap crude column (Cytiva, 5 mL) is equilibrated to a pH between 6.5 and 9, preferably equilibrated to pH 8. This type of column can be obtained from Cytiva under the full name "HisTrap FF crude histidine-tagged protein purification column".

[0118] The equilibration buffer can be Tris, Bis-Tris, phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also known as HEPES), or any other buffer solution within the pH range of 6.5 to 9. The buffer solution may contain salts (NaCl, KCl) between 0 - 500 mM, but preferably 50 mM.

[0119] - Once the column has been equilibrated, the clarified supernatant can be filtered and then loaded onto the column via a peristaltic pump, via a "Fast Protein Liquid Chromatography" system which typically includes a pump, a UV detector, a conductivity measuring device, a fraction collector, and a valve that enables passage from one column to another. Such a system is particularly available from Bio-Rad. Another chromatography system can also be purchased from Cytiva under the name "AKTA pure protein purification system".

[0120] Gravity separation techniques can also be used.

[0121] The column is washed in the presence of 0 to 40 mM imidazole, usually using 20 mM imidazole.

[0122] Surprisingly, and never observed before, β-xylosidase from Trichoderma reesei is immobilized onto the solid IMAC phase and can be easily purified on this type of resin, although it does not have any histidine tag.

[0123] To elute β-xylosidase from Trichoderma reesei, a gradient or elution must be carried out in the presence of 500 mM imidazole, or the pH must be lowered to reduce the affinity of the enzyme for the solid phase.

[0124] β-Xylosidase from Trichoderma reesei purified under these conditions has high purity (verified by mass spectrometry) and is active (activity assays show that the enzyme is active against 4-nitrophenyl β-D-xylopyranoside (4-NPX) and releases p-nitrophenol pNP).

[0125] The specific activity of β-xylosidase is evaluated using 4-nitrophenyl β-D-xylopyranoside (4-NPX) as a substrate, and its activity at 50 °C is between 10 and 100 μmol p-nitrophenol · min -1 · mg enzyme -1between, usually at least 20 to about 35 μmol p-nitrophenol·min -1 ·mg enzyme -1 (or higher). Use the same protocol as that used for measuring β-glucosidase activity. Only change the substrate (p-nitrophenyl β-D-xylopyranoside (pNP)), and use β-xylosidase. The principle of measuring specific activity with this type of reagent is well known in the literature. Examples

[0126] Example 1

[0127] The enzyme mixture produced by Trichoderma reesei according to the above procedure was experimented on in the laboratory using strain CL847, which has been cited and also described in the publication "A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains" by Jourdier E. et al. (Microb. Cell Fact. May 30, 2012; 11:70. doi: 10.1186 / 1475-2859-11-70. PMID: 22646695; PMCID: PMC3434075.).

[0128] Example 2

[0129] The enzyme mixture produced by Trichoderma reesei according to the above procedure was experimented on in the laboratory using the strain described under reference number 130G9 in Table 1 of patent EP3174979 (SEQ ID no: 7 as nucleic acid, SEQ ID no: 8 as polypeptide).

[0130] The following description relates to the treatment of the culture media obtained in each of these 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 Tris-Cl 50 mM pH 8, NaCl 50 mM.

[0131] This step has the dual advantage of removing low molecular weight molecules present in the medium and bringing the pH to 8. The protein extract was then centrifuged at 5000 rpm for 10 minutes and filtered at 0.2 μm using a syringe (PES filter, VWR, 514-2073).

[0132] The protein solution was then loaded onto the HisTrap column (Cytiva, HisTrap TM FF and HisTrap Crude, 5 mL) specified above, which has a stationary phase based on cross-linked agarose coupled to nickel ions via chelating groups.

[0133] The column was pre-equilibrated with 7 column volumes of buffer A Tris-Cl 50 mM pH 8, NaCl 50 mM. A linear gradient of 10 column volumes was then applied towards a solution of buffer B Tris-Cl 50 mM pH 8, NaCl 50 mM, imidazole 500 mM. The protein was eluted in a homogeneous and symmetric peak. It was then concentrated and washed three times in buffer A by centrifugation at 5000 rpm using an ultrafiltration unit that can be purchased under the name Vivaspin (10 kDa) from Sartorius to remove imidazole. The pure protein was then analyzed on an SDS-PAGE gel and by mass spectrometry. These analyses made it possible to clearly demonstrate that the purified protein / separated from the rest of the enzymes in the starting mixture was β-xylosidase. It should be noted that, on the other hand, the CBH2 with a histidine tag was not separated, which is doubly surprising.

[0134] Figure 1 FPLC (Fast Protein Liquid Chromatography) chromatogram representative of the purification of β-xylosidase according to Example 1, which is related to a polyacrylamide electrophoresis gel containing sodium dodecyl sulfate (SDS PAGE, Bio-Rad, Mini-PROTEAN TGX Stain-Free Precast Gel 10%-456-8035). In this figure, band 1 can be seen, corresponding to the enzyme purified by this chromatographic technique, which has been excised from the gel and analyzed by mass spectrometry. The protein identified by mass spectrometry corresponds to protein XP_006964075.1 in NCBI and has been mentioned above, with the UniProtKB reference number: Q92458_HYPJE. The same type of results were obtained with Example 2.

[0135] Figure 2It is an image of the SDS-PAGE gel of Example 1, which makes it possible to separate proteins according to their molecular weights after the proteins have been denatured. To indicate the molecular weight, the left-hand lane has markers of various 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 lane, the purified β-xylosidase is loaded and then analyzed. The results of the SDS-PAGE gel electrophoresis of β-xylosidase show that the molecular weight 1 corresponds to the molecular weight predicted by the DNA sequence, i.e., between 75 kDa and 100 kDa. In addition, the protein was found to be pure. The same type of results was obtained with Example 2.

