Method for separating β-xylosidase enzyme from an enzyme mixture

Abstract

The present invention relates to a method for separating a beta-xylosidase enzyme from a mixture of enzymes comprising the beta-xylosidase enzyme and other enzymes, wherein the beta-xylosidase enzyme to be separated does not have a histidine moiety, and wherein the beta-xylosidase enzyme is separated from the remainder of the enzyme mixture by immobilized metal affinity chromatography (IMAC).

Description

[Technical Field]

[0001] The present invention relates to the production of enzymes of cellulolytic and / or hemicellulolytic type, in particular involving the enzymatic hydrolysis of cellulosic or lignocellulosic materials to produce sugars from these materials, which can be used / profited as such or can be followed by their conversion to alcohol, in particular ethanol, by fermentation. [Background technology]

[0002] Since the 1970s, the conversion of lignocellulosic materials into ethanol, following hydrolysis of the constituent polysaccharides into fermentable sugars, has been the subject of numerous studies, reference may be made, for example, to benchmark studies by the National Renewable Energy Laboratory (NRL, 2003).

[0003] Lignocellulosic materials are cellulosic materials, i.e. materials consisting of cellulose, hemicellulose and also lignin, hemicellulose being a polysaccharide essentially consisting of pentoses and hexoses, and lignin being a macromolecule of complex structure and high molecular weight, based on phenolic compounds, which for the sake of brevity will be grouped together in this text under the generic name "biomass".

[0004] Wood, straw, and corncobs are the most commonly used lignocellulosic materials, but other sources, dedicated forestry harvests, residues from alcohol-producing, sugar-producing, and cereal plants, products and residues from the paper industry, and products from the conversion of lignocellulosic materials can be used. They are composed, for the most part, of about 35%-50% cellulose, 20%-30% hemicellulose, and 15%-25% lignin.

[0005] A method for the biochemical conversion of lignocellulosic material into sugars and then, optionally, into alcohols of the ethanol type, comprises a physicochemical pretreatment step followed by a step of enzymatic hydrolysis using an enzyme cocktail. This may be followed by a step of ethanol fermentation of the released sugars, which may be carried out simultaneously, followed by a step of ethanol purification. One example of such a method for converting biomass to ethanol is described in U.S. Pat. No. 5,623,799, the reference of which can be made for further details.

[0006] The enzyme cocktail used for hydrolysis is a mixture of cellulolytic enzymes (also known as cellulases) and / or hemicellulolytic enzymes. Cellulolytic enzymes have three main types of activity: endoglucanases, exoglucanases, and cellobiases, also known as β-glucosidases. Hemicellulolytic enzymes, in particular, have xylanase activity.

[0007] The most commonly used cellulolytic microorganism for industrial enzyme cocktail production is the fungus Trichoderma reesei. Wild-type strains are capable of secreting enzyme cocktails that are thought to be best suited for cellulose hydrolysis in the presence of a carbon-based inducer substrate, such as cellulose. Other proteins with essential properties for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, such as xylanases. The presence of a carbon-based inducer substrate is essential for the expression of cellulolytic and / or hemicellulolytic enzymes. The nature of the carbon-based substrate has a strong influence on the composition of the enzyme cocktail. This is particularly true for xylose, which allows for significant improvements in xylanase activity when combined with a carbon-based inducer substrate, such as cellulose or lactose.

[0008] More specifically, in the context of "second-generation" (2G) bioethanol production from lignocellulosic biomass, one of the major challenges is the decomposition of cellulose and hemicellulose fibers by pretreatment of the biomass (e.g., treatment with acidic or basic liquor followed by cooking or steam explosion) and then the action of cellulolytic and hemicellulolytic enzymes that depolymerize the fibers. Cellobiohydrolases (CBH1 and CBH2) allow the production of sugar oligomers, such as cellobiose, cellotriose, and other oligomers of glucose derived from cellulose. β-Glucosidase allows the decomposition of cellobiose (and other oligomers) into glucose, which can be directly assimilated by yeast for bioethanol production. Hemicellulose degradation is carried out by xylanases or xylobiohydrolases, allowing the formation of xylose oligomers (xylobiose, xylotriose, and other oligomers of xylose). The action of β-xylosidase allows the degradation of these xylose oligomers to yield xylose. Xylanases are generally inhibited by xylobiose and short-chain xylooligosaccharides, and the absence of β-xylosidase is involved in the rate-limiting step in xylan hydrolysis.

[0009] Patent application (US Pat. No. 5,629,499) teaches that in a method for simultaneous hydrolysis and fermentation of biomass (Simultaneous Saccharification and Fermentation (SSF)), enhancing the β-xylosidase activity of the enzyme cocktail used for enzymatic hydrolysis has a beneficial effect on the enzymatic hydrolysis of certain biomass, as it allows a reduction in the amount of enzyme required, which also allows alkyl xylosides to be hydrolyzed.

