WO2016120764A1

WO2016120764A1 - Aspergillus oryzae prolyl endopeptidases and use thereof in degradation of polypeptides

Info

Publication number: WO2016120764A1
Application number: PCT/IB2016/050312
Authority: WO
Inventors: Michel Monod; Eric Grouzmann
Original assignee: Centre Hospitalier Universitaire Vaudois (Chuv)
Priority date: 2015-01-26
Filing date: 2016-01-22
Publication date: 2016-08-04

Abstract

The invention relates to a novel enzyme composition comprising Aspergillus oryzae prolyl endopeptidases having unique catalytic properties. The invention further relates to methods for producing the enzyme composition as well as a pharmaceutical composition and a food supplement containing the enzyme composition and its use in the degradation of polypeptides. The invention further relates to fungus Aspergillus oryzae strains deposited under accession number IHEM 26503 and IHEM 26504.

Description

ASPERGILLUS ORYZAE PROLYL ENDOPEPTIDASES AND USE THEREOF IN

DEGRADATION OF POLYPEPTIDES

Field of the Invention

The invention relates to a novel enzyme composition comprising Aspergillus oryzae prolyl endopeptidases having unique catalytic properties. The invention further relates to methods for producing the enzyme composition as well as a pharmaceutical composition and a food supplement containing the enzyme composition and its use in the degradation of polypeptides. The invention further relates to fungus Aspergillus oryzae strains deposited under accession number IHEM 26503 and IHEM 26504.

Background of the Invention

Celiac disease (CD) is a digestive genetically determined disorder that damages the small intestine and interferes with absorption of nutrients from food. People who have CD cannot tolerate a protein called gluten, which is found in wheat, rye and barley. The disease has a prevalence of about 1 :200 in most of the world's population groups and the only treatment for CD is to maintain a life-long, strictly gluten-free diet. For most people, following this diet will stop symptoms, heal existing intestinal lesions, and prevent further damage. The disease is more frequent in the paediatric population. Patients are suspected of having CD when they are presenting gastrointestinal or malabsorption symptoms. The principal toxic components of wheat gluten are a family of proline- and glutamine- rich proteins called gliadins, which are resistant to degradation in the gastrointestinal tract and contain several T-cell stimulatory epitopes (33 mer and 31-49 (p31-49) peptides). The 33-mer peptide is an excellent substrate for the enzyme transglutaminase 2 (TG2) that deamidates the immunogenic gliadin peptides, increasing their affinity to human leucocyte antigen (HLA) DQ2 or DQ8 molecules and thus activating the T cell-mediated mucosal immune response leading to clinical symptoms. The toxicity of these fragments may be due to an overexpression of transferrin receptor in CD allowing intestinal transport of intact peptide across the enterocyte. Thus the peptides can escape degradation by the acidic endosome-lysosomal pathway only in patients with active CD and can reach the serosal border unchanged.

Since in patients with coeliac disease the gastrointestinal tract does not possess the enzymatic equipment to efficiently cleave the gluten-derived proline-rich peptides, driving the abnormal immune intestinal response, another therapeutic approach relies on the use of orally active proteases to degrade toxic gliadin peptides before they reach the mucosa. Oral therapy by exogenous prolyl-endopeptidases able to digest ingested gluten was therefore propounded as an alternative treatment to the diet.

It has been demonstrated (Shan et al, Science 2002) that an exogenous PEP (prolyl endoprotease) derived from Flavobacterium meningosepticum helps to digest gliadin peptides. The addition of PEP either in vitro in the presence of brush border membrane (BBM) extracts or during in vivo perfusion of rat small intestine caused a rapid degradation of the 33 mer peptide and a loss of its capacity to stimulate gliadin-specific T cells. A randomized, double-blind, crossover study in twenty asymptomatic patients with histologically proven celiac sprue involving two 14-day stages has been performed using gluten pretreated with recombinant PEP from F. meningosepticum. The result of this study was not very satisfactory mainly because PEP from F. meningosepticum exhibits pH optima near neutrality and is not active in the stomach.

To circumvent this problem, PEP was associated to a glutamine-specific endoprotease B, isoform 2 from Hordeum vulgare (EP-B2), a cysteine-protease derived from germinating barley seeds that is activated at acidic pH and by pepsin and can efficiently hydrolyse gliadin in vitro in conditions mimicking the gastric lumen (Bethune et al, Chem. Biol, 2006). Another study proved that the combination of EP-B2 with PEP from F. meningosepticum improve the breakdown of gluten. Also another reports that a PEP deriving from Aspergillus niger, deploying its main activity under acid conditions in the stomach, can start to degrade gliadin before it reached the intestinal lumen. (Stepniak et al, Am J. Physiol. Gastrointest. Liver Physiol., 2006). WO2005/019251 (Funzyme Biotechnologies SA) provides leucine aminopeptidase (LAP) of two different fungal species, Trichophyton rubrum and Aspergillus fumigatus in combination with dipeptidyl peptidase IV (DppIV). These enzymes have been evaluated for cleavage of the 33 mer under neutral pH condition since the optimal activity of LAPs were estimated around 7.0 with a range of activity between pH 6 and 8. However, a limitation of these enzymes relies on their optimum activity at neutral pH precluding a possible breakdown of gliadin in the gastric fluid.

WO 2011/077359A2 (Centre Hospitalier Universitaire Vaudois (CHUV) provides an oral enzyme therapy approach combining a dual enzyme composition, the A. fumigatus prolyl protease AfuS28 and at least one tripeptidylpeptidase of the S53 family (sedolisin), to degrade proline rich peptides, in particular those generated by endoprotease digestions of gluten. The production of an enzyme extract by a fungus species used in food fermentation would be the ideal choice to allow an application as food supplement in man. Therefore, Aspergillus oryzae was chosen to produce large amount of AfuS28 and one tripeptidylpeptidase of the sedolisin family (SedB). However, AfuS28 is produced with a low yield as a recombinant enzyme in A. oryzae.

The problem to be solved to confer a potential therapeutic value to an enzyme or enzyme composition are the following: the enzymes must be resistant to degradation by other gastrointestinal enzymes, efficient in the environment where the 33-mer is produced, must present a high proteolytic activity toward gluten peptides, should be active at a large range of acidic pH to neutral pH (for example pH 3 to pH 8) to enable 33-mer degradation in the gastric and intestinal fluids, should be able to access a complex composition of gluten hindered by other components of normal foodstuffs eventually baked or cooked, have fast degradation kinetics and be produced in large amounts during the fermentation process.

The present invention was able to solve this problem in the present invention by providing an enzyme composition having unique catalytic properties.

