IT201900012942A1 - METHOD FOR THE PRODUCTION OF AN ENZYMATIC COMPOSITION INCLUDING A RECOMBINANT ENDOPEPTIDASE - Google Patents

Description

Application for an Italian Patent entitled:

METHOD FOR THE PRODUCTION OF AN ENZYMATIC COMPOSITION INCLUDING A RECOMBINANT ENDOPEPTIDASE

DESCRIPTION

Celiac disease (CD) is a chronic autoimmune enteropathy triggered by gluten <1>. Gluten is a heterogeneous mixture of insoluble proteins, composed of gliadins and glutenins, present in cereals <2> wheat, rye and barley; gluten proteins are largely inaccessible to human gastrointestinal tract proteases, so large proline / glutamine-rich peptides can reach the small intestine and trigger both humoral and T-cell-mediated adaptive immune responses in patients with CD. To date, several T cell stimulatory peptides, resistant to gastrointestinal digestion, have been identified in gliadins and glutenin proteins; among them, a 33-mer peptide of α-gliadin with sequence LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 12) is currently considered the most immunogenic peptide, as it carries multiple copies of immunogenic epitopes in patients with CD <5>.

Permanent adherence to a strict gluten-free diet (GFD) is the current treatment for patients with CD <6>. However, total avoidance of gluten is practically impossible and new therapies have been intensively studied, leading to the development of new enzymes, called "glutenases", capable of digesting gluten and toxic gluten fragments in the gastrointestinal environment.

WO2013083338 discloses a group of new glutenases, i.e. endopeptidases with glutenase activity, identified by the inventors of the present application in Actinoallomurus A8 acidophilic actinomycete. Said Actinoallomurus endopeptidases are very fast and efficient in degrading gluten peptides into non-toxic peptides, being active throughout the pH range of the gastric and intestinal environment (enzymatic activity is shown in the pH range 3-6 with a excellent at pH 5) and being also resistant to degradation by endogenous gastrointestinal enzymes. This makes these new Actinoallomurus endopeptidases very suitable for use in the treatment and / or prevention of CD and CD-associated disorders. Among said Actinoallomurus endopeptidases described in WO2013083338, endopeptidase 40 (E40) was of particular interest. The native protein E40 consists of 398 amino acid residues (see SEQ ID NO: 3), belonging to the serinacarboxyl peptidase S53 family with the catalytic triad formed by aspartic acid, glutamic acid and serine in positions 156, 160 and 329, respectively. The N-terminal signal peptide was identified between positions 27 and 28 by analysis of server signal P 4.1, and the mature form was predicted to start at position 74, resulting in a mature enzyme of 32.5 kDa, such as expected. The mature form of native E40 is a SEQ ID NO: 1 sequence polypeptide.

There is a strong interest in the development of efficient and economical methods for the production of said Actinoallomurus endopeptidases with glutenase activity, in particular E40 of sequence comprising or consisting of SEQ ID NO: 1.

E40 is hereinafter used to identify a sequence endopeptidase comprising SEQ ID NO: 1, i.e. a sequence endopeptidase consisting of SEQ ID NO: 1 or a derivative thereof having a sequence comprising SEQ ID NO: 1; an exemplary derivative of E40 is a tagged E40, such as an endopeptidase having a sequence comprising or consisting of SEQ ID NO: 2.

Recombinant DNA (rDNA) technology offers a very powerful set of technical platforms for the controlled and scalable production of polypeptides of interest using relatively inexpensive procedures. Today, recombinant proteins are obtained in Escherichia coli, Saccharomyces cerevisiae, in insect, hamster and mammalian cells. However, there is a demand to improve the production of large quantities of recombinant proteins; furthermore, there are very strict requirements to be met when proteins are produced for human use, for example for use as food supplements and / or as drugs in the prevention and / or treatment of human diseases.

The present invention therefore has the purpose of providing an improved method for the efficient and economical production of an enzymatic preparation comprising at least one of the Actinoallomurus endopeptidases described in WO2013083338, as recombinant proteins, said enzymatic preparation being suitable for human use, optionally following correct wording.

Streptomycetes are considered a safe source of protein for human food use. Two examples of food enzymes from Streptomyces spp are: glucose isomerase used for the production of fructose syrup <12> and the widely exploited transglutaminase from S. mobaraensis, used in the food industry for its properties in improving texture and general quality of final food products, such as processed meat and fish products, as well as dairy products and baked goods <13>.

The present inventors have discovered that Streptomyces lividans is a particularly advantageous host cell for use in the improved method of the invention.

The present invention therefore provides a method for the production of an enzymatic preparation, which comprises at least one recombinant Actinoallomurus endopeptidase with glutenase activity, in S. lividans host cells, the method being characterized by a culture step of said host cells, which express the recombinant endopeptidase (s) of Actinoallomurus of interest, under fermentation conditions, followed by a recovery step of the culture medium supernatant of the host cell, comprising the recombinant endopeptidase (s) produced; preferably said recovery step is followed by a purification step from said supernatant of a final enzymatic preparation comprising the recombinant endopeptidase (s) of Actinoallomurus with glutenase activity of interest.

S. lividans cells are known to be efficient host cells for the production of recombinant proteins, since the recombinant proteins expressed by these cells can be directly secreted and released into the culture medium. However, different proteins are obtained at very different yields in S. lividans, this result being unpredictable <12>. Furthermore, S. lividans cells produce high levels of secondary metabolites, particularly antibiotics <12>, thus jeopardizing the possibility of establishing S. lividans as an appropriate cell factory for the production of enzyme preparations aimed at supplementing digestive enzymes. physiological.

The method of the invention provides an enzymatic preparation which comprises high amounts of the desired recombinant glutenase. Surprisingly, despite the culture of the host cells under fermentation conditions, the enzymatic preparation obtained with the method of the invention is substantially devoid of potentially harmful secondary metabolites released by S. lividans, such as for example antibiotics, which would be unacceptable from the point of view. regulatory for human intake in food, food supplements or pharmaceutical formulations; the enzymatic preparation obtained with the method of the invention is suitable for human use (optionally following an appropriate formulation thereof).

Brief description of the invention

The present invention is directed to a method for producing an enzymatic preparation comprising at least one recombinant Actinoallomurus endopeptidase with glutenase activity, characterized in that a batch of S. lividans host cells capable of expressing a recombinant Actinoallomurus endopeptidase is cultured under conditions of fermentation in a medium comprising at least 30% (w / v) sucrose, the enzymatic preparation then being recovered and purified from the supernatant of the host cell culture. The enzymatic preparation is obtained at high yields by the method of the invention and is substantially free of antibiotics, therefore being suitable for human use. The expression "substantially free" of antibiotics means that no antibiotic is detected in the enzymatic preparation, neither in microbiological assays nor in quantitative HPLC.

The present invention is further directed to said enzymatic preparation obtained by said method, to formulations of the enzyme preparation, suitable for human use, to a recombinant expression vector carrying a nucleic acid encoding the recombinant endopeptidase (s) of Actinoallomurus. of interest and to a host cell of S. lividans comprising said recombinant expression vector and stably expressing said recombinant Actinoallomurus endopeptidase (s). Furthermore, the present invention is directed to the clinical uses of the enzyme preparation and its formulations. Preferably, at least one Actinoallomurus endopeptidase of interest, with glutenase activity, is E40 or a derivative thereof, having a sequence comprising SEQ ID NO: 1.

Brief description of the figures

Figure 1. Activity of the supernatant fraction of the Actinoallomurus A8 culture containing native E40. A: relative activity towards the substrate sucAlaAlaProPhe-AMC (sucAAPF-AMC) at various pH values (the optimal activity of 100% is at pH 5). B: digestion of gliadin after 2 hours of incubation at 37 ° C, pH 3. Gel colored with brilliant blue Coomassie R250; Lane 1: gliadin alone, lane 2: gliadin fraction of supernatant containing E40.

Figure 2. Map of the pIJ86 vector bearing the gene encoding E40 (recombinant expression vector pIJ86 / e40)

Figure 3. Recombinant E40 (SEQ ID NO: 2) obtained by submerged fermentation of S. lividans TK24 / plJ86 / e40, in flask scale (boxes A-C) or in a 15 L bioreactor, followed by purification (boxes D-E). A: activity against the substrate sucAAPF-AMC at pH 5 of supernatant samples taken at different fermentation times (♦ 72, ▲ 96, ● 168, ■ 192 hours of fermentation) from S. lividans TK24 / plJ86 / E40 producer (E40 , solid line) and absence of activity of supernatant samples taken from the control strain with blank pIJ86 (ctr, dashed lines). Activity is shown as relative fluorescent units (rfu, Y axis) produced over time (X axis). B: Relative activity (%) shown by the E40 supernatant sample at 168 hours of fermentation, at different pH: the optimum (100% activity) is at pH 5. C: Zymography of the E40 supernatant sample of 168 h a pH 5, using the same substrate as in panel A and viewed under UV light. D: profile of an enzymatic preparation comprising recombinant E40 purified by fermentation of 15 L; lane 1: final enzymatic preparation including recombinant E40 stained with Coomassie (arrow indicates recombinant E40), lane 2: zymography as in C, lane 3: gelatin hydrolysis band after 2 hours of digestion at 37 ° C and pH 5, displayed after Coomassie staining of the gelatin substrate.