[0136] After various purifications, the activity of different batches of the purified enzyme was measured, and the activity results are shown in the Figure 3 bar graph. The graph shows that the β-xylosidase purified by Examples 1 and 2 has a specific activity of approximately 35 to 38 μmol p-nitrophenol·min -1 ·mg -1 of enzyme.

[0137] In summary, this IMAC purification technique for β-xylosidase makes it possible to obtain this enzyme quickly, simply, and very efficiently without using a histidine tag. The affinity of β-xylosidase for the IMAC column was an unexpected finding. The β-xylosidase isolated according to the present invention was identified by mass spectrometry, and its specific activity evaluated with p-nitrophenyl β-D-xylopyranoside (pNP) as a substrate is at least 15 or 20 to 35 μmol p-nitrophenol·min -1 ·mg of enzyme or more.

[0138] This enzyme has industrial significance for stimulating the degradation of xylan or xylose oligomers with various degrees of polymerization (DP), such as cellobiose, xylotriose, or higher xylose DP.

[0139] Depending on the needs and applications, it can be profitably utilized alone or in combination with other enzyme cocktails / mixtures.

Claims

1. A method for separating β-xylosidase (1) from an enzyme mixture containing β-xylosidase and other enzymes, characterized in that The β-xylosidase to be separated contains no histidine groups and is separated from the rest of the enzyme mixture by immobilized metal ion affinity chromatography (IMAC).

2. The separation method according to the preceding claim, characterized in that The other enzymes in the mixture include at least one enzyme selected from cellulases and / or at least one enzyme selected from hemicellulases.

3. The separation method according to any one of the preceding claims, characterized in that The other enzymes in the mixture include β-glucosidase, endoglucanase, hemicellulase and possibly cellobiohydrolase.

4. The separation method according to one of the preceding claims, characterized in that The β-xylosidase (1) constitutes at least 1% by weight, particularly 2% to 15% by weight, or 3% to 8% by weight of all the enzymes present in the mixture.

5. The separation method according to one of the preceding claims, characterized in that The immobilized metal ion affinity chromatography (IMAC) uses: - a solid stationary phase comprising a matrix on which metal ions are immobilized by a chelating agent, - and a liquid mobile phase called an eluent.

6. The separation method according to the previous claim, characterized in that The matrix of the stationary phase is selected from at least one of the following compounds: agarose gel, Sephadex gel, silica.

7. The separation method according to any one of claims 5 and 6, characterized in that The chelating agent is selected from at least one of the following compounds: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris[carboxymethyl]ethylenediamine (TED).

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

9. The separation method according to one of the preceding claims, characterized in that The enzyme mixture is obtained by producing enzymes through microorganisms, particularly through filamentous fungi, such as fungi of the genus Trichoderma, particularly Trichoderma reesei or Trichoderma citrinoviride or Trichoderma orientale or Trichoderma longibrachiatum or Trichoderma arundinaceum species, or fungi of the genus Aspergillus, particularly Aspergillus niger, Aspergillus japonicus, Aspergillus oryzae, Aspergillus clavatus, Aspergillus aculeatus, Aspergillus awamori or Aspergillus flavus species.

10. The separation method according to one of the preceding claims, characterized in that It includes a preliminary step of separating the medium, which comprises an enzyme mixture and the microorganisms called mash that produce the mixture, and this preliminary step aims to separate the mash from the liquid enzyme mixture, particularly including filtration of the medium or several consecutive filtrations.

11. The separation method according to the previous claim, characterized in that It also includes a step of treating the mash that may have been separated from the rest of the medium or may not have been separated from the rest of the medium, and this treatment includes cooling the mash and then separating the mash and the liquid containing an additional amount of the enzyme mixture.

12. The separation method according to one of the preceding claims, characterized in that Chromatography is continuously carried out in a chromatographic column with a solid stationary phase through which a liquid mobile phase called an eluent can continuously pass.

13. The separation method according to any one of claims 1 to 11, characterized in that The chromatographic separation is carried out batchwise - by contacting the immobilized chromatographic phase with a mixture containing β-xylosidase and other enzymes in a liquid medium for a given time to form a reaction medium in a container, and then by eluting the solid part of the reaction medium to extract β-xylosidase therefrom.

14. The separation method according to the preceding claim, characterized in that Said separation comprises the steps of mixing the stationary phase with the dissolved enzyme mixture, then an optional decantation step, then the step of separating the solid phase from the reaction medium, then an optional washing step, and then the step of eluting the separated solid phase to extract β-xylosidase therefrom.

15. The separation method according to one of the preceding claims, characterized in that Said chromatographic separation immobilizes β-xylosidase (1) on the stationary phase at a pH between 6.5 and 9, and elutes β-xylosidase (1) by changing the nature, composition or concentration of the eluent. β-xylosidase (1) obtained by the separation method according to one of the preceding claims, characterized in that They have a specific activity of at least 10 μmol p-nitrophenol·min -1 ·mg -1 enzyme, especially at least 20 or at least 30 μmol p-nitrophenol·min -1 ·mg -1 enzyme.

17. A β-xylosidase (1) obtained by the separation method according to any one of claims 1 to 15, characterized in that They have a purity of greater than or equal to 90%, in particular greater than or equal to 95% or 97%.

18. Use of β-xylosidase (1) obtained by a method for enriching β-xylosidase from an enzyme mixture produced by microorganisms according to one of claims 1 to 15.

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