Summary of the Invention

In one aspect, the invention provides an enzyme composition comprising a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and/or a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

In a further aspect, the invention provides a pharmaceutical composition comprising an effective amount of the enzyme composition of the invention and at least one

pharmaceutically acceptable excipient, carrier and/or diluent.

In another aspect, the invention provides a food supplement comprising the enzyme composition of the invention.

In another aspect, the invention provides the enzyme composition of the invention for use in a method for treating and/or preventing a condition selected from the group comprising celiac disease, digestive tract bad absorption, an allergic reaction, an enzyme deficiency, a fungal infection, mycoses, Crohn disease, sprue and wound healing. In another aspect, the invention provides a method of degrading a polypeptide substrate, wherein said method comprises contacting the polypeptide substrate with the enzyme composition of the invention.

In another aspect, the invention provides a method of detoxifying gliadin, wherein said method comprising contacting gliadin containing food product with an effective dose of the enzyme composition of the invention. In another aspect, the invention provides a method for improving food digestion in a mammal, wherein said method comprising oral administration to the said mammal of the enzyme composition of the invention.

In another aspect, the invention provides a method for producing the enzyme composition of the invention, wherein said method comprises

(a) introducing into a host cell a nucleic acid encoding for

i. a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 4, and/or

ii. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5, (b) cultivating the cell of step (a) in a culture medium under conditions suitable for producing the enzyme composition; and

(c) recovering the enzyme composition.

(a) cultivating Aspergillus oryzae strain having accession number IHEM 26504 and/ 'or Aspergillus oryzae strain having accession number IHEM 26503 in a culture medium under conditions suitable for producing the enzyme composition; and

(b) recovering the enzyme composition.

In further aspects, the invention provides a recombinant Aspergillus oryzae strain, wherein the strain is fungus Aspergillus oryzae deposited under accession number IHEM 26503 and a recombinant Aspergillus oryzae, wherein the strain is fungus Aspergillus oryzae deposited under accession number IHEM 26504. Brief description of figures

Figure 1 shows sum of the MS peak intensities of all detected peptides of size equal to nine of more amino acids, the minimal size of immuno stimulatory epitopes for CD4+ T cells in CD patients (a, b) and to 7-9 amino acids (c, d). Incubations were performed at pH 3.8 (a, c) and pH 7.8 (b, d), using Aos28A+B (red squares), Aos28A (yellow circles), and Aos28B (blue triangles). According to (a), all immunotoxic peptides were completely digested after 20 min at acidic pH by the combination of both enzymes, while 30 to 60 min were necessary when only one enzyme was used.

Figure 2 shows (A) Protein extract from 200 μΐ, of A. oryzae KS36.9 (IHEM 26504) producing AoS28A in GP (1) and GS (2) medium showing ~ 5 and 10 μg of AoS28A, respectively; (B) Purified His₆-tagged AoS28A from an equivalent of 1 ml and 0.3 ml of oryzae culture supernatant in GS medium; (C) Protein extract from 100 iL of A. oryzae KS 1 producing AfuS28 in MM (3) and GP (4) medium showing ~ 0.5 μg of AfuS28. In lane 2 was loaded 10 μΙ_, of culture supernatant of a P. pastoris clone producing AfuS28 (about 2 μg of AfuS28).

Figure 3 shows western blot detection of native and deglycosylated AoS28A (a, b) and AoS28B (c, d) in preparations from A. oryzae RIB40 (a, c) and NRRL2220 (b, d) culture supernatants: Aspergillus oryzae was grown in acidic soy meal protein medium for 48 h. Desalted culture supernatant was filtered through a HPT column, and adsorbed prolyl peptidase activity was subsequently released with phosphate buffer. 1-3 : Protein profile of desalted culture supernatant, HPT column filtrate containing prolyl peptidase that was not adsorbed to HPT, and pooled active fractions eluted from hydroxyl apatite, respectively. 4- 6: Same fractions as 1-3 after endoH treatment. 7-8: AoS28A and AoS28B before (7) and after (8) deglycosylation as controls (0.1 μg of purified enzyme was loaded per lane). The proteins of an equivalent of 100 μΐ of culture supernatant were loaded per lane of the SDS- PAGE gel. Immunolabelling was performed with antiserum raised against AoS28A and antiS28B large peptides as described in the Examples.

Figure 4 shows AoS28A (a) and AoS28B (b) extracted from culture supernatants of A. oryzae overproducing transformants before (1) and after (2) deglycosylation. SDS-PAGE gels (10%) were stained with Coomasie Blue.

Figure 5 shows peak intensities of the 33-mer measured after incubation with enzymes Aos28A+B (red squares), Aos28A (yellow circles), and Aos28B (blue triangles), at pH 3.8 (a) and pH 7.8 (b). The 33-mer was found to be stable for one hour in these experimental conditions without enzyme (data not shown). The 100% values correspond to peak intensities at 0 min.

Figure 6 shows peptides with > 10 amino acids size detected by Orbitrap after digestion for 0 to 60 minutes by Aos28A or Aos28B at pH 3.8 and 7.8. Detected peptides with < 10 amino acids size.

Figure 7 shows degradation of the 33-mer of gliadin by AoS28A+B at pH 3.8 and 7.8: the ion of gliadin is absent while several fragments of MW < 800 (7 amino acids or less) are present.

Figure 8 shows degradation of the 33-mer of gliadin (m/z 1305, z = 3) by AfuS28+SedB or AoS28A+B : the reactional medium (100 μΐ,) contains 40 μg of gliadin and 1.88 + 2.30 μg, or 2.85 + 2.13 μg of enzymes, respectively, on a 20 mM pH 3.8 or 7.8 buffer at 37°C. Every 2.5 or 5.0 minutes, 10 μΙ_, of medium were taken and diluted in 990 μΙ_, of acetonitrile 50% to stop the reaction. All samples were infused in the Xevo TQ-S triple quadrupole.

Figure 9 shows production of an unknown fragment of the 33-mer of gliadin (m/z 1 1 12.05, z = 1) by AfuS28+SedB or AoS28A+B: the reactional medium (100 μΐ.) contains 40 μg of gliadin and 1.88 + 2.30 μg, or 2.85 + 2.13 μg of enzymes, respectively, on a 20 mM pH 3.8 or 7.8 buffer at 37°C. Every 2.5 or 5.0 minutes, 10 μΙ_, of medium were taken and diluted in 990 μΙ_, of acetonitrile 50% to stop the reaction. All samples were infused in the Xevo TQ- S triple quadrupole.