E: relative activity (%) of recombinant E40 in the preparation of purified enzymes at different pH values (from 2 to 8), evaluated with the suc-Ala-Ala-Pro-Phe-pNA substrate.

Figure 4. Recombinant E40 activity evaluated in the absence or presence of the digestive proteases pepsin (P) (boxes A-C) at pH 4, 4.5 and 5, and trypsin (T) (boxes D-E, E ') at pH 6 and 7. The reactions started with the addition of the substrate to the enzyme preparation samples comprising the enzyme E40 alone (lane ①), the enzyme and P (lane ②) and T (lane ④), and the addition of P or T alone (lanes ③ and ⑤ respectively). Each reaction condition was tested in duplicate, the error bars represent the standard deviation. Box E 'is an enlargement of E. Axis X: incubation time (minutes), axis Y: absorbance units read at 410 nm. (R <2> values are 0.9948, 0.9976, 0.9978, 0.9639, 0.9994 for E40 alone and 0.993, 0.9975, 0.9982: 0.9661, 0.9994 for E40 P / T at pH 4, 4.5, 5, 6 and 7, respectively).

Figure 5. LC / MS profile of digestion of 33-mer peptide by recombinant E40 at pH 5 and 37 ° C, with E40 in a molar ratio of 1:48 to 33-mer peptide. A: whole peptide alone (triple charged ion [(M <+> 3H <+>) <3+> = 1304.68]). B-E: time-course of the 33-mer cut through the activity of E40. The peptide fragments formed during digestion are indicated by arrows. F: schematic view of the E40 cleavage sites deduced from the final residual peptides.

Figure 6. HPLC analysis of gliadin incubated with recombinant E40 at different incubation times, up to 240 min. At time point 0, gliadin is eluted based on hydrophobicity in ω-, α- and γgliadin.