[0010] Therefore, it is advantageous to isolate β-xylosidase present in an enzyme mixture produced by a microorganism, for example, to enrich a given enzyme cocktail with β-xylosidase. To achieve this, various techniques have been proposed, in particular, first separating the fungus from the enzyme produced in the culture medium. Thus, Patent Document 3 teaches the isolation and purification of cellulase produced by the fungus Trichoderma reesei: the fungus is separated from the enzyme by filtration through a rotary filter under vacuum. The enzyme is then isolated by passing it through a cotton column and eluting it with a basic solution.

[0011] Patent document 4 proposes isolating the enzyme from the fungus through a series of steps in the treatment of the medium, including a step of filtration of the medium through a filter press, followed by a step of tangential microfiltration of the resulting liquid phase.

[0012] It is also known to separate β-xylosidase from the enzyme mixture by fractional distillation with ethanol, as described in the publication by V. Cortez et al. (Non-Patent Document 2). Although this is an advantageous technique, it is not without drawbacks insofar as it requires the use of solvents and involves multiple steps, making it expensive and complicated to carry out.

[0013] The present invention aims to develop improved techniques for separating enzymes from mixtures of enzymes, more particularly techniques for separating β-xylosidase in mixtures containing β-xylosidase and other types of enzymes. More particularly, it is directed to separation techniques that are highly efficient and can be employed on an industrial scale. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] European Patent Application Publication No. 3484945 [Patent Document 2] International Publication No. 2011 / 079048 [Patent Document 3] U.S. Patent No. 3,398,055 [Patent Document 4] International Publication No. 2018 / 015228 [Non-patent literature]

[0015] [Non-Patent Document 1] Humbird et al., "Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol", NREL / TP-5100-57764, May 2011 [Non-patent document 2] V. Cortez et al. "Xylanase and β-xylosidase separation by fractional precipitation", Process Biochemistry, Volume 35, Issues 3-4, 1999, Pages 277-283 Summary of the Invention [Means for solving the problem]

[0016] (Summary of the Invention) A first object of the present invention is a method for separating a β-xylosidase enzyme from an enzyme mixture containing the β-xylosidase enzyme and other enzymes, which comprises: - the β-xylosidase enzyme to be isolated lacks a histidine group, and - the β-xylosidase enzyme is separated from the rest of the enzyme mixture by immobilized metal ion affinity chromatography (hereinafter also designated by the acronym IMAC for Immobilized Metal Affinity Chromatography).

[0017] IMAC chromatography is known to separate proteins that have histidine groups exposed on their surface, whether naturally occurring or as a result of genetic modification; in the latter case, reference is made to histidine "tags" or histidine "clusters" added to the protein. Reference may also be made 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.

[0018] It has now been found, quite surprisingly, in the context of the present invention that this chromatographic technique was nevertheless able to separate enzymes lacking a histidine group, very particularly the β-xylosidase that the inventors were trying to separate in the microbially produced enzyme cocktail.

[0019] This has many advantages: - β-xylosidase enzymes can be isolated by this technique without the need to previously modify them to carry these histidine tags or clusters. By avoiding their modification, the method for obtaining / isolating them is naturally simplified by eliminating the genetic modification step, but any risk of performance loss due to modification of their behavior / change in their activity due to the presence of these histidine groups is limited (the literature describes many cases of enzymes whose activity has changed after the addition of a histidine tag, especially in the case of metalloenzymes and multimeric enzymes). - the technique of separation by IMAC chromatography is highly 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 harsh, the reactants used are generally reusable and therefore economically advantageous, and the results are excellent in terms of selectivity for the enzymes separated, in particular the β-xylosidase in this case, The separation can be carried out in a single step, resulting in an easier and faster method to carry out.

[0020] Generally, the other enzymes in the mixture may include at least one enzyme selected from cellulases, hemicellulases, and / or hemicellulases.

[0021] Generally, other enzymes in the mixture may include β-glucosidases, endoglucanases, and optionally cellobiohydrolases.

[0022] The β-xylosidase may constitute at least 1% by weight, particularly 2% to 15% or 3% to 8% by weight, of the total enzymes present in the mixture, which is the content typically encountered in enzyme cocktails produced by Trichoderma reesei, although the invention naturally applies in the same way to enzyme mixtures containing higher proportions of β-xylosidase.

[0023] Preferably, immobilized metal ion affinity chromatography IMAC uses: - a solid immobile phase; comprising a matrix in which metal ions are immobilized by a chelating agent; and - A liquid mobile phase called the eluent.

[0024] The matrix of the immobile phase may advantageously be chosen from at least one of the following compounds: agarose gel, cross-linked dextran gel, silica.

[0025] The chelating agent may advantageously be chosen from at least one of the following compounds: iminodiacetic acid IDA, nitrolotriacetic acid NTA, tris[carboxymethyl]ethylenediamine TED.

[0026] The metal ions may advantageously be chosen from metal ions of transition metals, and in particular from: - divalent ions Cu(II), Ni(II), Zn(II), Co(II), trivalent metal ions, in particular trivalent metal ions chosen from the trivalent ions Fe(III), Al(III), Ga(III), or - Tetravalent metal ions, especially the metal ion Zr(IV).