Figure 10 shows production of the YPQPQPF (SEQ ID NO: 29) fragment of the 33-mer of gliadin (m/z 876.8, z = 1) by AfuS28+SedB or AoS28A+B: the reactional medium (100 μΐ,) contains 40 μg of gliadin and 1.88 + 2.30 μg, or 2.85 + 2.13 μg of enzymes, respectively, on a 20 mM pH 3.8 or 7.8 buffer at 37°C. Every 2.5 or 5.0 minutes, 10 μΙ_, of medium were taken and diluted in 990 μΙ_, of acetonitrile 50% to stop the reaction. All samples were infused in the Xevo TQ-S triple quadrupole.

Figure 11 shows production of the YPQPQLP (SEQ ID NO: 30) fragment of the 33-mer of gliadin (m/z 842.98, z = 1) by AfuS28+SedB or AoS28A+B: the reactional medium (100 μΐ,) contains 40 μg of gliadin and 1.88 + 2.30 μg, or 2.85 + 2.13 μg of enzymes, respectively, on a 20 mM pH 3.8 or 7.8 buffer at 37°C. Every 2.5 or 5.0 minutes, 10 μΙ_, of medium were taken and diluted in 990 μΙ_, of acetonitrile 50% to stop the reaction. All samples were infused in the Xevo TQ-S triple quadrupole.

Figure 12 shows plasmid pKS l (pAfuS28) sequence (SEQ ID NO: 23).

Figure 13 shows pAoS28A (pKS36) sequence (SEQ ID NO: 24) Figure 14 shows determination of specific activities of Aos28A (yellow circles) and Aos28B (blue triangles) at different pH values using AAP-pNA as a substrate. Absorbance was measured at 405 nm at 37°C. Detailed description of the Invention

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components. In addition, as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of " and/or "consisting essentially of .

As used in the specification and claims, the term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A", and "B" .

As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

The term "enzyme composition" is equivalent and interchangeable with the term "enzyme cocktail" or "enzyme combination" and refers to a composition containing at least one or more than one enzyme (endopeptidase in the context of the present invention) that digests for example proline rich peptides, proteins or polypeptides, such as gluten.

As used herein, the term "endopeptidases" is synonymous with peptidase, proteases, proteolytic enzyme and peptide hydrolase. The endopeptidases include all enzymes that catalyse the cleavage of the peptide bonds (CO-NH) of proteins, digesting these proteins into peptides or free amino acids. Exopeptidases act near the ends of polypeptide chains at the amino (N) or carboxy (C) terminus. Those acting at a free N terminus liberate a single amino acid residue and are termed aminopeptidases.

As used herein the term "derived" encompasses the terms "originated from", "obtained" or "obtainable from", and "isolated from" and as used herein means that the polypeptide, for example a endopeptidase, encoded by a nucleic acid is produced from a cell in which the nucleic acid is naturally present or in which the nucleic acid has been inserted in supplementary copies.

As used herein, the term "an extract from Aspergillus oryzae" or "an extract derived from Aspergillus oryzae" refers to a clarified aqueous solution that formerly comprised Aspergillus oryzae for example, a suspension of Aspergillus oryzae which was centrifuged at 1000 x G for 10 minutes to sediment the fungi. The culture supernatant fluid is "an extract from Aspergillus oryzae" . "An extract from Aspergillus oryzae" can also mean a clarified cell lysate of Aspergillus oryzae, wherein the fungi are lysed in a suitable buffer and the lysate is centrifuged at 20,000 x G for 30 minutes to sediment the cell debris. Ultracentrifugation clarified cell lysate of Aspergillus oryzae is also "an extract from Aspergillus oryzae" . "An extract from Aspergillus oryzae" can also mean a chromatography fraction containing a 70 kDa protein or a 140kDA protein with gluten-degrading activity (e.g., a prolyl endopeptidase activity) as assayed by for example gliadin zymography.

Aspergillus species grow well at neutral and acidic pH in media containing protein as the sole nitrogen source. These fungi produce different sets of endo- and exopeptidases that degrade extracellular protein sources into amino acids, and di- and tripeptides that are assimilable via transporters. During this process, the main function of endopeptidases is to produce a large number of free ends on which exoproteases or exopeptidases may act. Exoproteases or exopeptidases secreted at neutral or alkaline pH are leucine aminopeptidases (Laps) of the M28 family and a X-prolyl peptidase (DppIV) of the S9 family (see MEROPS peptidase database). Exoproteases or exopeptidases secreted at acidic pH are tripeptidyl-peptidases of the sedolisin family and prolyl peptidases of the S28 family. Leucine aminopeptidases and tripeptidyl-peptidases are non-specific amino peptidases but are not able to cut before and after a proline residue. Conversely, X-Pro sequences can be removed at neutral pH by DppIV, and X-X-Pro sequences are trimmed off at acidic pH by prolyl peptidases of the S28 family. Therefore, DppIV and prolyl peptidases are key enzymes in the digestion of proline-rich proteins by removing roadblocks to Laps and tripeptidylpeptidases, respectively, during the sequential degradation of large peptides into amino acids and short peptides.

Prolyl peptidases are especially interesting in the degradation of proline rich proteins, in particular gliadins which are components of gluten, a protein source found in wheat, rye and barley. Gluten is a complex protein consisting of a mixture of numerous gliadin and glutenin polypeptides. Gluten proteins are rich in proline (15%) and glutamine (35%) residues, a feature that is especially notable among gluten epitopes that are recognized by disease-specific T cells. The principal toxic components of gliadins are proline- and glutamine-rich peptide sequences [e.g. peptides 31-49 and 57-89 (= 33-mer) of a2-gliadin] which are resistant to degradation in the gastrointestinal tract and contain several T-cell stimulatory epitopes. Proline rich nutriments such as glutens in cereals are highly resistant to proteolytic degradation in the gastrointestinal tract by pepsin, trypsin, chymotrypsin and the like. Gliadins are the proximate environmental cause of inflammation in people suffering coeliac disease or gluten intolerance. An enzyme therapeutic approach to prevent the immunotoxicity of peptides derived from gliadin requires that these peptides should be rapidly degraded in the gastric mucosa before entering the intestine.