Figure 7. A: E40-treated gliadin recognition in gliadin-reactive T cell lines obtained from fasting biopsies of 5 different celiac subjects (samples A-H). Positive controls: digestion of untreated gliadin (samples I and L) and phytohemagglutinin (PHA). Each box is a representative experiment in three performed for each T cell line. B: Immunostimulatory activity of purified gliadin from hexaploid wheat on human intestinal T cells, after digestion with E40, shown as a percentage of T cell response to digestion products of gliadin treated with E40 alone (samples A-B) or with E40 in the presence of gastrointestinal proteases (samples C-H), calculated on gliadin digestates not treated with E40 (sample I), under the conditions specified in Table 1. The enzymatic digestates of gliadin are been deamidated by tTG treatment and tested for stimulating capacity on intestinal T cells. Activation of T cells was determined by measurements of IFN-γ production. All gliadin digestates were analyzed at 50 and 100 µg / ml. Data are mean responses of T cell lines from 5 different CD patients. The unpaired student T test was used to assess statistical significance. * = p <0.05 Figure 8: SDS-PAGE of the enzyme preparation comprising recombinant E40. The numbered bands were cut and analyzed by proteomic analysis. ; Figure 9: A: SDS-PAGE showing degradation of peanut proteins by recombinant E40; B: HPLC analysis of peanut proteins incubated with recombinant E40 (enzyme 1:20: substrate, e: s) at different incubation times, up to 120 minutes; for each time point the HPLC of the untreated samples is shown in the upper panel, while the lower panel shows the samples treated with E40. ; Detailed description of the invention; The present invention is directed to a method for producing an enzymatic preparation comprising the mature form of at least one recombinant Actinoallomurus endopeptidase with glutenase activity, serially comprising: culturing a recombinant host cell of Streptomyces lividans, preferably of the strain TK24, in a culture medium under fermentation conditions, said recombinant host cell comprising a recombinant expression vector for the heterologous expression of the at least one recombinant Actinoallomurus endopeptidase, the recombinant expression vector comprising a polynucleotide coding for said at least one recombinant Actinoallomurus endopeptidase, functionally linked to regulatory sequences capable of directing the expression of said at least one recombinant Actinoallomurus endopeptidase in the recombinant host cell; recovering the supernatant of the culture medium and purifying from said supernatant an enzymatic preparation comprising the mature form of the at least one recombinant endopeptidase of Actinoallomurus. ; The at least one recombinant endopeptidase of Actinoallomurus in the enzymatic preparation obtained by the method of the invention is preferably selected from the group consisting of: endopeptidases 40 (E40) of sequence comprising SEQ ID NO: 1; a biologically active fragment of E40; a natural allelic variant of E40; and a sequence endopeptidase having at least 60%, 70%, 80%, 90% or 95% identity with respect to SEQ ID NO: 1. ; The expression "enzymatic preparation (or enzymes)" is used herein to identify the product obtained at the end of the method of the invention, comprising (enriched with) at least one recombinant Actinoallomurus endopeptidase with glutenase activity; said product can be a composition comprising furthermore other components. ; The expression "biologically active fragment" refers to portions of the endopeptidases of the invention that maintain the specific glutenase activity. ; A "biologically active fragment" of an endopeptidase according to the invention can be identified for example: by isolating a polynucleotide encoding a fragment of an endopeptidase of sequence SEQ ID NO: 3 or 4 (for example a polynucleotide portion of the polynucleotides of sequence SEQ ID NO: 5 or 6), which express the encoded endopeptidase fragment (for example, by recombinant expression in vitro) and verifying by means of an adequate assay whether said endopeptidase fragment has the same glutenase activity as the endopeptidases; a suitable assay is for example any of the enzymatic activity assays described in the present examples. The method of the invention also comprises obtaining the enzymatic preparation comprising the at least one recombinant Actinoallomurus endopeptidase with glutenase activity by introducing a recombinant expression vector comprising a polynucleotide having a sequence that differs from the sequences of nucleic acid SEQ ID NO: 5 or 6 due to the degeneration of the genetic code and therefore encodes the same endopeptidases encoded by a polynucleotide of sequence SEQ ID NO: 5 or 6.; The endopeptidases obtained by the method of the invention can have a sequence which includes variations in amino acid residues which are not essential for the biological activity of the endopeptidase. The "biological activity" is in this context the natural or normal function of the native Actinoallomurus endopeptidases of sequence SEQ ID NO: 3, for example, it is the ability to degrade gluten proteins. The method of the invention comprises obtaining an enzymatic preparation comprising at least one sequence recombinant endopeptidase having at least 60%, 70%, 80%, 90% or 95% identity with SEQ ID NO: 1. The terms "identity" and "homology" when referring to a nucleotide or amino acid sequence are used interchangeably herein and refer to the degree to which two polynucleotide or polypeptide sequences are identical or homologous on a residue-by-residue basis in reference to a particular comparison region. Alignment and percent identity or homology can be determined using any suitable software program known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel FM et al., "Commercially available Software", Current Protocols in Molecular, 1987, Supplement 30, section 7.7.18, table 7.7.1). Favorite programs include the GCG Pileup program, FASTA (Pearson R. and Lipman D.J. "Improved Tools for Biological Sequence Analysis" Proc. Natl., Acad. Sci. USA, 1988, 85, 2444-2448) and BLAST (Altschul SF, Gish W., Miller W., Myers EW, Lipman DJ "Basic local alignement search tool" J. Mol. Biol., 1990, 215, 403–410). ; The expression "allelic variant" indicates one of the two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation occurs naturally through mutation and can lead to phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or they can encode polypeptides with altered amino acid sequence. The allelic variant expression also refers to a protein encoded by an allelic variant of a gene. The at least one endopeptidase of the enzyme preparation obtained with the method of the invention can also be functionally fused to another polypeptide, for example a tag; preferably the at least one endopeptidase of the enzyme preparation obtained by the method of the invention is a tagged endopeptidase, more preferably a histidine tagged endopeptidase, more preferably tagged at the C-terminus of the protein, such as the endopeptidase of the sequence comprising or consisting from SEQ ID NO: 2. ; Note that the at least one Actinoallomurus endopeptidase of the enzyme preparation obtained by the method of the invention is in mature form, since the host cell of Streptomyces is able to process the expressed endopeptidase and to secrete it into the culture medium in the form mature. For example, native E40 has sequence SEQ ID NO: 3 and its mature form has sequence SEQ ID NO: 1. As a recombinant endopeptidase, produced in the host cell of S. lividans according to the method of the invention, E40 is secreted in the culture medium as mature E40 of sequence comprising or consisting of SEQ ID NO: 1, despite the entire coding sequence (SEQ ID NO: 3) has been introduced into the host cell. The enzymatic preparation of the invention is preferably enriched in said at least one recombinant endopeptidase of Actinoallomurus; more preferably the enzyme preparation does not comprise endopeptidases other than the at least one recombinant Actinoallomurus endopeptidase; for example, the enzymatic preparation preferably does not comprise endopeptidases of S. lividans origin. The host cell of S. lividans is cultured under fermentation conditions in a suitable culture medium. By "fermentation conditions" we mean the culture conditions of the host cell strain (composition of the medium, parameters of agitation, aeration and temperature) suitable for the growth of the strain and for the production of the compound of interest (recombinant endopeptidase). In particular, the culture medium of the fermentation conditions of the method of the invention comprises suitable nutrients of non-animal origin, sources of carbon and nitrogen and inorganic salts, and is characterized in that it comprises at least 30% (w / v) of sucrose (i.e. at least 30 g of sucrose per 100 ml of medium), preferably about 34% sucrose (w / v). The fermentation conditions according to the present invention therefore comprise the culture of the recombinant host cell in a suitable culture medium comprising at least 30% (weight / vol) of sucrose; the fermentation of the recombinant host cells is carried out at a temperature of between 25 ° C and 30 ° C, preferably between 28 ° C and 30 ° C, more preferably of about 30 ° C; fermentation is preferably continued until the pH of the culture medium is greater than 7 and / or until the glucose in the medium is completely consumed and / or until the recombinant protein secreted in the medium has an activity greater than 8 au / min per mL of supernatant, measured as described herein in Example 5. Preferably, the fermentation is carried out for at least 48 hours, preferably for at least 72 hours, before recovering the supernatant of the culture medium for subsequent purification. Preferably, the culture medium further comprises yeast extract and soy peptone. A particularly preferred culture medium used in the method of the invention is Medium P comprising sucrose (about 340 g / L), glucose (about 20 g / L), yeast extract (about 3 g / L), soy peptone ( about 5 g / L), malt extract (about 3 g / L) and having a pH of about 6.7. Preferably, the method of the invention comprises a first step of culturing a spore suspension of recombinant S. lividans host cells in a first medium comprising glucose, yeast extract and soy peptone at about 30 ° C for 3-7 days, preferably for about 4 days, before the recombinant host cell culture step under fermentation conditions. More preferably, said first medium is medium V comprising glucose (about 20 g / L), yeast extract (about 5 g / L), soy peptone (about 10 g / L), NaCl (about 1 g / L) and with a pH of about 6.7. Preferably, a suitable amount of an antibiotic is added to the culture medium for the selection of recombinant host cells, preferably Apramycin, more preferably at 50 mg / L; optionally said antibiotic is added only to the medium of the first culture phase of the host cells and not to the culture medium of the fermentation phase. Host cells can be cultured by small-scale or large-scale fermentation in laboratory or industrial fermenters. ; The recombinant endopeptidase (s) can / can be recovered directly from the culture medium, as it is / are secreted into it. The supernatant of the culture medium containing the recombinant endopeptidase can be recovered by methods known in the art, for example following centrifugation of the collected culture. In addition to the recovery of the supernatant, the method of the invention preferably comprises one or more steps of purification of the enzymatic preparation from said supernatant. The culture medium can for example be filtered to separate the microbial bodies and the filtrate then processed for the collection of the recombinant endopeptidase (s) by one or more of different procedures, such as: ultrafiltration, concentration at reduced pressure, desalination, precipitation by means of organic solvent, dialysis, gel filtration, adsorption chromatography, ion exchange chromatography, electro-focusing and lyophilization. Preferably, the purification step (s) of the method of the invention comprises / comprise at least one purification step of the enzymatic preparation from the supernatant recovered by affinity chromatography; more preferably it also comprises an ultrafiltration step; more preferably the purification of the enzymatic preparation from the supernatant of the culture medium comprises in series the steps of: filtering the supernatant, preferably through a paper filter and / or through capsule filters having a nominal pore size between 1.0 µm and 0 , 3 µm, to obtain a clarified solution comprising the recombinant endopeptidase (s) of interest; concentrate the clarified solution by ultrafiltration; purifying the enzymatic preparation by affinity chromatography, preferably by affinity chromatography for immobilized metals (IMAC), of the clarified ultrafiltered solution, thus obtaining a final enzymatic preparation comprising (enriched in) the recombinant endopeptidase (s) of interest. In preferred embodiments, the purification further comprises: depigmentation of the eluted fractions by affinity chromatography, preferably by DEAE anion exchange chromatography, concentration and desalting of the depigmented samples, preferably by ultrafiltration of the same, thus obtaining a final enzymatic preparation comprising (enriched in) the recombinant endopeptidase (s) of interest. Representative examples of a preferred method of producing recombinant endopeptidases