[0027] According to a preferred embodiment of the present invention, the enzyme mixture is obtained from the production of enzymes by a microorganism, in particular a filamentous fungus, for example a filamentous fungus of the genus Trichoderma, in particular of the species Trichoderma reesei.

[0028] The separation method according to the invention may include a preliminary step of separating the culture medium containing the enzyme mixture and the microorganisms that produced said mixture, which step is aimed at separating the microorganisms from the enzyme mixture and in particular involves filtration or several successive filtrations of the culture medium. This preliminary separation may be carried out, for example, as described in patent WO 2018 / 015228. After solid / liquid separation, what is thus obtained is, firstly, the microorganisms that produced the enzymes in solid / semi-solid form (also called must) and, secondly, the soluble enzymes in a liquid (aqueous) phase. This liquid phase may optionally be concentrated, so that it can then be treated according to the invention.

[0029] The separation method according to the invention may also comprise a step of treating the must, which may or may not be separated from the remainder of the culture medium, said treatment comprising cooling the must and then separating the must from an "additional" liquid phase containing an additional amount of the enzyme mixture, as taught in patent EP 3 174 979.

[0030] If this additional separation is carried out on an already separated must, the liquid phase obtained after the above solid / liquid separation can be mixed with this additional liquid phase and the process according to the invention can be carried out on a mixture of these two liquid phases.

[0031] The method according to the invention may of course also be carried out on a liquid phase containing a pre-concentrated enzyme mixture.

[0032] According to a first variant of the separation method of the invention, the chromatography is carried out continuously in a chromatography column containing a solid stationary phase through which a liquid mobile phase, called the eluent, can pass continuously.

[0033] According to another variant, the chromatographic separation according to the invention is carried out batchwise by contacting an immobile chromatographic phase with a mixture containing the β-xylosidase enzyme and other enzymes in a liquid medium to form a reaction medium in a container for a given period of time, and then eluting the solid portion of the reaction medium to extract the β-xylosidase from that portion.

[0034] In this variant, the separation may comprise the steps of mixing the immobile phase with a mixture of enzymes in solution, followed by an optional decantation step, followed by isolating the solid phase from the reaction medium, followed by an optional washing step, followed by eluting the isolated solid phase to extract the β-xylosidase therefrom.

[0035] In the chromatographic separation according to the present invention, the β-xylosidase is immobilized on a stationary phase, preferably at a pH of 6.5 to 9, and the β-xylosidase is preferably eluted by varying the nature, composition or concentration of the eluent, which makes it possible, in particular, to vary the pH of the stationary phase.

[0036] The subject of the present invention is also a β-xylosidase enzyme, which is in particular produced by a fungus, for example Aspergillus or Trichoderma, and in particular obtained by the above-mentioned isolation method, and whose specific activity is at least 10 μmol, in particular at least 20 or at least 30 or at least 35 μmol p-nitrophenol min-1 per minute and mg of enzyme. -1 mg -1 This demonstrates the efficient isolation resulting from the high specific activity and high purity of the β-xylosidase thus isolated.

[0037] The method for measuring the specific activity is known to those skilled in the art and consists of 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 by spectroscopy and the specific activity is calculated using a PNP standard range.

[0038] One example of a β-xylosidase targeted by the present invention is the xylan 1,4-β-xylosidase protein, which is obtained from Trichoderma reesei, which has the reference number XP_006964075.1 in NCBI (the acronym for National Center for Biotechnology Information) under the reference number 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) and in the Uniprot database under the reference number 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) Gene: bxl1, -organism: Hypocrea jecorina (Trichoderma reesei) listed below.

[0039] Also targeted are all β-xylosidases having a sequence that has at least 50% identity with this β-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 at least 99% identity with this β-xylosidase.

[0040] The present invention more generally targets any β-xylosidase, which may in particular be obtained from fungi of the genus Trichoderma, in particular the species Trichoderma reesei or the genera citrinoviride or orientale or longibrachiatum or arundinaceum, or from fungi of the genus Aspergillus, in particular the species Aspergillus niger, japonicus, oryzae, clavatus, aculeatus, awamori, flavus.

[0041] The subject of the present invention is also the β-xylosidase, which is in particular obtainable by the above-described isolation method, the purity of which is greater than or equal to 90%, generally greater than or equal to 95% or greater than or equal to 97%.

[0042] Purity was assessed by electrophoresis on SDS-PAGE gels (polyacrylamide gels containing sodium dodecyl sulfate) in a known manner, followed by analysis with Image-Lab software available from Bio-Rad.

[0043] Enzymes of very high purity are thus obtained, allowing for very profitable utilization, a result all the more remarkable since the separation according to the invention can be carried out on enzyme cocktails which may contain tens or even about a hundred different enzymes, for example those produced by microorganisms such as Trichoderma.

[0044] The subject of the present invention is also the use of a β-xylosidase enzyme, in particular a β-xylosidase enzyme obtained according to the method described above, for enriching an enzyme mixture produced by a microorganism with the β-xylosidase enzyme.