The present invention identifies two major prolyl endopeptidases of the MEROPS S28 protease family, AoS28A and AoS28B, secreted by A. oryzae when this fungus was growing at acidic pH in a medium containing protein as the sole source of nitrogen. The genes encoding AoS28A and AoS28B were overexpressed in A. oryzae strains under the control of a strong promoter, and transformants producing large amounts of AS28 endopeptidases were selected for further characterization of the enzymes. The proteolytic activity of both enzymes was tested on a proline rich 33-mer of gliadin, which is known to be highly resistant to digestion and the most representative immunotoxic peptide involved in the pathogenesis of coeliac disease. AoS28A and AoS28B efficiently degraded the proline-rich 33-mer of gliadin, especially in acidic conditions. Both enzymes were found to act as strict prolylendopeptidases by preferably cleaving peptide bonds from the N-terminus region of this 33-mer. AoS28A and AoS28B produced similar but not identical patterns of peptides fragments, with different kinetic of apparition of some individual digestion products. The concept of an oral endopeptidase approach to detoxify the gluten is based on the rapid digestion of immunotoxic proteins in smaller non-antigenic peptides of less than 9 amino acids. Figure 1 shows that all peptides equal or larger than 9 amino acids in size were completely digested in the acidic conditions corresponding to those of the stomach by the combination of both enzymes after 20 min. Conversely, 30 to 60 min were necessary when only one of the enzymes was used in the same experimental conditions. A combination of both endopeptidases would be of particular interest in the development of an oral enzyme therapy product for patients suffering intolerance to gluten.

In addition, the digestion of the 33-mer of gliadin, by the combination of AoS28A + B is faster than by the combination of AfuS28 + SedB of the prior art. This faster kinetic is advantageous for the degradation of gliadin in the frame of celiac disease. Therefore, extracellular enzyme extracts from the generated A. oryzae transformants secreting large amounts of AoS28A or AoS28B could be of particular interest in the development of an oral enzyme therapy product for patients suffering intolerance to gluten. Both prolyl peptidases could be for example concomitantly overproduced with a battery of other secreted proteases using a protein fungal growth medium. These additional proteases in a crude fungal extract could increase the efficiency of both prolyl peptidases for gluten breakdown. Thus in addition to a purified enzyme, a fungal extract rich in prolyl peptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest as a cheap preparation to administer to patients suffering from coeliac disease or gluten intolerance. An aspect of the invention discloses an enzyme composition of prolyl endopeptidases, which exhibits a proteolytic activity toward peptides, such as proline rich peptides, at acidic pH, which corresponds to the pH of the gastric fluid, and found that this enzyme composition is also able to degrade the 33-mer of the gliadin. In one embodiment of the invention, an enzyme composition comprises a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and/or a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2. In another embodiment, the enzyme composition of the invention comprises a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and a prolyl

endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

In some embodiments of the invention, the prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 is derived from Aspergillus oryzae deposited under accession number IHEM 26504, and the prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2 is derived from Aspergillus oryzae deposited under accession number IHEM 26503. In another embodiment, the enzyme composition of the invention consists of an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and/or an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2. In another embodiment, the enzyme composition of the invention consists of an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2. In some embodiments of the invention, the host cell overexpressing the prolyl

endopeptidase AoS28A contains a suitable promoter and a nucleic acid encoding for a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 4 and wherein the host cell overexpressing the prolyl endopeptidase AoS28B contains a suitable promoter and a nucleic acid encoding for a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5.

In further embodiments of the invention, the host cell overexpressing the prolyl

endopeptidase AoS28A is Aspergillus oryzae strain deposited under accession number IHEM 26504, and the host cell overexpressing the prolyl endopeptidase AoS28B is

Aspergillus oryzae strain deposited under accession number IHEM 26503.

In a further embodiment, the invention provides an enzyme composition, comprising i. a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 1, and/or

ii. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 2.

In a further embodiment, the enzyme composition of the invention can further comprise only a prolyl endopeptidases AoS28A comprising or consisting of SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 1.

In another embodiment, the enzyme composition of the invention can comprise only a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 2.

In another embodiment, the enzyme composition of the invention is a mixture of extracellular enzyme extracts from Aspergillus oryzae strains deposited under accession number IHEM 26503 and IHEM 26504 overproducing and secreting large amount of a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

In some embodiments, overexpressing, overproducing or secreting large amounts of the prolyl endopeptidases of the invention refers to at least 20 μg/ml or at least 1000 U/ml of secreted prolyl endopeptidases of the invention.

The enzyme composition of the invention has an activity at pH values 2.0 to 8.0. The optimum activity of the enzyme composition of the invention corresponds to the pH of the gastric fluid. In certain embodiments, the enzyme composition of the invention has an optimal activity at pH 4 - 5.

The term "acidic pH" or "low pH" corresponds to pH values below 7, which indicate an acid.

As herein used the term "endopeptidase of the invention" or "endopeptidases of the invention" is a endopeptidase or endopeptidases of the enzyme composition of the present invention.

Compositions of the invention include, but are not limited to, an isolated enzyme AoS28A or AoS28B, a combination of AoS28A and AoS28B, extracts from Aspergillus oryzae strains deposited under accession number IHEM 26503 and IHEM 26504, or even Aspergillus oryzae strains deposited under accession number IHEM 26503 and IHEM 26504 themselves.

The following sequences are considered in the present invention:

SEQ ID NO: 1 (AoS28A)

AIHPPRPVPPPVSRPVSTQSSVVEGNATFEQLLDHHDSSKGTFSQRYWWSTEYWGGP GSPVVLFTPGEASADGYEGYLTNNTLTGLYAQEIQGAVILIEHRYWGDSSPYEELTA ETLQYLTLEQSILDLTHFAETVQLEFDTSNSSNAPKAPWVLVGGSYSGALAAWTAAV APETFWAYHATSAPVQAIDDFWQYFDPIRHGMAPNCSRDVSLVANHIDTVGKNGSA ADQLALKELFGLEALEHYDDFAAALPTGPYLWQSNTFVTGYSNFFAFCDAVENVEA GAAVVPGPEGVGLQKALTGYANWFNSTVIPGYCASYGYWTDNRTVACFDTHNPSS AIFTDTSVDNAVDRQWQWFLCNEPFFWWQDGAPEGVPTIVPRTINAEYWQRQCSLY FPEVNGYTYGSAKGKTAATVNTWTGGWSDSKNTSRLLWVNGQYDPWRDSGVSST HRPGGPLTSTADEPVQVIPGGFHCSDLCLKAYFANAGVKQVVDNAVAQIKSWVAEY YK

SEQ ID NO: 2 (AoS28B)

ALSFLPGIKANNLQLASVLGIDGHTARFNPEKIAETAISRGSGSEVPARRISIPIDHEDP SMGTYQNRYWVSADFYKPGGPVFVLDAGEGNAYSVAQSYLGGSDNFFAEYLKEFN GLGLVWEHRYYGDSLPFPVNTSTPNEHFKYLTNSQALADLPYFAEKFTLNGTDLSPK SSPWIMLGGSYPGMRAAFTRNEYPDTIFASFAMSAPVEARVNMTIYFEQVYRGMVA