according to the invention are given below in Examples 2-4 and 15. Preferably, the recombinant expression vector which is introduced into the host cell in order to express the endopeptidases of interest comprise / comprise a polynucleotide which codes for E40 of sequence SEQ ID NO: 1 or for a derivative thereof, preferably the polynucleotide being of sequence SEQ ID NO: 5 or SEQ ID NO: 6, encoding respectively for E40 with sequence consistent in SEQ ID NO: 3 or for an E40 with histidine tag on the C-terminal having a sequence consisting of SEQ ID NO: 4; the polynucleotide can also be of sequence having at least 60%, 70%, 80%, 90% or 95% identity with respect to SEQ ID NO: 5 or SEQ ID NO: 6. The recombinant host cell of S. lividans comprising said expression vector is able to produce the endopeptidase (s) of interest and secrete its mature form into the culture medium. For example, the mature form of E40 of sequence SEQ ID NO: 3 is the endopeptidase of sequence SEQ ID NO: 1 and the mature form of E40 tagged with histidine of sequence SEQ ID NO: 4 is the endopeptidase of sequence SEQ ID NO: 2. It is of particular interest to produce by the method of the invention an enzymatic preparation comprising a histidine-tagged endopeptidase, such as a histidine-tagged E40. Therefore, in a preferred embodiment, the invention is directed to a method according to claim 1, wherein the recombinant expression vector comprises a polynucleotide encoding a histidine-tagged E40 of sequence comprising SEQ ID NO: 2, preferably in wherein the polynucleotide is of sequence SEQ ID NO: 6 and the enzyme preparation purified from the culture medium supernatant comprises the mature form of E40 tagged histidine is of sequence SEQ ID NO: 4. Preferably, the recombinant expression vector used in the method of the invention is the recombinant pIJ86 expression vector. The present invention is therefore also directed to a recombinant pIJ86 expression vector, comprising a polypeptide having sequence comprising SEQ ID NO: 5 or SEQ ID NO: 6, preferably to the recombinant pIJ86 expression vector having sequence SEQ ID NO: 8. The present invention is also directed to a recombinant host cell of S. lividans, preferably of the TK24 strain, comprising said recombinant expression vector; more preferably said host cell is of the DSM 33207 strain, obtained as described herein according to the invention, and deposited on 17 July 2019 at the Leibniz DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstraße 7B 38124 Braunschweig - GERMANY, under the provisions of the Budapest Treaty. The present invention is also directed to the enzymatic preparation obtained with the method of the invention, comprising the mature form of the at least one recombinant endopeptidase of Actinoallomurus with glutenase activity. In a preferred embodiment, the enzyme preparation is in powder form. In a preferred embodiment, the recombinant endopeptidase (s) of Actinoallomurus included in the enzyme preparation is / are isolated from the culture medium supernatant or the enzymatic preparation obtained after purification of the culture medium. The present invention is therefore also directed to an isolated recombinant endopeptidase of Actinoallomurus, obtained from the supernatant recovered from the culture medium of the recombinant host cell of S. lividans in the method of the invention or from the final enzymatic preparation obtained after one or more purification steps according to the method of the invention. Preferably, the isolated recombinant of Actinoallomurus is selected from the group consisting of: sequence recombinant endopeptidase 40 (E40) comprising SEQ ID NO: 1; a biologically active fragment of E40; a natural allelic variant of E40; and a sequence endopeptidase having at least 60%, 70%, 80%, 90% or 95% identity with respect to SEQ ID NO: 1. More preferably, it is an endopeptidase of sequence consisting of SEQ ID NO: 1 or 2, or of sequence having at least 60%, 70%, 80%, 90% or 95% identity with SEQ ID NO: 1 or 2. Optionally, the enzymatic preparation obtained with the method of the invention can be administered orally directly after purification to a subject who needs it. Preferably, the enzyme preparation or the isolated recombinant endopeptidase of Actinoallomurus is formulated in a pharmaceutical formulation suitable for human use. The present invention therefore also relates to a pharmaceutical formulation comprising as an active proteolytic ingredient the enzyme preparation or the isolated recombinant endopeptidase (s) of Actinoallomurus obtained with the methods of the invention. A preferred pharmaceutical formulation is compatible with its intended route of administration, which according to the present invention is preferably oral administration. The pharmaceutical formulation of the invention is therefore preferably an oral pharmaceutical formulation. The pharmaceutical formulations according to the invention can be prepared with the appropriate ingredients to generate a preparation in liquid form, for example in the form of solution, emulsion or in solid form, such as tablets, capsules, semi-solid or powder. The pharmaceutical formulation of the enzyme preparation can be administered in various ways, including those particularly suitable for mixing with foods. The recombinant endopeptidase (s) of Actinoallomurus with glutenase activity present in the pharmaceutical formulation can / can be active before or during ingestion and can / can be treated, for example, with an appropriate encapsulation, to control the times of activity. To prepare an appropriate pharmaceutical formulation according to the present invention, any method for stabilizing chemical or biological material known in the art, including those based on irradiation or temperature modulation or combinations thereof, can be used. The pharmaceutical formulations of the invention are more preferably formulated to release their active ingredients into the gastric fluid. This type of formulations will provide optimal activity in the right place, for example by releasing the enzyme preparation of the invention into the stomach of an individual in need. The present invention also includes a food or a food supplement which comprises as an active proteolytic ingredient the enzyme preparation or the isolated recombinant endopeptidase of Actinoallomurus obtained with the methods of the present invention. The enzyme preparation or the isolated recombinant endopeptidase, obtained by the method of the invention, can also be formulated as a food supplement, prepared, supplied and distributed as described in other previous documents concerning the field of the present invention (WO2011 / 077359, WO2003 / 068170, WO2005107786). For example, the food supplement of the invention can be an enzyme coated or uncoated granulated product that can be readily mixed with food components; alternatively, the food supplements of the invention can form a component of a premix; alternatively, the food supplements of the invention can be a stabilized liquid, an aqueous or oil-based suspension. The pharmaceutical composition or food supplement of the invention can be administered before meals, immediately before meals, during meals or immediately after meals, so that the endoproteases of the enzyme composition of the present invention are released or activated in the lumen. of the upper gastrointestinal tract where endoproteases can integrate gastric and pancreatic enzymes to detoxify ingested gluten and prevent harmful peptides from passing the enterocyte layer. The enzyme preparation can also be formulated as a food; in a preferred embodiment said food is a flour which has been put in contact with said enzymatic preparation or said isolated recombinant endopeptidase of Actinoallomurus capable of degrading gluten. Alternatively, the enzyme preparation and the isolated recombinant endopeptidases of Actinoallomurus of the invention can also be used to produce protein hydrolyzates for food and / or beverages. Preferably, the enzyme preparation, the isolated recombinant endopeptidase of Actinoallomurus, the pharmaceutical formulations, the food or dietary supplement of the present invention are used in the treatment and / or prevention of celiac disease or a disorder associated with celiac disease or non-celiac sensitivity to gluten, preferably any disorder associated with intolerance to gliadin peptide of sequence SEQ ID NO: 6. Disorders associated with celiac disease include: celiac sprue, herpetiform dermatitis, damage to the mucous membrane from celiac disease and diseases resulting from damage to the celiac mucosa, such as iron deficiency anemia, osteoporosis, type 1 diabetes, autoimmune thyroiditis, and enteropathy-associated T-cell lymphomas. Furthermore, the Actinoallomurus endopeptidase with glutenase activity is also able to prevent and / or treat allergies and / or intolerances to nuts and / or peanuts. The present invention is therefore further directed to said use. ; Disorders selected from the group consisting of: celiac disease, a disorder associated with celiac disease, non-celiac sensitivity to gluten and allergy or intolerance to nuts and / or peanuts, can therefore be prevented and / or treated by administering them to a person who needs them an effective amount of the enzyme preparation or the isolated recombinant endopeptidase of Actinoallomurus, optionally formulated as a pharmaceutical formulation, dietary supplement or food of the invention, more preferably by oral administration. ; The present invention is therefore further directed to a method of treating and / or preventing a disorder selected from the group consisting of: celiac disease, a disorder associated with celiac disease, non-celiac sensitivity to gluten and allergy or intolerance to nuts and / or peanuts, administering to a subject in need an effective amount of the required enzyme preparation or the recombinant isolated Actinoallomurus endopeptidase claimed, or the pharmaceutical formulation, food or dietary supplement claimed. Depending on the patient and the condition to be treated and the route of administration, the active amount can be a dosage corresponding to 0.01 mg - 0.5 mg of recombinant endopeptidase per kg body weight per day, e.g. about 20 mg / day. day for an average person. A typical dose of glutenase in patients will be at least about 1 mg / adult, more generally at least about 10 mg; and preferably at least about 50 mg; generally not more than about 5 g, more generally not more than about 1 g and preferably not more than about 500 mg. Dosages will be appropriately adjusted for the pediatric formulation. In children, the effective dose may be lower, for example at least about 0.1 mg or 0.5 mg. Experts will readily appreciate that dose levels may vary as a function of the specific enzyme, the severity of symptoms, and the subject's susceptibility to side effects. ; EXAMPLES; Reagents used in the examples; The actinomycete strain Actinoallomurus A8 came from the collection of the "Insubric Institute of Research for Life Foundation". N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (suc-Ala-Ala-Pro-PhepNA) and N-Succinyl-Ala-Ala-Pro-Phe-7-aminomethyl coumarin (suc-Ala-Ala-Pro -Phe-AMC) came from Bachem (Bubendorf, Switzerland); pepsin, trypsin, nalidixic acid and apramycin came from Sigma-Aldrich (Milan, Italy); mannitol came from Carlo Erba (Cornaredo, Italy); the soy flour came from Cargill (Padua, Italy); soybean agar and peptone came from Conda (Madrid, Spain); sucrose and glucose came from VWR (Leuven, Belgium); the yeast and malt extracts came from BD (Franklin Lakes, NJ, USA) and Costantino (Favria, TO, Italy) respectively; gliadin a 33-mer peptide was synthesized by Biotem (Apprieu, France); S. lividans TK24 strain and pIJ86 expression vector originated from the John Innes Center (Norwich, UK); the strain of the type Saccharomyces cerevisiae X4004-3A (access no. KR348914) was provided by Prof. L. Pollegioni. ; Example 1: Native E40 activity; Native E40 of sequence SEQ ID NO: 3 was purified from the supernatant fraction of a culture of Actinoallomurus A8 isolated from soil (native E40) as described in WO2013083338. Native E40 shows marked glutenase activity at environmental conditions of pH 3-6, with an optimal level at pH 5 (Figure 1, panel A). Under these conditions, gliadins are completely digested (Figure 1, panel B). ; Example 2 - Cloning of the gene encoding E40 in the pIJ86 expression vector and insertion in the host cell of TK24 S. lividans. ; A polynucleotide of sequence SEQ ID NO: 6, encoding a histidine-tagged E40 was amplified by genomic DNA of Actinoallomurus A8 using the Phusion High Fidelity polymerase (Thermo Scientific, Rodano, MI, Italy) and 0.5 µM of primer oligonucleotides (SEQ ID NO: 9 and 10), which introduced restriction sites BclI and HindIII on the 5 'and 3' ends of the polynucleotide sequence and a glycine followed by six histidine residues on the C-terminus of the endopeptidase. The conditions of the PCR cycles were: 3 minutes at 98 ° C, followed by 10 cycles of 10 seconds at 98 ° C, 20 seconds at 67 ° C and 50 seconds at 72 ° C, 20 cycles of 10 seconds at 98 ° C, 70 seconds at 72 ° C, with a final extension at 72 ° C for 5 minutes. The PCR products (SEQ ID NO: 11) were purified from an agarose gel, digested with HindIII and BclI and ligated into an empty pIJ86 vector (SEQ ID NO: 7) digested with HindIII and BamHI to produce the recombinant vector pIJ86 / e40 of sequence SEQ ID NO: 8 (Figure 2). The absence of PCR-generated mutations in the recombinant vector was verified by DNA