[0045] They can therefore be added in a controlled manner to processes for converting different types of lignocellulosic biomass, with varying recalcitrance to sugars or alcohols (ethanol type), into one or more sugars (saccharification, including enzymatic hydrolysis of the biomass) or into alcohol (saccharification and fermentation).

[0046] Another use consists in utilizing these β-xylosidase enzymes advantageously as they are for applications that specifically require β-xylosidase activity.

[0047] The β-xylosidase may be purified and sold in pure form for biotechnological uses, whether for degrading or producing xylooligosaccharides.

[0048] β-Xylosidase may be added to β-xylosidase deficient enzyme cocktails for industrial applications in the field of lignocellulosic biomass degradation, with the aim of generating sugars that can be advantageously utilized for bioproducts, or for the production of alcohols, including in particular bioethanol. DETAILED DESCRIPTION OF THE INVENTION

[0049] (List of drawings) FIG. 1 depicts an FPLC profile (acronym for Fast Protein Liquid Chromatography, which is a known technique for the rapid chromatography of proteins in a liquid phase) for the separation of β-xylosidase from an enzyme mixture according to one exemplary embodiment of the present invention.

[0050] FIG. 2 shows the results of SDS-PAGE gel electrophoresis of β-xylosidase after FPLC purification (described below).

[0051] FIG. 3 presents a graph in the form of a bar graph of the activity of β-xylosidases isolated according to two exemplary embodiments of the present invention, with the identity of Examples 1 and 2 shown on the x-axis and their activity expressed in μmol of p-nitrophenol per time (min) and weight of enzyme (mg) on ​​the y-axis.

[0052] (Description of the embodiment) The present invention will be described in detail below with the help of figures and examples, which are given by way of illustration and therefore without any limitation.

[0053] According to the present invention, a method is proposed which makes it possible to separate a specific enzyme: the β-xylosidase enzyme, from a mixture of enzymes.

[0054] The present invention is more particularly directed to the isolation of this enzyme from an enzyme cocktail produced by microorganisms, more particularly by the fungus Trichoderma, especially Trichoderma reesei, to which the following examples and detailed description relate.

[0055] However, the invention applies equally to the separation of this enzyme from any mixture of enzymes containing it, particularly any enzyme cocktail produced by a microorganism containing this enzyme in various proportions.

[0056] The method according to the invention allows β-xylosidase to be purified simply and quickly from T. reesei, regardless of the type of T. reesei strain used.

[0057] This method is a single-step method for purifying the target enzyme (β-xylosidase) from a complex mixture of enzymes (approximately 100 enzymes), which requires the production of the enzyme and prior isolation of the mycelium.

[0058] The following description details a variation of the invention using a continuously operating chromatography column, however, the invention may also be practiced in a similar manner in batch mode without a column.

[0059] (pre-process) To carry out the separation method of the present invention, a preliminary separation of a medium containing the enzyme cocktail and the fungus Trichoderma reesei is first performed. To do this, the medium is subjected to separation on a filter press lined with a cloth having a porosity of 3-20 μm within a period of less than 24 hours after production has stopped, obtaining a filtrate with a corrected optical density (OD) at 600 nm of less than 2.5. The resulting liquid phase is subjected to tangential microfiltration on a ceramic membrane with a cutoff threshold of 0.5-1.4 μm, so that the corrected optical density (OD) at 600 nm does not exceed 0.1. Separation and microfiltration on the filter press are carried out at 20-30°C, preferably 22-27°C.

[0060] At the end of the separation on the filter press, a solid residue ("cake") of 5 to 10% by weight and a filtrate of 90 to 95% by weight are generally obtained. Advantageously, the microfiltration of the filtrate obtained at the end of the filter press is carried out within a period of up to 30 hours, preferably up to 24 hours.

[0061] Preferably, tangential microfiltration is carried out on a ceramic membrane with a cut-off threshold of 0.8 to 1.4 μm.

[0062] The liquid phase obtained after microfiltration may be subjected to ultrafiltration, preferably on a ceramic membrane, even more preferably on a ceramic membrane with a cut-off threshold of 5 to 15 kDa.

[0063] The filtration method described herein employs the teachings of patent WO 2018 / 015228, to which reference is made for further details.

[0064] The retentate obtained is then passed through an IMAC affinity column, which makes it possible to separate enzymes that have a polyhistidine tag (also called a "His tag"), an amino acid motif in proteins 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".

[0065] However, it will be clearly noted that the β-xylosidase purified from the cocktail does not contain a histidine tag (as do any of the other enzymes in the enzyme mixture here).

[0066] (Purification on an IMAC column) The supernatant containing the enzyme (permeate from microfiltration or retentate from ultrafiltration), i.e., cellulase produced by Trichoderma reesei, is stored at 4°C to 30°C, but preferably below 10°C.