NGLGGCAKDLKAINDYIDSQLDKKGQAADAIKTLFLGKEGIHNSNGDFTAALGSIYN LFQSYGVDGGEESLSQLCSYLDKGASPNGIARKIGVKELTEKFAAWPPLLYLINQWG SQVGNGDSNCKGQNNSTETVCELGGQFTDPDTISWTWQYCTEWGYLQADNVGPHS LLSKYQSLEYQQSLCYRQFPGAKESGLLPEHPEANETNAETGGWTIRPSNVFWSAGE FDPWRTLTPLSNETFAPKGVQISTNIPKCGVETPENVLFGYVIPRAEHCFDYDLSYKP ADKSRKLFSLALKKWLPCWRSEHAPKGVQRKWM

A endopeptidase of the invention includes a endopeptidase comprising the amino acid sequence comprising SEQ ID NOs: 1 and/or 2. The invention also includes a mutant or variant endopeptidases any of whose residues may be changed from the corresponding residues shown in SEQ ID NOs: 1 and/or 2 while still maintaining its activity and physiological functions, or a biologically active fragment thereof. The present invention is also directed to variants of endopeptidases of the invention. The term "variant" refers to a polypeptide or protein having an amino acid sequence that differs to some extent from a native SEQ ID NOs: 1 and 2 and which is an amino acid sequence that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, deletions, side-chain modifications and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly

II. Polar, positively charged residues: His, Arg, Lys

III. Polar, negatively charged residues: and their amides: Asp, Asn, Glu, Gin

IV. Large, aromatic residues: Phe, Tyr, Trp

V. Large, aliphatic, nonpolar residues: Met, Leu, He, Val, Cys. In another aspect, the present invention is directed to isolated endopeptidases of the invention, and biologically active fragments thereof (or derivatives, portions, analogs or homologs thereof). Biologically active fragment refers to regions of the endopeptidases of the invention, which are necessary for normal function, for example, prolyl, pepsin, glutamic or carboxypeptidase like endopeptidases activities. Biologically active fragments include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of SEQ ID NOs: 1 and 2, that include fewer amino acids than the full-length endopeptidase, and exhibit at least one activity of a endopeptidase of the invention. Typically, biologically active fragments comprise a domain or motif with at least one activity of the endopeptidase of the invention. A biologically active fragment of a endopeptidase of the invention can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acid residues in length. Moreover, other biologically active fragments, in which other regions of the endopeptidase are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native endopeptidase of the invention.

In a further embodiment, the endopeptidase of the invention is a endopeptidase that comprises an amino acid sequence having at least 70%, 80%, 90%, 95% or 99%, preferably 95%), identity to the amino acid sequence comprising SEQ ID NOs: 1 and 2 and retains the activity of the endopeptidase comprising SEQ ID NOs: 1 and 2.

To determine the percent of identity or homology of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e. , as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18). Preferred programs include the GCG Pileup program, FASTA (Pearson et al. (1988) Proc. Natl, Acad. Sci USA 85 :2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., (1997) NAR 25 :3389-3402). Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), preferably using default parameters. Another sequence software program that finds use is the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).

The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.

The invention also provides endopeptidases of the invention as chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" of endopeptidases of the invention comprises a endopeptidase of the invention operatively-linked to another polypeptide. A endopeptidase of the invention refers to a polypeptide having an amino acid sequence corresponding to a SEQ ID NOs: 1 and 2, whereas "another polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the endopeptidase of the invention, e.g., a protein that is different from the endopeptidase of the invention and that is derived from the same or a different organism. Within a fusion protein, the polypeptide can correspond to all or a portion of a endopeptidase of the invention. In one embodiment, a fusion protein comprises at least one biologically active fragment of a endopeptidase of the invention. In another embodiment, a fusion protein comprises at least two biologically active fragments of a endopeptidase of the invention. In yet another embodiment, a fusion protein comprises at least three biologically active fragments of a endopeptidase of the invention. Within the fusion protein, the term "operatively-linked" is intended to indicate that the polypeptide of a endopeptidase of the invention and another polypeptide are fused in-frame with one another. Another polypeptide can be fused to the N-terminus and/or C-terminus of the polypeptide of endopeptidase of the invention. In one embodiment, the fusion protein is a GST fusion protein in which the sequences of the endopeptidase of the invention are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant endopeptidase of the invention. In another embodiment, the fusion protein is a endopeptidase of the invention containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g. , mammalian host cells), expression and/or secretion of endopeptidases of the invention can be increased through use of a heterologous signal sequence. A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques or conventional techniques including automated DNA synthesizers. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g. , by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.

In further embodiments of the invention, AoS28A (SEQ ID NO: l) and AoS28B (SEQ ID NO:2) are fused to the signal sequence of AfuS28 (MRTAAASLTLAATCLFELASAR (SEQ ID NO: 3) for better secretion of AoS28A and AoS28B, providing amino acid sequences SEQ ID NO: 3 + SEQ ID NO: 1 and SEQ ID NO:3 + SEQ ID NO: 2.

A further aspect of the invention provides a pharmaceutical composition comprising the enzyme composition of the invention and at least one pharmaceutically acceptable excipient, carrier and/or diluent. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, which is preferably the oral administration. According to some embodiments, crude preparations of cell culture medium from Aspergillus oryzae or transgenic (recombinant) Aspergillus oryzae strains overproducing the enzyme composition of the invention, or extracts purified from Aspergillus oryzae, for example from Aspergillus oryzae strains deposited under accession numbers IHEM 26503 and IHEM 26504, can be administered orally since the endopeptidases of the invention are secreted.

In other embodiments, the enzyme composition of the invention or the prolyl endopeptidase AoS28A and/or the prolyl endopeptidase AoS28B derived or isolated from Aspergillus oryzae starins deposited under accession number IHEM 26504 and 26503 is a lyophilized preparation. Lyophilization or freeze-drying is a means of drying achieved by freezing a wet substance and causing the ice to sublime directly to vapor by exposing it to a low partial pressure of water vapor. In practice, the substance may not be completely frozen, especially if non-aqueous solutions are present, and most lyophilization processes are completed by a period of desorption drying. The purpose of freeze-drying is to increase the shelf life, or preserve a specimen, be it food, microbial organisms, or, in some circumstances to decrease the size of the product. For various purposes, such as stable storage, the extract, bacteria or isolated enzyme can be lyophilized. Lyophilization is preferably performed on an initially concentrated preparation, e.g. of at least about 1 mg/ml for extract or isolated enzyme preparation and 1000 bacteria/ml. PEG can be added to improve enzyme stability, if so desired. In some embodiments, lyophilization of an extract, fungi or isolated enzyme can be performed without loss of specific activity (e.g., prolyl endopeptidase activity). Lyophilized extracts are useful in the production of enteric-coated capsules, enteric-coated tablets, capsules, or tablets.