sequencing. The vector pIJ86 / e40, bearing e40 of sequence SEQ ID NO: 6 under the control of the strong constitutive promoter ermE, was used to transform E. coli ET12567 / pUZ8002. The conjugations were performed according to Flett et al <14> using E. coli ET12567 as donor and S. lividans TK24 <17> as recipient, thus obtaining a recombinant S. lividans TK24 host cell carrying the gene encoding E40 (host cell T24 / pIJ86 / e40). The binding mixture was spread onto agar, mannitol and soy meal plates (8 g / L mannitol, 8 g / L soy meal, 8 g / L agar, MS) containing 10 mM MgCl2 and incubated at 28 ° C . After 16-20 hours, 1 ml of distilled water containing 50 µg / ml of nalidixic acid and 30 µg / ml of apramycin was added to the surface of each plate and spread with a glass rod and further incubated at 28 ° C until when colonies of exconjugant have appeared. Exconjugants were repeatedly plated on MS agar containing nalidixic acid and apramycin; a final spore suspension, prepared according to Kieser et al <15>, was stored at -80 ° C in 20% glycerol. ; Example 3 - Expression of E40 by recombinant S. lividans TK24; The recombinant TK24 / plJ86 / e40 host cells of S. lividans of Example 2 were inoculated in Erlenmeyer flasks (50 ml) containing 20 ml of Medium V (as defined above) with the addition of apramycin 50 mg / L and incubated on a rotary shaker at 200 rpm at 30 ° C. After 5 days of growth, the culture was inoculated in an Erlenmeyer flask (500 ml) containing 100 ml of Medium P (as defined above) with the addition of 50 mg / L Apramycin and incubated under the same conditions for 8 days. Alternatively, for a 15 L scale fermentation, the 5 day culture cultured in Medium V was first inoculated into three 500 ml Erlenmeyer flasks containing 100 ml of Medium V and incubated under the same conditions; then, after 72 hours, the cultures of the flasks were collected, grouped and used to inoculate a fermenter (Biostat Cplus, Sartorius Stedim, Gottingen, Germany) containing 15 liters of Medium P with the addition of apramycin 50 mg / L. Fermentation was carried out at 30 ° C under stirring conditions of 450 rpm and the production of recombinant E40 was monitored over time by measuring the enzymatic activity in the culture supernatant, obtained after centrifugation at 11000 g for 6 minutes. Fermentation was stopped after 94 hours, when the enzymatic activity reached 1430 U per ml of supernatant (see Example 5 for the enzyme activity test used here). ; Example 4 - Purification of the enzymatic preparation from the supernatant of the culture medium of S. lividans TK24 / plJ86 / e40; The culture TK24 / plJ86 / e40 of S. lividans collected from Example 3 was centrifuged for 90 minutes at 4120g and the recovered supernatant was collected and filtered on Rapida A paper (Enrico Bruno, Turin, Italy) and further clarified twice on opticap polyethersulfone capsules (nominal pore sizes 1.0 µm and 0.5 µm) (Merck, Vimodrone, Italy ). Subsequently, the clarified solution was concentrated 10 times with the ultrafiltration system (TFF1) using TFF Pellicon 3 Ultracel cellulose cassettes (nominal MW cut-off 10 kD) and added to 0.5 M NaCl and 50 mM Na2HPO4 pH 7.2. The enzyme was purified by affinity chromatography for immobilized metals (IMAC) using Ni Sepharose® 6 Fast Flow resin (GE Healthcare, Milan, Italy). The IMAC column was equilibrated with 5 volumes of phosphate buffer (pH 8.0), elution was performed with 5 volumes of 250 mM imidazole, 50 mM phosphate buffer (pH 8.0), the eluted fractions (330 ml) were adjusted at pH 6.3 with formic acid and depigmented by DEAE (GE Healthcare) anion exchange chromatography. The depigmented samples were concentrated and desalted using an ultrafiltration system (TFF2, Pellicon, nominal cut-off of 10 kD MW). Before lyophilization, mannitol and trehalose (15 and 5 mg / ml respectively) were added to the sample to improve the crystallization process <16>. ; The protein content of the purified sample was 6% (measured by BCA assay) with 41000 U / mg of protein, equivalent to approximately 8 mg of protein / ml of culture supernatant. The efficiency of the purification process was demonstrated by SDS-PAGE, in which recombinant E40 migrated in the main band at ~ 40 kDa (Figure 3, inset D, lane 1, arrow). Analysis of the N-terminal sequence of the SDS-PAGE band at 40 kDa confirmed the predicted mature form of recombinant E40. ; Example 5 - Enzymatic activity of the enzymatic preparation. Enzyme activity assays were performed in 96-well microtiter plates (clear, flat bottom) using the Infinite 200 PRO plate reader (Tecan, Männedorf, CH). Twenty microliters of samples comprising E40 in a concentration range of 10-50 nM, were pre-incubated for 5 minutes at 37 ° C, and then added to 180 µl of suc-Ala-Ala-Pro-Phe-pNA 220 µM in phosphate citrate buffer (0.1 M citric acid, 0.2 M disodium hydrogen phosphate, pH 5). The samples were incubated for 20 minutes at 37 ° C. The pNA was detected at 410 nm, reading at 5 min intervals. ; Enzyme activity is determined by the linear increase in absorbance over time: 1 activity unit (AU) is defined as the amount of enzyme capable of releasing 1 arbitrary absorbance unit (aau) in 1 minute and measured as " arbitrary absorbance units produced per minute "(aau / m): ;; It is usually calculated in the range of 5-15 '(T1 = 5 and T2 = 15 minutes, respectively). To correctly evaluate E40 production, each sample must be diluted to provide a linear increase in absorbance over time; 1:40 dilution is used for supernatant samples from the currently used E40 manufacturer. ; The production of E40 is expressed in AU / mL of supernatant. ; The same E40 quantification method is used for samples derived from all downstream process steps and expressed in AU / mL or AU / mg for liquid or solid samples respectively. 1 unit (U) is defined as the amount of enzyme that released 1 µmole of pNA per minute. Under our assay conditions: au per µmole of pNA = 0.006; 1 U = AU / 0.006. The activity of the sample was expressed in Enzyme Units per ml (in the case of supernatant samples) or per mg of solid material (in the case of the final preparation of enzyme powder containing recombinant E40). ; The optimal pH was determined using the same substrate that was prepared in 0.2 M ammonium citrate for pH 2 or in phosphate citrate buffer at the pH range of 3-8, respectively. ; For zymographic analysis, samples containing E40 were run by non-reducing SDS-PAGE (12% polyacrylamide) at 100 V using a Tetra-cell Mini-PROTEAN system (Bio-Rad, Milan, Italy). The gel was washed twice with citrate-phosphate / phosphate buffer (pH 5.0) and then incubated with the same buffer containing 100 µM suc-Ala-Ala-Pro-Phe-AMC. E40 activity was visualized in the gel exposed to UV light. ; The proteolytic activity of E40 was also evaluated using gelatin as substrate, after electrophoretic run, the gel was washed with phosphate citrate buffer pH 5.0 and superimposed on a 10% zymogram gelatin gel (Bio-rad) balanced with the same swab for 10 minutes. The two overlapping gels were incubated at 37 ° C for 2 hours, then the gelatin gel was stained with Coomassie Brilliant R250. Gelatin digestion was visualized after gel discoloration as clear lysis bands, due to the proteolytic action of proteases diffused from the PA gel to that of the zymogram. ; The monitoring of the enzymatic activity in the supernatant of the cultures grown in flasks in Example 3 demonstrated a stable and continuous production of recombinant E40, up to 8 days of fermentation (Figure 3, panel A). As with native E40, recombinant E40 was active in the pH range of 3-6, with an optimal level at pH 5 (Figure 3, panel B). Zymographic analysis, conducted at pH 5, showed that recombinant E40 was the only active protein in culture supernatants (Figure 3, panel C). ; Recombinant E40 is also the only protein in the purified enzyme preparation obtained in Example 4 that is active against the chromogenic substrate suc-Ala-Ala-Pro-Phe-AMC (Figure 3, inset D, lane 2) and also against a generic protein substrate such as gelatin (Figure 3, panel D, lane 3), thus excluding the presence of any endoprotease other than recombinant E40 in the preparation of the final purified enzyme. The activity of the purified enzyme preparation comprising the enzyme preparation of recombinant E40 at different pH values was evaluated with the substrate suc-Ala-Ala-Pro-Phe-pNA, pH range 2 to 8 (Figure 3, panel E ). It was found to be maximally active at pH 5 and a significant residual activity (~ 40%) was maintained at pH 3. These results predict optimal action in a postprandial gastric environment, when the pH is above 3 for more than a ' now <8.9>. Interestingly, recombinant E40 remains significantly active (approximately 50%) at pH 6, also suggesting a prolonged action in the postprandial duodenal compartment where pH is less than or equal to 6 for a long time, after a meal <9 >. Overall, the results support a perfect similarity between the native and recombinant form of E40. The purified enzymatic preparation comprising the recombinant E40, obtained in Example 4 with a method according to the invention, has in fact chemical-physical properties and bioactivity comparable to the native E40 isolated from the Actinoallomurus strain. ; Example 6 - Recombinant E40 is resistant to digestive proteases. ; The resistance of recombinant E40, obtained with the method of the invention, to the proteolytic activity of gastric (pepsin) and duodenal (trypsin) digestive proteases was further evaluated (Figure 4), using suc-Ala-Ala-Pro-Phe -pNA as a substrate. The aqueous solutions of enzyme E40, alone or in combination with pepsin and / or trypsin (0.2 mg / ml of E40, 0.1 mg / ml of pepsin or trypsin), were diluted in citrate-phosphate buffer containing suc -Ala-Ala-Ala-Pro-Phe-pNA at pH 4, 4.5 and 5 for pepsin and at pH 6 and 7 for trypsin. The final concentrations in the reaction mixture were: 200 µM of substrate, 1-4 nM of E40 for incubations at pH 4 to 5, or 4 nM and 7 nM for incubations at pH 6 or 7, respectively (the concentration of E40 has been adjusted to the optimal pH). Digestive protease was maintained in the ratio 1: 2 (w / w) with respect to E40. The reactions (volume of 200 µl in 96-well flat-bottom µ-titol plates) were continued for 110 minutes at 37 ° C. Absorbance was monitored every 10 minutes to determine enzyme activity. Pepsin and trypsin without E40 were processed in the same way and tested under the same conditions as a reference control. Each analysis was performed in duplicate. It should be noted that neither pepsin (Figure 4, boxes A-C) nor trypsin (Figure 4, boxes D-E, E ') hydrolyze the chromogenic substrate. In particular, the cleavage activity of E40 was not affected either by the addition of pepsin / trypsin or by the acidic pH. Conversely, slightly improved E40 activity was observed in the presence of pepsin (Figure 4, panels A-C), possibly due to the unmasking of the E40 catalytic site by pepsin. Overall, these results confirm E40's resistance to major human digestive proteases under physiological conditions. Example 7 - Digestion of 33-mer immunodominant gliadin peptides by E40. The degradation of the 33-mer peptide of gliadin was monitored by LC-MS / MS, before (Figure 5, panel A) and after (Figure 5, panels BE) digestion by recombinant E40. Digestion was performed in a 96-well U-bottom microtiter plate; 33-mer was diluted in phosphate citrate buffer (pH 5) at 5 mg / ml, mixed with the enzyme preparation comprising recombinant E40 diluted in the same buffer (1:48 mole ratio enzyme to substrate) and incubated at 37 ° C. At time points of 0, 15, 30 and 60 minutes, aliquots (10 µl) were taken and the reactions were stopped by boiling for 3 minutes before being analyzed by LC-MS / MS. ; Samples were analyzed using the Accela Instrument HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled to both the UV detector and the LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific). In particular, a slight hydrolysis of 33-mer was observed as soon as the incubation began (Figure 5, panel B, 0 minutes); the native 33-mer peptide signal completely disappeared after only 15 minutes (Figure 5, panel C). More specifically, MS / MS spectra analysis demonstrated that E40 activity degraded all 33-mer immunotoxic sequences. Cleavage of E40 occurs between the F-P and Q-L residues of the gliadin, leading to short peptides lacking the stimulating capacity of T cells (Figure 5, F) <3>. This result was also observed at a more diluted E40 concentration (1:96, molar ratio): in this case the complete degradation of 33-mer took 30 minutes (not shown). ; Example 8 - Digestion of complete gliadin by E40. The ability of E40 to hydrolyze harmful peptides into complete gliadin proteins was evaluated by HPLC analysis (Figure 6). Gliadin was extracted from whole wheat flour of Triticum aestivum (Cultivar Sagittario) according to Mamone et al <4>. Gliadin (1 mg) was dissolved in 1 ml of 0.1 M ammonium acetate pH 4 and incubated with E40 enzymes (enzyme: substrate, 1:20), at 37 ° C for different time points (0.15, 30, 60, 120, 240 minutes). Enzymatic hydrolysis was stopped by boiling the samples for 5 minutes. The samples were freeze-dried and stored at -40 ° C until further chemical analysis. ; SDS-PAGE was performed on a Mini-PROTEAN Tetra-cell system (Bio-Rad, Milan, Italy). Samples of digested gliadin were dissolved in Laemli buffer (Tris – HCl 0.125 M pH 6.8, 5% SDS, 20% glycerol, 0.02% bromophenol blue) and loaded onto precolate 12% acrylamide gel (Bio-Rad). Electrophoresis was performed under non-reducing