[0067] The resulting supernatant is loaded onto an immobilized metal ion affinity chromatography column, also known as IMAC (Immobilized Metal Affinity Chromatography). This type of affinity chromatography is based on the mechanism of chelation of immobilized metal cations. This allows the purification of histidine-tagged proteins from supernatants that generally contain a complex mixture of various proteins of biological origin.

[0068] Chelation of (generally divalent) metal ions is a method that allows the formation of complexes between metal cations and ligands immobilized on a solid phase. Thanks to this chelation, the metal ions remain immobilized in the column onto which the enzyme mixture desired to be fractionated or purified is applied. The bond between the metal ion and the ligand is generally formed within a pH range of 7-8. To maintain this pH, the column is pre-equilibrated with a buffer.

[0069] A solution in which the sample can ideally be solvated has high ionic strength to reduce nonspecific electrostatic interactions, but these ions do not themselves need to bind to the metal. The solution is preferably also neutral or slightly alkaline, since in the presence of protons occupying binding sites on the amino acids, the interaction between the histidine group and the metal is inactivated. An example of this type of solution is 50 mM Tris acetate (tris(hydroxymethyl)aminomethane acetate (CHCOO - )) or 20-50 mM sodium phosphate. TrisHCl (tris(hydroxymethyl)aminomethane HCl) allows the purification of enzymes with very strong protein-metal interactions.

[0070] For the eluent, an acidic solution with a pH gradient from 7 to 4 can be chosen, which protonates the amino acids that interact with the IMAC matrix, thereby inducing a drastic decrease in the affinity of the enzyme for the resin. Alternatively, an imidazole solution can be used to displace proteins on the binding sites (exchange the ligand). Finally, metal ions can be extracted with strong chelating agents, such as ethylenediaminetetraacetic acid (EDTA), which are often used for column regeneration.

[0071] (Production process) The enzyme cocktail for which the separation method according to the invention is carried out is produced by Trichoderma reesei by aerobic fermentation in the conventional production chain. Examples of methods for producing enzyme cocktails using this fungus are described in patents FR 3 024 463, FR 3 049 957, FR 3 085 961 and FR 3 088 934. An improved method for increasing the β-glucosidase and / or β-xylosidase content by cooling the must obtained at the end of production is described in patent EP 3 174 979.

[0072] The process for producing the enzyme cocktail begins with a growth phase, which is carried out in small reactors of generally increasing size in order to grow the filamentous fungi and limit the duration of the lag phase and the risk of contamination.

[0073] When this production is deemed sufficient (fungal concentration is 10 g / L or more, preferably 15 g / L or more), the medium is transferred to a large-volume final reactor.

[0074] The enzyme production method includes two steps and is therefore detailed as follows according to a preferred embodiment. - step a) growing the microorganism in the presence of at least one carbon-based growth substrate in an aerated closed reactor; said growing being carried out at a carbon-based growth substrate concentration of 10 to 90 g / L; - step b) production of an enzyme cocktail, introducing at least one carbon-based inducer substrate, said carbon-based inducer substrate being chosen from the group formed by lactose, cellobiose, sophorose, residues obtained after the ethanolic fermentation of monosaccharides of enzymatic hydrolysates of cellulosic biomass and / or crude extracts of water-soluble pentoses originating from the pretreatment of cellulosic biomass; said production step being carried out at a carbon-based production substrate concentration of 150 to 400 g / L.

[0075] The microorganism used in the method for producing the enzyme cocktail according to the present invention is a fungal strain belonging to the species Trichoderma reesei.

[0076] The most effective industrial strains belong to the species Trichoderma reesei, which have been engineered to improve the enzyme cocktail by mutation-selection methods.

[0077] Strains improved by genetic engineering may also be used, which are cultivated in stirred and aerated reactors under conditions suitable for their growth and enzyme production.

[0078] As examples of strains and methods for obtaining them, it can be recalled that conventional genetic mutation techniques have made it possible to select strains of Trichoderma reesei that overproduce cellulases, such as MCG77 (Gallo-Patent US 4 275 167), MCG 80 (Allen, AL and Andreotti, RE, Biotechnol.-Bioeng. 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 Genetique des microorganismes industriels [genetics of industrial microorganisms]. Improvements have made it possible to obtain hyperproductive strains that are less sensitive to catabolic repression, especially for monosaccharides, such as glucose, compared to wild-type strains.

[0079] Recombinant strains were also obtained from Trichoderma reesei strains, such as Qm9414, RutC30, and CL847, by cloning a heterologous gene, e.g., an invertase from Aspergillus niger, which enabled Trichoderma reesei to use sucrose as a carbon source. These strains retained their hyperproductivity and their ability to be cultivated in fermentors.

[0080] The carbon-based growth substrate for the microorganisms used in the growth step a) of the method according to the invention is advantageously chosen from industrial soluble sugars, preferably glucose, lactose, xylose, liquid residues obtained after ethanolic fermentation of monosaccharides of enzymatic hydrolysates of lignocellulosic material, and extracts of hemicellulose fractions in monomeric form originating from pretreated lignocellulosic substrates, used alone or in mixtures.