For oral administration, the enzyme composition of the invention may be formulated for example in the form of capsules (coated or non-coated) containing powder, coated or non-coated pellets, granules or micro-/mini-tablets or in the form of tablets (coated or non- coated) pressed from powder, coated or non-coated pellets, dragees or micro- /mini-tablets, hydrogels, liposomes, nanosomes, encapsulation, PEGylation. The enzyme composition of the invention may also be formulated for example in the form of gel caps or in liquid form as solution, drops, suspension or gel also be formulated e.g. as dried or moist oral supplement. The formulation of the enzyme composition according to the present invention as powder is particularly suitable for admixing with foodstuff. The powder may be sprinkled onto a meal or mixed into a pulp or beverage. It is particularly beneficial, if the enzyme composition offered as bulk powder is packaged in single dosage amounts, such as in single bags or capsules, or if it is provided in a dosing dispenser.

Suitable excipients, carriers and/or diluents include maltodextrin, cyclodextrines, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, dextrose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gums, lactose, methylcellu- lose, povidone, carboxymethyl cellulose, corn starch, modified starch, fibersol, gelatine, hy-droxypropylmethyl cellulose and the like (including mixtures thereof). Preferable carriers include calcium carbonate, magnesium stearate, maltodex- trin, dicalcium phosphate, modified starch, microcrystalline cellulose, fibersol, gelatine, hydroxypropylmethyl cellulose and mixtures thereof.

The various ingredients and the excipient, carrier and/or diluent may be mixed and formed into the desired form using common methods well known to the skilled person. The administration form according to the present invention which is suited for the oral route, such as e.g. tablet or capsule, may be optionally coated. According to an embodiment, a coating is resistant against low pH values (approximately pH 1 to 2.5) and which dissolves at a pH value of approximately 3.0 to 8.0, preferably at a pH value of 3.0 to 6.5 and particularly preferable at a pH value of 4.0 to 6.0. An optionally used coating should be in accordance with the pH optimum of the enzyme composition used and its stability at pH values to which the formulation will be exposed. Also a coating may be used which is not resistant to low pH values but which delays the release of the enzyme composition at low pH values. It is also possible to prepare the enzyme composition according to the present invention as coated (see above) pellets, granules or micro-/mini-tablets which can be filled into coated or non- coated capsules or which can be pressed into coated or non-coated tablets. Suitable coatings are, for example, cellulose acetate phthalate, cellulose deri- vates, shellac, polyvinylpyrrolidone derivates, acrylic acid, poly-acrylic acid derivates and polymethyl methacrylate (PMMA), such as e.g. Eudragit® (from Rohm GmbH, Darmstadt, Germany), in particular Eudragit® L30D-55. The coating Eudragit® L30D-55 is dissolved, for example, at a pH value of 5.5 and higher. If it is desired to release the enzyme composition already at a lower pH value, this may be achieved e.g. by the addition of sodium hydroxide solution to the coating agent Eudragit® L30D-55, because in this case carboxyl groups of the methacrylate would be neutralised. Therefore, this coating will be dissolved, for example, already at a pH value of 4.0 provided that 5 % of the carboxyl groups are neutralised. The addition of about 100 g of 4 % sodium hydroxide solution to 1 kg of Eudragit® L30D-55 would result in a neutralisation of about 6 % of the carboxyl groups. Further details about formulation methods and administration methods can be found in the 21^st edition of "Remington: The Science & Practice of Pharmacy", published 2005 by Lippincott, Williams & Wilkins, Baltimore, USA, in the Encyclopedia of Pharmaceutical Technology (Editor James Swarbrick) and in Prof. Bauer "Lehrbuch der Pharmazeutischen Technologie", 18th edition, published 2006 by Wissenschaftliche Verlagsgesellschaft (ISBN 3804-72222-9). The contents of these documents are incorporated herein by reference. Other suitable acceptable excipients, carriers and/or diluents for use in the present invention include, but are not limited to water, mineral oil, ethylene glycol, propylene glycol, lanolin, glyceryl stearate, sorbitan stearate, isopropyl my ri state, isopropyl palmitate, acetone, glycerine, phosphatidylcholine, sodium cholate or ethanol.

The pharmaceutical compositions for use in the present invention may also comprise at least one co-emulsifying agent which includes but is not limited to oxyethylenated sorbitan monostearate, fatty alcohols, such as stearyl alcohol or cetyl alcohol, or esters of fatty acids and polyols, such as glyceryl stearate.

The enzyme composition according to the present invention may be provided in a stabilized form. Generally, stabilization methods and procedures which may be used according to the present invention include any and all methods for the stabilization of chemical or biological material which are known in the art, comprising e.g. the addition of chemical agents, methods which are based on temperature modulation, methods which are based on irradiation or combinations thereof. Chemical agents that may be used according to the present invention include, among others, preservatives, acids, bases, salts, antioxidants, viscosity enhancers, emulsifying agents, gelatinizers, and mixtures thereof. In cases of treating the celiac disease, the pharmaceutical compositions employed are preferably formulated so as to release their activity in gastric fluid. This type of formulations will provide optimum activity in the right place, i.e. the release of the endopeptidases of the invention in stomach The dosage unit form of the pharmaceutical composition may be chosen from among a variety of such forms. In the case of tablets, capsules etc. the weight of each dosage unit is usually less than 0.5 g, these dosage units being intended for administration in an amount of say 1 to 2 tablets (to be ingested before, during or after meals) e.g. 2 to 3 times per day. The pharmaceutical composition according to the invention will normally contain the enzyme composition of the invention in an amount of from 0.0001 to 100% (w/w), e.g. from 0.001 to 90% (w/w). The exact amount will depend on the particular type of composition employed and on the specific protease activity per mg of protein. As regards the protease (peptidase) activity in the pharmaceutical composition, this will often be within a range of from 0.1 to 0.0001 enzyme units per mg; but in some cases other activity per mg ranges may be obtained, depending on the purity of the enzyme preparation.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Another aspect of the invention provides a food supplement comprising the enzyme composition of the invention. The term "food supplement" in the context of the invention is equivalent and interchangeable with the terms food additive, a dietary supplement, alicament, and nutritional supplement.