conditions, omitting βmercaptoethanol in Laemli's buffer. Protein bands were visualized with silver blue (Coomassie Brilliant Blue G-250) and scanned using a LABScan scanner (Amersham Bioscience / GE Healthcare, Uppsala, Sweden). RP-HPLC was performed on an RP-HPLC using an Agilent Technology HP 1100 modular system (Palo Alto, CA, USA). The digested gliadin samples were suspended in 0.1% TFA and separated by C18 column (Aeris PEPTIDE, 3.6 µm, 250 × 2.10 mm i.d., Phenomenex, Torrance, CA, USA). Eluent A was 0.1% TFA (v / v) in Milli-Q water, eluent B was 0.1% TFA (v / v) in acetonitrile. The column was equilibrated to 5% B. The peptides were separated by applying a linear gradient of 5-70% B for 90 minutes at a flow rate of 0.2 ml / min. Chromatographic separation was performed at 37 ° C, using a thermostatic column holder. Column effluent was monitored at 220 and 280 nm with a UV-Vis detector. Due to the complex mixture of complete gliadins, digestion with the enzyme preparation comprising recombinant E40 (1:48, molar ratio) was extended up to 240 minutes of incubation (Figure 6, panel B-G). The undigested gliadin sample was run as a reference control (Figure 6, panel A). As expected, the chromatographic profile was drastically changed, as the peaks assigned to α-, β- and γ-gliadin were significantly reduced after 30 minutes (Figure 6, panel C) and disappeared between 60 and 120 minutes of E40 digestion. (Figure 6, boxes D and E). The profiles of the digested products at 180 min and 240 min (Figure 6, panels F and G) were similar, indicating that no further degradation occurred. ; Example 9 - E40 proteolytic degradation of gluten epitopes of T and humoral cells. ; It was subsequently evaluated whether the enzymatic activity of recombinant E40 effectively neutralized: i) the ability of gluten proteins to bind G12 antibodies and ii) the stimulation of an immune response mediated by T cells, measured by the release of IFN-γ . For this purpose, gliadin proteins were suspended in 1 ml of 0.1 M ammonium acetate and digested with E40 alone or with E40 co-incubated with gastrointestinal proteases, pepsin and trypsin / chymotrypsin, at pH and time points. different, as described in Table 1. ;; ; ;; Table 1.; All enzymes were added in the ratio w / w 1:20 (enzyme: protein) and incubated at 37 ° C, with indicated pH and incubation time. Gliadins digested with E40 / pepsin / chymotrypsin were deamidated by tTGase (Sigma Aldrich), as previously described <4>. Briefly, the enzymatic digestion products of gliadin (500 µg / ml) were incubated with 2 U of guinea pig tTG (T-5398, Sigma, St. Louis, MO, USA) at 37 ° C for 2 hours in PBS with 1 mmol / L of CaCl2. ; To estimate the effect of digestion with E40 on the immunological potential of wheat, the gluten content of the A-L samples was determined by monoclonal antibodies specific for the QPQLPY sequence (Elisa - G12) <10>. Compared to the untreated gliadin samples (samples I and L), the digested sample of E40 (samples A to H) showed a drastic reduction in the gluten content, well below 20 ppm (Table 2). ;; ;; Table 2; In order to evaluate the ability of gliadin preparations to activate a T cell response, the different gliadin enzymatic digestion products (samples A-L in Figures 7A and 7B) deamidated by tTG treatment, were analyzed on cell lines T CD4 + previously established from intestinal biopsies of patients with CD <11> (Table 3). ;; ;; Table 3; Briefly, intestinal mononuclear cells were stimulated with autologous irradiated peripheral blood mononuclear cells (PBMCs) and deamidated gliadin digested with pepsin-chymotrypsin extracted from hexaploid grain. Growing T cells were maintained in culture by repeated stimulation with heterologous irradiated PBMCs and PHAs and IL-2 as growth factor. The peptide specificity of TCLs was assessed by analyzing their reactivity towards a group of gluten immunogenic peptides and revealed a large repertoire of peptide recognition. In functional assays, the immune response of TCLs to gliadin enzymatic digestates (samples A-L, Table 1 and Figure 7A and B) was analyzed by detecting IFN-γ production. T cells (3 × 10 <4>) were co-incubated with autologous irradiated EBV-transformed B lymphoblastoid cell lines, (B-LCL, 1 × 10 <5>), in 200 µL of complete medium (X-Vivo15 supplemented with 5% human serum and penicillin-streptomycin antibiotics, all reagents supplied by Lonza-BioWhittaker, Verviers, Belgium) in 96-well U-bottom plates. After a 48-hour incubation, cell supernatants (50 µL ) were collected for the determination of IFN-γ. Each antigen preparation was tested in duplicates and in at least three independent experiments for each T cell line. IFN-γ was measured by a classic sandwich ELISA using purified biotin-conjugated anti-IFN-γ antibodies purchased from Mabtech ( Nacka Strand, Sweden). The sensitivity of the assay was 32 pg / mL. As expected, all T cell lines produced a high level of IFN-γ when exposed to gliadin digested with pepsynachymotrypsin / trypsin (samples I and L of Table 1, Figure 7A and Figure 7B). In contrast, under all experimental conditions examined, no detectable IFN γ production was measured in E40-digested gliadins-exposed T cells (samples A-H of Table 1 and Figure 7A and Figure 7B). ; Example 10: in vitro digestion of gluten-based food products (bread); In order to evaluate the efficiency of the enzyme E40 as an oral supplement to eliminate gluten intake in patients, an oral digestion model was applied -gastric in vitro. The digestion step of bread (T. aestivum) was performed under simulated physiological conditions, which include oral chewing and the presence of digestive gastric enzymes. The digestion time was selected based on the average transit time of the chyme in the gastric compartments. The E40 enzyme preparation was added at the start of the simulated gastric phase. The destruction of T cell epitopes and toxic peptides was tested using monoclonal antibodies (Elisa-R5 competitive), according to the guidelines of the manufacturer and AOAC. Compared to the samples not treated with E40, the digested bread sample that includes E40 showed a drastic reduction in the gluten content, well below 20 ppm (Table 4). ;; ;; Table 4; Example 11 - In vitro digestion of peanut proteins incubated with recombinant E40 (1:20, e: s) The degradation of peanut proteins incubated in the presence of recombinant E40 was evaluated by SDS-PAGE and HPLC analysis. The results are shown in Figure 9 (A: SDS-PAGE; B: HPLC analysis). ; Example 12 - Proteomic analysis of recombinant E40. A detailed proteomic analysis of an enzyme preparation, obtained in S. lividans according to the method of the invention, was also performed. A sample of the enzyme preparation was run on SDS-PAGE (Figure 8), then the lanes were excised and digested with trypsin, and the peptide mixture was analyzed by LS-MS / MS. The mass spectra were processed by Proteome Discoverer software which revealed the identity of the protein (s) within each gel band (Table 5). ; ; Table 5; Proteomic analysis confirmed that the recombinant E40 protein migrates in the main band at ~ 45 kDa (Figure 8, band 4). SDS-PAGE also highlighted the presence of additional, albeit less intense, bands. These bands were assigned to Streptomyces lividans proteins that were not completely eliminated during the purification process. Some of these contaminants are proteases, but none of these have glutenase properties, confirming the fact that recombinant E40 is the only protein active against gluten in the final enzyme preparation. ; Example 13 - Microbiological assays on enzymatic preparation powders, in the fermentation process and downstream stages; As known, S. lividans TK24 could potentially produce actinorodine (ACT), undecylprodigiosin (UDP) and calcium-dependent antibiotic (CDA), especially if cultivated under fermentation conditions. The broth microdilution assay, standardized by the Clinical and Laboratory Standards Institute (CLSI), was then used to evaluate the presence of antibiotics in samples deriving from enzymatic preparations obtained with the method of the invention. Antimicrobial activity has been tested against test microorganisms (test-mo), known to show no resistance to antibiotics: Staphylococcus aureus ATCC6538 (representative of Gram +); Escherichia coli L47 (Lepetit collection; representative of Gram-); candida albicans L145 (Lepetit collection; representative of the yeast). A two-fold serial dilution of the samples to be tested was performed in sterile 96-well microplates, then inoculated with the test-mo in their respective medium (100 µl / final well volume); each determination was made in triplicate. The inoculated microplates were then incubated for 18, 20 and 24 hours for E. coli, S. aureus and C. albicans, respectively, at 35 ° C. The antimicrobial activity of samples of unknown “antimicrobial” concentration was measured as the end point, ie the maximum dilution that inhibits the growth of the test strain; was determined by visual examination of the microplates with the aid of a magnifying glass. Standard assay control compounds were used: ciprofloxacin (activity against G + and G-), daptomycin (Gramm + only), amphotericin B (C. albicans only); furthermore, apramycin (G and G-) was tested as it is used in the fermentation process and also imidazole, as it can be used to elute E40 from Nickel resin. The standard compounds were tested in the concentration range from 128 to 0.125 µg / ml, with the exception of the imidazole used in the concentration range from 0.35 mM to 250 mM; their antimicrobial activity expressed as MIC (minimum inhibitory concentration) is shown in the following Table 6. ;; ;; Table 6; The activity of three batches of E40 powder enzyme preparation (F30, F34 and F35) was then verified: all three batches analyzed did not show any antimicrobial activity at the highest concentration tested (dilution of sample 1: 4, corresponding to 2.5 mg / ml final powder concentration), demonstrating that no antibiotics are present in the preparation of the enzyme. Control wells containing powdered E40 samples in uninoculated media (i.e. without the mo test) showed no microbial growth. ; The antimicrobial activity of samples deriving from a fermentation cycle of TK24 / pIJ86 / e40 of S. lividans on a 15L scale, with subsequent purification steps, according to the invention, was further tested (Tables 7-9). ;; ;; Table 7 ;; ; Medium with Apra 1: 4, approximately 12.5 µg / ml of Apra; ; Table 8 ;; Table 9; The crop culture supernatant (HV) (obtained by centrifugation at 17664 g for 30 minutes), showed slight activity against E. coli and C. albicans which could be related to the presence of undecylprodigiosin (UDP ), as expected from its known activity pattern. The anticandida activity shown by E1 eluted with IMAC and the flow of DEAE is certainly due to imidazole; the low IMAC eluate activity against E. coli is probably still due to the residual presence of UDP. The final diafiltered TFF2 sample was found to be devoid of any detectable antimicrobial activity. Regarding apramycin, its presence was not detected at the time of crop harvest, although it is added to the production medium at 50 µg / ml and its MIC results are 8 µg / ml towards both S. aureus and E. coli. This is probably due to the decomposition and / or transformation by the action of enzymes. ; Example 14 - Testing for the presence of secondary metabolites in enzymatic preparations of E40 and downstream intermediates; For the evaluation of safety, the search for secondary metabolites produced by TK24 of S. lividans is essential. It is therefore necessary to measure the potential molecules identified in the fermentation supernatant and in the formulated product. For the purpose of quantification, HPLC / MS was selected as the best technique to assess the presence of ACT, UDP and Daptomycin (DAP, belonging to the CDA class). Extraction of antibiotics using solid phase techniques (SPE), with Diaion HP-20 resin (styrenedivinylbenzene), was applied, which allows for better quantitative analysis. Diaion HP-20 is generally used for the extraction of small molecules such as secondary metabolites, pigments etc. with a wide range of polarity. Typically, the resin is contacted with the aqueous solution in a 1:20 v / v ratio for 2 hours, then transferred to a column, washed and eluted with an increasing gradient of methanol in water. The eluted extracts are dried and then analyzed by HPLC / MS. A more appropriate column for small molecules and metabolites was used (Phenomenex Luna C18, 5µm, 2.1x250 mm). The fractions E1 (50% methanol), E2 (100% methanol) and E3 (methanol / isopropanol 90:10) are collected. Daptomycin is eluted in E1 and E2, while Undecylprodigiosin (UDP) is eluted in E2 and E3. The recovery of daptomycin is quantitative, while UDP has a tendency to bind tightly to resins, filters and membranes, so the recovery was partial (20-25%). ; Since apramycin does not bind to HP-20, an alternative extraction method for apramycin was applied. The weak cationic resin of Amberlite IRC 50 in NH4 form was tested and found to bind apramycin quantitatively in a single column transit step. Then, elution with 0.1 M ammonium hydroxide provided complete recovery.; The Luna C18 HPLC column was used by default for samples extracted with a 5 to 90% phase B gradient in 30 minutes and a flow rate of 1.0 ml / min. ; Final tests with 50 mg of E40 powder enzyme preparations (lots F-30, F-34, F-35) were completed and gave the following results (Table 10): ;; ;; Table 10 (* based on analysis of 20 mg of final powder)