[0081] Depending on its nature, the carbon-based growth substrate is either introduced into the closed reactor before sterilization, or is sterilized separately and introduced into the closed reactor after sterilization of the reactor.

[0082] The carbon-based growth substrate is typically used in the growth step a) at an initial concentration of 20-90 g of carbon-based substrate per liter of reaction volume.

[0083] Preferably, said growth step a) is carried out over a period of 30 to 70 hours, preferably 30 to 40 hours.

[0084] Preferably, the growth step a) is carried out at a pH of 4.8 and at a temperature of 20 to 30°C, generally 22 to 27°C, preferably about 27°C.

[0085] The carbon-based inducer substrate used in the production step b) is advantageously fed in a fed-batch mode at a limited flow rate of 30-80 mg per gram of cell weight per hour (hour). The temperature is generally the same as in step a).

[0086] At the end of the enzyme production process, a medium containing solids at a concentration of 10-45 g / L (equivalent to dry fungus) is typically obtained; the enzymes are all water-soluble. The pellet, measured after centrifugation (4000 rpm, 5 min), is greater than 15%, often greater than about 30%, and up to 60%. It corresponds to the percentage of the volume occupied by solids relative to the total volume of the sample.

[0087] The objective of the present invention is to isolate the fungal enzymes and then purify the β-xylosidases and either sell them or add them specifically to enzyme mixtures if they are limiting in biochemical processes involving the enzymes.

[0088] The present invention may be applied to any mixture of enzymes produced by microorganisms, called enzyme cocktails, including enzyme cocktails obtained from a reaction medium containing microorganisms that has been treated, in particular by cooling, as described in patent EP 3174979.

[0089] (Solid / liquid separation process to separate fungi from liquid) The liquid contains enzymes and residual salts. 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 to 9, preferentially to pH 8 (the buffer can be changed via desalting columns, filtration, ultrafiltration under pressure (stirred cells marketed by Merck under the name Amicon, or ultrafiltration cells marketed under the name Pellicon with an Ultracel 10 kD membrane, also from Merck)). - Once the cocktail of cellulases is buffered, the IMAC column described above is used.

[0090] It should be noted that the tested enzyme mixture had the following composition (the indicated contents are expressed in large amounts and are approximate data, but give an idea of ​​the distribution of the enzymes in the mixture): - CEL7A=CBH1 content: approx. 35% - CEL6A = CBH2 content: approx. 30% - BGL1 = β-glucosidase 1 content: approximately 5% - content of endoglucanase I = Cel7B: about 10%; - Content of endoglucanase II = Cel5A: about 10% - Other (enzymes and / or other compounds) content: approx. 10%.

[0091] For an example of an analysis of the secretome of the modified Trichoderma reesei strains RUT-C30 and CL847, reference may be made to the publication by I. Herpoel-Gimbert et al., "Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains," Biotechnology for biofuels, article number 18 (2008), published November 23, 2008, in particular Table 1 therein.

[0092] Nickel ion Ni 2+ A pre-loaded His Trap Crude column (Cytiva, 5 mL) is equilibrated to pH 6.5-9, preferentially pH 8. This type of column is available from Cytiva under the full name "HisTrap FF Crude Histidine-Tagged Protein Purification Column."

[0093] The equilibration buffer may be Tris, Bis-TriS, phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also known as HEPES), or any other buffer falling within the pH range of 6.5 to 9. The buffer solution may contain salt (NaCl, KCl) at 0 to 500 mM, but preferably 50 mM. Once the column has been equilibrated, the clarified supernatant may be filtered and then loaded onto the column via a peristaltic pump via a "fast protein liquid chromatography" system, which generally includes a pump, a UV detector, a conductivity measuring device, a fraction collector, and valves that allow, among other things, passage from one column to another. Such systems are commercially available, among others, from Bio-Rad. Another chromatography system is also available from Cytiva under the name "AKTA High Purity Protein Purification System."

[0094] Gravity separation techniques may also be used.

[0095] The column is washed in the presence of 0-40 mM imidazole, typically with 20 mM imidazole.

[0096] Surprisingly, and never before observed, the β-xylosidase from T. reesei can be easily purified on this type of resin, despite being immobilized on a solid IMAC phase and not having any histidine tag.

[0097] To elute the β-xylosidase from T. reesei, it is necessary to perform a gradient or elution in the presence of 500 mM imidazole or to lower the pH to reduce the affinity of the enzyme for the solid phase.

[0098] The β-xylosidase enzyme from T. reesei purified under these conditions is highly pure (verification was performed by mass spectrometry) and it is active (activity tests show that the enzyme is active towards 4-nitrophenyl β-D-xylopyranoside (4-NPX) and releases para-nitrophenol pNP).