In the food supplement of the invention, a carrier material is commonly added, although not essential, to the enzyme composition. Suitable carrier materials include maltodextrins, modified starches, direct compression tablet excipients such as dicalcium phosphate, calcium sulfate and sucrose. A particularly preferred carrier ingredient is the 10 DE Maltrin Ml 00 maltodextrin from Grain Processing Corporation. Carriers can be added in concentrations ranging from 50 to 95 weight percent of the total composition.

The enzyme composition according to the present invention may contain the enzymes without further additives. However, it is preferable that the enzyme composition according to the present invention further contains additives that are pharmaceutically acceptable and/or acceptable for food supplements, such as for example extenders, binders, stabilizers, preservatives, flavourings, etc. Such additives are commonly used and well known for the production of pharmaceutical compositions, medical devices, food supplements, and special food supplements and the person skilled in the art knows which additives in which amounts are suitable for certain presentation forms. The enzyme composition according to the present invention may for example contain as additives dicalcium phosphate, lactose, modified starch, microcrystal- line cellulose, maltodextrin and/or fibersol.

The food supplement of the invention may be a granulated enzyme product which may readily be mixed with food components. Alternatively, food supplements of the invention can form a component of a pre-mix. The granulated enzyme composition product of the invention may be coated or uncoated. The particle size of the enzyme granulates can be compatible with that of food and pre-mix components. This provides a safe and convenient mean of incorporating enzymes into food supplements. Alternatively, the food supplements of the invention may be a stabilized liquid composition. This may be an aqueous or oil-based slurry.

In another aspect, enzyme composition of the invention can be supplied by expressing the enzymes directly in transgenic food crops (as, e.g., transgenic plants, seeds and the like), such as grains, cereals, corn, soy bean, rape seed, lupin and the like. For example transgenic plants, plant parts and plant cells can comprise nucleic acids encoding the endopeptidases of the invention. In one aspect, the nucleic acid is expressed such that the enzyme (e.g., AoS28A and/or AoS28B) of the invention is produced in recoverable quantities. The enzyme composition of the invention can be recovered from any plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide can be used as such for improving the quality of a food, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

The pharmaceutical composition or the food supplement of the invention can be provided at a time of a meal so that the endopeptidases of the enzyme composition are released or activated in the upper gastrointestinal lumen where the endopeptidases can complement gastric and pancreatic enzymes to detoxify ingested gluten and prevent harmful peptides to reach the mucosal surface. The enzyme composition according to the present invention can be taken orally prior to meals, immediately before meals, with meals or immediately after meals, so that it can exert its proteolytic effect on proline-rich nutriments in the food pulp. For example the extract from an engineered (recombinant) strain of Aspergillus, such as Aspergillus oryzae strains deposited under accession numbers IHEM 26503 and IHEM 26504, to produce the enzyme composition of the invention could be used as a food supplement before a gluten rich meal in celiac disease. In an embodiment, the extract used as a food supplement is an extract comprising an extract rich in AoS28A of the invention from Aspergillus oryzae strain deposited under accession number IHEM 26504 and an extract rich in AoS28B of the invention from Aspergillus oryzae strain deposited under accession number IHEM 26503.

Another aspect of the invention also relates to a kit for degrading a polypeptide product comprising the enzyme composition of the present invention. The kit of the invention can also include reagents necessary for carrying out the degradation of a polypeptide product. Said reagents can be buffers, for example sodium citrate buffer, Tris-HCl buffer, and/or acetate buffer; precipitation reagents, such as trichloroacetic acid; and/or the reagents for stopping the enzyme activity, such as acetic acid and/or formic acid. The kit featured herein can further include an information material describing how to perform the degradation of a polypeptide product. The informational material of the kit is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. Of course, the informational material can also be provided in any combination of formats. The kit can also contain separate containers, dividers or compartments for the reagents and informational material. Containers can be appropriately labeled.

Since in patients with celiac disease the gastrointestinal tract does not possess the enzymatic equipment to efficiently cleave the gluten-derived proline-rich peptides, driving the abnormal immune intestinal response, another therapeutic approach relies on the use of orally active endopeptidases to degrade toxic gliadin peptides before they reach the mucosa. Oral therapy by prolyl-peptidases able to digest ingested gluten is therefore propounded as an alternative treatment to the diet.

A further aspect of the invention provides the enzyme composition of the invention for use in a method for treating and/or preventing a condition selected from the group comprising celiac disease, digestive tract bad absorption, an allergic reaction, an enzyme deficiency, a fungal infection, Crohn disease, mycoses, sprue and and wound healing. In an embodiment, the allergic reaction is a reaction to gluten or fragments thereof. Preferably a fragment of gluten is gliadine.

In another embodiment, the invention relates to a method for treating and/or preventing a condition in a subject suffering therefrom comprising administering a therapeutically effective amount of the enzyme composition of the invention or the pharmaceutical composition of the invention, said condition being selected from the group comprising celiac disease, digestive tract bad absorption, an allergic reaction, an enzyme deficiency, a fungal infection, Crohn disease, mycoses and sprue. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder, such as celiac disease, digestive tract bad absorption, an allergic reaction, an enzyme deficiency, a fungal infection, Crohn disease, mycoses, sprue and wound healing. In other embodiments, the subject is a subject in need to improve food digestion. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, the term "condition" or "a medical condition" denotes any illness, injury, disorder or disease.

In another embodiment the invention relates to a method of detoxifying gliadin comprising contacting gliadin containing food product with an effective dose of the enzyme composition of the invention. The term "food product", "foodstuff or "food" encompasses also any proline rich nutriment, such as gluten.

In one aspect, treating food products using the enzyme composition of the invention can help in the availability of nutrients, e.g., starch, protein, and the like, in the food product. By breaking down difficult to digest proteins, such as gluten, or indirectly or directly unmasking starch (or other nutrients), the enzyme composition of the invention makes nutrients more accessible to other endogenous or exogenous enzymes. The enzyme composition of the invention can also simply cause the release of readily digestible and easily absorbed nutrients and sugars. When added to food products, the enzyme composition of the invention improve the in vivo breakdown of plant cell wall material partly due to a reduction of the intestinal viscosity (see, e.g., Bedford et al., Proceedings of the 1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77), whereby a better utilization of the plant nutrients by the mammal is achieved. In a further embodiment, the invention provides the use of the enzyme composition of the invention for the degradation of proteins, for the degradation of by-products, toxic or contaminant proteins; for the degradation of prions or viruses; for the degradation of proteins for proteomics; for the degradation of cornified substrate; for the hydrolysis of polypeptides for amino acid analysis; for wound cleaning; for wound healing; for cosmetology such as peeling tools, depilation, dermabrasion and dermaplaning; for prothesis cleaning and/or preparation; for fabric softeners; for soaps; for tenderizing meat; for the controlled fermentation process of Soja or cheese; for cleaning or disinfection of septic tanks or any container containing proteins that should be removed or sterilized; and for cleaning of surgical instruments.