These data confirm that none of the analyzed antibiotics can be found in measurable quantities in any of the powders of the three batches of E40 enzyme preparation.

With the application of this methodology, the detection of the same antibiotics in the fermentation culture and in the subsequent downstream purification steps was monitored. The fermentation culture named F-46 was set up for this purpose. The fermentation culture was subjected to the following purification steps, according to a preferred embodiment of the method of the invention:

1. Centrifugation (30 'at 17664 g) to recover the supernatant-HV sample

2. Clarification (PES Opticap capsules of 1.0 and 0.5 micron nominal size)

3. TFF1 concentration (with regenerated cellulose cassette with 10kD cutoff)

4. Ni-based IMAC affinity chromatography. 5. Depigmentation by ion exchange with DEAE resin

6. TFF2 concentration and diafiltration (10 kDa cutoff)

7. Freeze-drying after addition of excipients to obtain the final powder.

Samples of each phase were analyzed by HPLC-MS, searching for the above-mentioned antibiotics by molecular weight. Small volumes (12.5 microliters) were injected. Only UDP was found in measurable quantities. No trace of the others was detected.

Regarding Undecylprodigiosin (UDP), the concentration in each phase was quantified by HPLC-MS (Table 11). As expected, UDP is successfully removed during the downstream process:

Table 11

In conclusion, the enzyme preparation powders comprising E40, obtained with a method according to the invention, do not contain measurable quantities of antibiotics.

Example 15

Two new 15 L fermentations according to the invention were carried out, starting from a starter culture in flasks incubated for about 42 hours. The two cultures, named F47 and F48, were harvested after 99 and 94 hours respectively under fermentation conditions. The collected cultures were subjected to centrifugation (Beckmann J-2-21; Rotor JA-10; 10,000 rpm / 17700 g, 15 min) and filtration on paper, and the supernatant was then microfiltered (clarification step) in three successive steps (starting from 1 µm filtration, up to 0.3 µm filtration). Ultrafiltration (TFF1) of the clarified solution was then applied, followed by IMAC nickel affinity chromatography of the ultrafiltration permeate, performed with Ni Sepharose 6 FastFlow resin (GE Healthcare) as previously described. An optional DEAE anion exchange depigmentation chromatography step follows: the elution fractions from IMAC were adjusted to pH 6.3 with formic acid, transferred to a DEAE Sepharose CL-6B column equilibrated with ammonium formate 20 mM pH 6.3 and collected for gravimetry. The ultrafiltration system for smaller volumes (TFF2 diaftraction) (nominal MW limit: 10 kD) was prepared for the ultrafiltration of the elution fraction from DEAE, then the desalting solution prepared with ammonium formate 10 was added in portions. mM pH 6.3 in order to remove residual imidazole. The solution may become cloudy and require centrifugation to discharge a precipitate. The solid removed does not include recombinant E40. This can be avoided by keeping the solution more diluted at this stage. A 3: 1 solution of mannitol and trehalose, corresponding to 8 micrograms of total sugar per AU, was added to the final solution of TFF2 and then lyophilized.

The final yield of the powder was 1437 mg. A sample of the final powder was dissolved at 10 mg / mL in a 20% EtOH aqueous solution and checked for enzymatic activity and protein content, then subjected to SDS-page.

The total protein content BCA assay is approximately 14% in the final batch powder (100% excluding added sugars).

The production of E40 is about 25 mg / L of supernatant (about 20 mg / L of fermentation volume).

The microbiological activity of the above preparations was tested at the highest concentration of 5 mg / ml (stock solution at 10 mg / ml in dH20) and after two serial dilutions. The activity of the dust generated by F47 / F48 is shown in Table 12: no activity was shown at the highest concentration tested compared to any of the tests analyzed.