[0099] The specific activity of β-xylosidase is assessed with 4-nitrophenyl β-D-xylopyranoside (4-NPX) as a substrate. At 50°C, the activity is between 10 and 100 μmol of p-nitrophenol per minute and mg of enzyme, generally at least 20 to about 35 μmol (or more) p-nitrophenol·min -1 mg -1 The enzyme is β-glucosidase. The same protocol is used for measuring β-glucosidase activity. Only the substrate (para-nitrophenyl β-D-xylopyranoside (pNP)) is changed, and β-xylosidase is used. The principles for measuring specific activity with this type of reagent are well known in the literature.

[0100] (Example) Example 1 Experiments were performed in the laboratory using the enzyme cocktail produced by Trichoderma reesei according to the procedure described above, using strain CL847. Strain CL847 has already been cited and is also described in the publication by Jourdier E. et al., "A new stoichiometric 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).

[0101] Example 2 Experiments were carried out in the laboratory on the enzyme cocktail produced by Trichoderma reesei according to the procedure described above, using the strain described under reference 130G9 in Table 1 of patent EP 3 174 979 (nucleic acid: SEQ ID NO: 7, polypeptide: SEQ ID NO: 8).

[0102] The following description concerns the treatment of the medium obtained in each of the two examples: the extracellular medium was separated from the mycelium by filtration. The extracellular medium containing the enzymes secreted by Trichoderma reesei was removed by filtration through a Pellicon membrane (10 kDa) and then diluted three-fold in buffer A (Tris-Cl 50 mM pH 8, NaCl 50 mM).

[0103] 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 syringe filtered to 0.2 μm (PES filter, VWR, 514-2073).

[0104] The protein solution is then loaded onto the above-mentioned HisTrap column (Cytiva, HisTrap® FF and HisTrap Crude, 5 mL), which has a stationary phase based on cross-linked Sepharose bound to nickel ions via chelating groups.

[0105] The column was pre-equilibrated with 7 column volumes in buffer A (50 mM Tris-Cl pH 8, 50 mM NaCl). A linear gradient of 10 column volumes was then applied to buffer B (50 mM Tris-Cl pH 8, 50 mM NaCl, 500 mM imidazole). The protein eluted in a uniform and symmetrical peak. It was then concentrated and washed three times in buffer A by centrifugation at 5000 rpm and an ultrafiltration unit (10 kDa) commercially available from Sartorius under the name Vivaspin to remove the 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 protein purified / separated from the remaining enzymes of the starting cocktail was β-xylosidase. It should be noted that CBH2 was not separated, which, however, is doubly surprising due to its histidine tag.

[0106] Figure 1 shows the FPLC (Fast Protein Liquid Chromatography) profile of the purification of β-xylosidase, correlated with a polyacrylamide electrophoresis gel containing sodium dodecyl sulfate (SDS PAGE, Bio-Rad, Mini-PROTEAN TGX Stain-Free Precast Gel 10% - 456-8035) according to Example 1. In this figure, band 1 can be seen, which corresponds to the enzyme purified by this chromatography, which was 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 the UniProtKB reference name: Q92458_HYPJE, already cited above. The same type of results are obtained according to Example 2.

[0107] Figure 2 is an image of an SDS-PAGE gel for Example 1, which allows separating proteins according to their molecular weight after denaturing them. To indicate molecular weight, the band on the left has markers for various molecular sizes: 15 kDa, 20 kDa, 25 kDa, 37 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa, and 250 kDa. The purified β-xylosidase was loaded onto the band on the right and then analyzed. The results of electrophoresis of β-xylosidase on an SDS-PAGE gel show that the molecular weight 1 corresponds to that predicted by the DNA sequence, i.e., 75 kDa to 100 kDa. Additionally, the protein is found to be pure. The same type of results are obtained for Example 2.

[0108] Following various purifications, activity was measured for different batches of purified enzyme, and the activity results are shown in the bar graph of Figure 3. The figure shows that the specific activity of the purified β-xylosidase enzyme from Examples 1 and 2 was approximately 35-38 μmol p-nitrophenol per minute per mg enzyme weight.

[0109] In conclusion, this technique for IMAC purification of β-xylosidase allows for rapid, easy, and highly efficient access to this enzyme without the use of a histidine tag. The affinity of β-xylosidase for IMAC columns is a finding that could not have been predicted. β-Xylosidase isolated according to the present invention was characterized by mass spectrometry, and its specific activity, assessed with para-nitrophenyl β-D-xylopyranoside (pNPX) as substrate, ranges from at least 15 or at least 20 μmol to 35 μmol or more of p-nitrophenol per minute per mg of enzyme weight.

[0110] This enzyme is of industrial interest to stimulate the degradation of xylans or oligomers of xylose with different degrees of polymerization (DP) or higher, such as xylobiose, xylotriose, etc., which have a xylose DP.

[0111] It may be advantageously utilized alone or in combination with other enzyme cocktails / mixtures depending on the needs and applications. [Brief explanation of the drawings]

[0112] [Figure 1] 1 depicts an FPLC profile for the separation of β-xylosidase from an enzyme mixture according to one exemplary embodiment of the present invention. [Figure 2] 1 shows the results of SDS-PAGE gel electrophoresis of β-xylosidase after FPLC purification (described below). [Figure 3] A graph of the activity of β-xylosidases isolated according to two exemplary embodiments of the present invention is presented in the form of a bar graph, with the identity of Examples 1 and 2 shown on the x-axis and their activity expressed in μmol of p-nitrophenol per time (min) and weight of enzyme (mg) shown on the y-axis.