Table 12

This result confirms that the final E40 preparation is free of detectable antibiotics.

Comparative example 1

The X4004-3A type strain of Saccharomyces cerevisiae (S. cerevisiae) was used as a host for the heterologous expression of E40. Two different synthetic E40 genes have been designed which differ in the presence or absence of the coding sequence for the pro-enzyme; in both genes the native E40 secretion signal sequence was replaced with the modified factor α preproleader sequence <20>. Furthermore, in both synthetic genes the use of the codon was optimized based on that of S. cerevisiae using the Codon Optimization Tool software. Sequences corresponding to XbaI and HindIII restriction sites Both genes were also added. The resulting genes, one coding for the inactive precursor (proE40, SEQ ID NO: 13) and the other for the mature enzyme (matE40, SEQ ID NO: 14) were cloned into both inducible pEMBL and pVT expression plasmids - Constitutive U, both carriers of the URA3 gene for auxotrophic selection in S. cerevisiae <19, 21> and then introduced into ElectroMAX DH10B E. coli cells by electroporation method. Transformants were selected on ampicillin-containing LB agar plates (100 µg / mL) incubated overnight at 37 ° C. The correctness of the constructs was confirmed by sequencing. Transformants were selected on ampicillin-containing LB agar plates (100 µg / mL) incubated overnight at 37 ° C. The pEMBL / E40 and pVT-U / E40 plasmids were transferred to S. cerevisiae host strain X4004-3A using the lithium acetate method (Elble, 1992). Cells were plated on selective plates (uridine-free medium) and incubated at 30 ° C for 5-6 days.

A single colony of S. cerevisiae clones containing the E40 genes was removed from the selective plate, inoculated in 100 ml of minimum medium (0.68% yeast nitrogen base, YNB, 2% glucose, 50 mg / L of amino acid L-Lys-L-Met-L-Trp, potassium phosphate 67 mM, pH 6.0) for 3 days at 30 ° C.

An aliquot of cells was removed and inoculated into a final volume of 50 mL of minimum medium in a 500 mL flask starting at OD600nm = 0.25. Incubation continued until OD600nm reached 0.5 (6 hours), then 8 ml were inoculated into a 500 ml flask containing 72 ml of expression medium (yeast extract 10 g / L, peptone 20 g / L and 2% glucose for pVT-U / E40 plasmid or 2% galactose for pEMBL / E40 inducible expression plasmid) for 10 days. For the protein expression tests, cells were cultured at 30 ° C and harvested at different times.

The expression of recombinant E40 was verified in culture supernatants by an activity assay using the synthetic substrate suc-Ala-Ala-Pro-Phe-pNA, following the absorbance at 410 nm during incubation at pH 5 and 37 ° C. Enzymatic activity was expressed in absorbance units (au) per minute per ml. The maximum activity was detected in the supernatant of cells transformed with the pVT-U / matE40 plasmid and grown for 6 days: a value of 0.9 au / min / mL was shown. A parallel fermentation test of S. cerevisiae transformed with the four constructs was performed, resulting in similar expression levels (0.73 ∆Abs min <-1> mL <-1>). On the basis of these results, a Western blot analysis was performed on supernatants of cells transformed with pVT-U / matE40 and pEMBL / matE40 plasmids collected at different times. Western blot analysis showed that when the pEMBL-matE40 plasmid was used, the protein was expressed after 6 days of growth; for the pVT-U / matE40 plasmid, protein expression was detected after 3 days.

S. cerevisiae can therefore be used as a recombinant producer of E40. However, the modest expression of recombinant E40 obtained (less than 1 mg of E40 per ml of supernatant) makes this production system currently unsuitable for industrial exploitation.

In conclusion, the recombinant expression of E40 in S. lividans host cells according to the method of the invention results in the secretion of active E40. All the physico-chemical characteristics and biological properties of the native form of Actinoallomurus A8 are maintained by the recombinant glutenase, eg. stability and activity at post-prandial gastric pH, resistance to digestion of pepsin, high efficiency in the extensive degradation of the most immunogenic peptide of α-gliadin, such as the 33-mer peptide enriched with Pro-Gln (SEQ ID NO: 12) , as well as complete gliadin proteins.

The enzymatic preparation obtained with the method of the invention therefore provides high quantities of active recombinant E40 and is also substantially free of antibiotics, therefore being suitable for human use, preferably after a correct formulation as a pharmaceutical formulation or food supplement.

Claims (11)

CLAIMS Method for producing an enzymatic preparation comprising at least one recombinant Actinoallomurus endopeptidase having glutenase activity, preferably in its mature form, preferably selected from the group consisting of: endopeptidase 40 (E40) of sequence comprising SEQ ID NO: 1; a biologically active fragment of E40; a natural allelic variant of E40; and a sequence endopeptidase having at least 60%, 70%, 80%, 90% or 95% identity at SEQ ID NO: 1; the method comprising in series: a) culturing a recombinant host cell of Streptomyces lividans, preferably of the TK24 strain, in a culture medium under fermentation conditions, said recombinant host cell comprising a recombinant expression vector for the heterologous expression of the at least one recombinant endopeptidase of Actinoallomurus, the recombinant expression vector comprising a polynucleotide coding for said at least one recombinant Actinoallomurus endopeptidase functionally linked to regulatory sequences capable of directing the expression of said at least one recombinant Actinoallomurus endopeptidase in the recombinant host cell; in which the culture medium comprises at least 30% (w / v) of sucrose; b) recovering the supernatant of the culture medium comprising at least one recombinant Actinoallomurus endopeptidase; And c) purifying from said supernatant the enzymatic preparation comprising at least one recombinant Actinoallomurus endopeptidase, preferably in which the purified enzymatic preparation comprises the mature form of E40 of sequence comprising or consisting of SEQ ID NO: 1, more preferably an E40 with histidine tag of sequence comprising or consisting of SEQ ID NO: 2; preferably in which the enzymatic preparation is in powder form. Method according to claim 1, wherein the fermentation conditions comprise the culture of the recombinant host cell at a temperature of between 28 ° C and 30 ° C, preferably of about 30 ° C. Method according to claim 1 or 2, wherein in step b) the culture under fermentation conditions is carried out for at least 48 hours, preferably for at least 72 hours. The method of claims 1 to 3, wherein the recombinant expression vector comprises an E40 encoding polynucleotide of sequence comprising SEQ ID NO: 1, preferably encoding E40 of sequence consisting of SEQ ID NO: 3 or SEQ ID NO: 4; where the polynucleotide is of sequence SEQ ID NO: 5 or SEQ ID NO: 6 or sequence with at least 60%, 70%, 80%, 90% or 95% identity at SEQ ID NO: 5 or SEQ ID NO : 6, more preferably wherein the polynucleotide is of sequence SEQ ID NO: 6 encoding a histidine tagged E40 of sequence consisting of SEQ ID NO: 2; more preferably wherein the enzymatic preparation purified from the culture medium supernatant comprises the mature form of histidine-tagged E40, said mature form having sequence SEQ ID NO: 2. Method of any one of claims 1 to 4 wherein the recombinant expression vector is recombinant pIJ86, preferably wherein the recombinant expression vector is of sequence SEQ ID NO: 8. 6. Enzymatic preparation obtained by the method of any one of claims 1 to 5. 7. Isolated recombinant Actinoallomurus endopeptidase having glutenase activity, isolated from the culture medium supernatant recovered in step c) of any one of claims 1 to 5 or from the enzymatic preparation of claim 5; preferably in which the isolated recombinant Actinoallomurus endopeptidase is selected from the group consisting of: endopeptidases 40 (E40) of sequence comprising SEQ ID NO: 1; a biologically active fragment of E40; a natural allelic variant of E40; and a sequence endopeptidase having at least 60%, 70%, 80%, 90% or 95% identity with respect to SEQ ID NO: 1. 8. Recombinant pIJ86 expression vector, comprising a polypeptide having sequence consisting of SEQ ID NO: 5 or SEQ ID NO: 6, preferably the recombinant pIJ86 expression vector having sequence SEQ ID NO: 8. 9. Recombinant host cell of Streptomyces lividans, preferably of the TK24 strain, comprising the recombinant expression vector of claim 8; preferably the recombinant host cell of Streptomyces lividans being of the DSM 33207 strain. 10. Formulation comprising as an active proteolytic ingredient the enzymatic preparation of claim 6 or the isolated recombinant Actinoallomurus endopeptidase of claim 7; preferably the formulation being a food or a food supplement or a pharmaceutical formulation; more preferably the formulation being a food supplement, or a flour which has been contacted with the enzymatic preparation of claim 6 or the isolated recombinant Actinoallomurus endopeptidase of claim 7. 11. Enzyme preparation according to claim 6 or the isolated recombinant Actinoallomurus endopeptidase of claim 7, or the wording of claim 10, for use in the treatment and / or prevention of celiac disease or a disorder associated with celiac disease or sensitivity to non-celiac gluten, preferably for use in the treatment and / or prevention of any disorder associated with gliadin peptide intolerance of sequence SEQ ID NO: 6, or for use in the treatment and / or prevention of allergies or intolerances to walnuts and / or peanuts.