Claims

1. A method for separating a β-xylosidase enzyme (1) from an enzyme mixture containing the β-xylosidase enzyme and other enzymes, characterized in that the β-xylosidase enzyme to be separated lacks a histidine group, and the β-xylosidase enzyme is separated from the remainder of the enzyme mixture by immobilized metal ion affinity chromatography (IMAC).

2. 2. The separation method according to claim 1, wherein the other enzymes in the mixture include at least one enzyme selected from cellulases and / or hemicellulases.

3. 3. The method of claim 1, wherein the other enzymes in the mixture include β-glucosidase, endoglucanase, hemicellulase, and optionally cellobiohydrolase.

4. 4. The method according to claim 1, wherein the β-xylosidase (1) constitutes at least 1% by weight, in particular 2% to 15% by weight or 3% to 8% by weight, of all enzymes present in the mixture.

5. Immobilized metal ion affinity chromatography IMAC is a separation method according to any one of claims 1 to 4, characterized in that it uses: a solid stationary phase comprising a matrix on which metal ions are immobilized by a chelating agent, and a liquid mobile phase, called the eluent.

6. 6. The separation method according to claim 5, wherein the matrix of the immobile phase is selected from at least one of the following compounds: agarose gel, cross-linked dextran gel, and silica.

7. 7. The method of claim 5, wherein the chelating agent is selected from at least one of the following compounds: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris[carboxymethyl]ethylenediamine (TED).

8. 8. The separation method according to claim 5, wherein the metal ions are selected from metal ions of transition metals, in particular from divalent ions Cu(II), Ni(II), Zn(II), Co(II), trivalent metal ions or tetravalent metal ions, the trivalent metal ions being selected in particular from trivalent ions Fe(III), Al(III), Ga(III), the tetravalent metal ion being in particular the metal ion Zr(IV).

9. 9. The method according to claim 1, wherein the enzyme mixture is obtained from the production of enzymes by a microorganism, in particular a filamentous fungus, such as a filamentous fungus of the genus Trichoderma, in particular of the species Trichoderma reesei, Trichoderma citrinovii, Trientale, Trichoderma longibrachiatum, Trichoderma arundinaceum, or a filamentous fungus of the genus Aspergillus, in particular of the species Aspergillus niger, Aspergillus japonicus, Aspergillus oryzae, Aspergillus clavatus, Aspergillus acutus, Aspergillus awamori, Aspergillus flavus.

10. 10. A method according to any one of claims 1 to 9, characterized in that it comprises a preliminary step of separating the culture medium containing the enzyme mixture and the microorganisms that gave rise to said mixture, called must, which preliminary step is aimed at separating the must from the liquid enzyme mixture and in particular comprises a filtration or several successive filtrations of the culture medium.

11. 11. The method of claim 10, further comprising a step of treating the must, which may or may not be separated from the remainder of the culture medium, said treatment comprising cooling the must and then separating the must from the liquid containing the additional amount of the enzyme mixture.

12. 12. A separation method according to any one of claims 1 to 11, characterized in that the chromatography is carried out continuously in a chromatography column containing a solid stationary phase through which a liquid mobile phase, called the eluent, can be passed continuously.

13. 12. The method of any one of claims 1 to 11, wherein the chromatographic separation is carried out batchwise by contacting an immobile chromatographic phase with a mixture containing the β-xylosidase enzyme and other enzymes in a liquid medium to form a reaction medium in a container for a given duration, and then eluting the solid portion of the reaction medium to extract the β-xylosidase therefrom.

14. 14. The method of claim 13, wherein the separation comprises mixing the immobile phase with a mixture of enzymes in solution, followed by an optional decantation step, followed by isolating the solid phase from the reaction medium, followed by an optional washing step, followed by eluting the isolated solid phase to extract the β-xylosidase therefrom.

15. The separation method according to any one of claims 1 to 14, characterized in that the chromatographic separation comprises immobilizing the β-xylosidase (1) on an immobile phase at pH 6.5 to 9, and eluting the β-xylosidase (1) by varying the properties, composition, or concentration of the eluent.

16. β-xylosidase enzymes (1) obtainable by the separation method according to any one of claims 1 to 15, characterized in that their specific activity is at least 10 μmol p-nitrophenol per minute per mg enzyme weight, in particular at least 20 or at least 30 μmol p-nitrophenol per minute per mg enzyme weight.

17. β-xylosidase enzymes (1) obtained by the separation method according to any one of claims 1 to 15, characterized in that their purity is 90% or more, in particular 95% or more or 97% or more.

18. Use of the β-xylosidase enzyme (1) obtained according to the method of any one of claims 1 to 15 for enriching an enzyme cocktail produced by a microorganism with the β-xylosidase enzyme.

Images