In another embodiment, the invention provides a method of degrading a polypeptide substrate, comprising contacting the polypeptide substrate with the enzyme composition of the invention. In the method of degrading a polypeptide substrate, the enzyme composition sequentially digests a full-length polypeptide substrate or a full-length protein. Preferably the polypeptide substrate is selected from the group comprising casein, gluten, bovine serum albumin or fragments thereof and the polypeptide substrate length is from 2 to 200 amino acids.

It is contemplated that the compositions described herein relating to composition for the treatment of celiac disease or a related disorder, for the treatment of gluten-containing foodstuff, and for the detoxifying gluten-containing foodstuff can comprises isolated enzyme AoS28A and/or AoS28B of invention, extract from Aspergillus oryzae strain having accession numbers IHEM 26504 or 26503, a plurality of extracts from Aspergillus oryzae strains having accession numbers IHEM 26504 and 26503.

The enzyme composition of the invention have numerous applications in food processing industry. For example, the endopeptidases of the invention can be used in the enzymatic treatment of various gluten-containing materials, e.g. from cereals, grains, wine or juice production, or agricultural residues such as vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp, and the like. The endopeptidases of the invention can be used to modify the consistency and appearance of processed fruit, vegetables or meat. The endopeptidases of the invention can be used to treat plant material to facilitate processing of plant material, including foods, facilitate purification or extraction of plant components. In an embodiment, the enzyme composition of the invention can be used in the manufacture of the food supplement of the invention.

The enzyme composition according to the invention can also be added to a food product before its consumption. It can already be added to the food product during production, with the aim that it exhibits its effect only after eating the food product. This could also be achieved by microencapsulation, for example. With this, for example the utilizable proline-rich materials, such as gluten, in the food product would be reduced without negatively affecting its taste. Therefore, preparations containing the enzyme composition according to the invention are useful, which release the enzyme composition only in the digestive tract of a human (or animal) or let it become effective in another way, especially in the stomach or small intestine. Therefore, the enzyme composition according to the present invention can be used, for example, in the production of desserts, fruit preparations, jam, honey, chocolate and chocolate products, bakery products (e.g. biscuits and cakes), breads, pastas, vegetable dishes, potato dishes, ice cream, cereals, dairy products (e.g. fruit yogurt and pudding), gluten-containing beverages, gluten- containing sauces and gluten-containing sweeteners. For dishes that are boiled or baked, the enzyme composition according to the present invention could, for example, be mixed into or sprinkled onto them after cooling. The enzyme composition according to the invention can also be added to a food product, to exert its effect after eating on the gluten originating from another food product. An example of this would be the addition of the enzyme composition according to the present invention to a spread so that the reduction of the gluten that is contained in the bread and that can be used by the body occurs after the intake of the bread, without impairing its taste.

In the modification of food product, the enzyme composition of the present invention can process the food product either in vitro (by modifying components of the food product) or in vivo. The enzyme composition of the invention can be added to food product containing high amounts of gluten, e.g. plant material from cereals, grains and the like. When added to the food product, the enzyme composition of the present invention significantly improves the in vivo break-down of gluten- containing material, e.g., wheat, whereby a better utilization of the plant nutrients by the human (or animal) is achieved. The enzyme composition according to the invention may also be used in immobilized form. This is especially useful for the treatment of liquid food products. For example, the enzyme composition of the invention can be embedded in a matrix which is permeable for gluten. If a gluten containing liquid food product is allowed to flow along the enzyme containing matrix, then gluten is extracted from the food product by the action of the enzymes and digested. The enzyme composition of the invention can also be used in the fruit and brewing industry for equipment cleaning and maintenance.

In a further embodiment, the invention provides a method for improving food digestion in a mammal, wherein said method comprising oral administration to the said mammal of the enzyme composition of the invention. Preferably, the food contains proline rich nutriments such as gluten and the mammal is a human. Thus in one aspect, the growth rate and/or food conversion ratio (i.e. the weight of ingested food relative to weight gain) of the human or animal is improved. For example a partially or indigestible proline- comprising protein is fully or partially degraded by the enzyme composition of the invention, resulting in availability of more digestible food for the human or animal. Thus, the enzyme composition of the invention can contribute to the available energy of the food. In addition, by contributing to the degradation of proline- comprising proteins, the endopeptidases of the invention can improve the digestibility and uptake of carbohydrate and non-carbohydrate food constituents such as protein, fat and minerals In a further aspect of the invention, the endopeptidases of the enzyme composition of the invention are produced by recombinant DNA techniques. As used herein, the term "recombinant" when used with reference to a cell indicates that the cell overexpresses homologues nucleic acid. In an embodiment, recombinant cells contain genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques. The person skilled in the art will recognize that these cells can be used for unicellular or multicellular transgenic organisms, for example transgenic fungi producing the enzyme composition of the invention.

Thus, an aspect of the present invention was to generate A. oryzae strains producing large amounts of prolyl peptidases, i.e. overexpressing prolyl endopeptidases. Transformants secreting high amounts of prolyl endopeptidase activity (1000 and 2600 U/ml) in a medium containing 1% peptone and 2% glucose were obtained by AoS28A and AoS28B overexpression, respectively. This activity corresponded to the secretion of 28 and 23 μg of AoS28A and AoS28B /ml of culture supernatant, respectively. For comparison, the maximal production of prolyl peptidase activity in culture supernatant of A. oryzae obtained by heterologue overexpression of AfuS28A, the orthologue of AoS28A in Aspergillus fumigatus, was 60 U/ml. This low amount could be due to the heterologue nature of the protein in A. oryzae, and possibly to the lower specific activity of AfuS28 in comparison to that of AoS28A and AoS28B. The transformants for high amounts of secreted AoS28A and AoS28B were obtained in culture supernatants are of particular interest in the development of an oral enzyme therapy product for patients suffering intolerance to gluten.

In an embodiment, the invention provides a method for producing the enzyme composition of the invention comprising the steps of:

(a) introducing into a host cell a nucleic acid encoding for

i. a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 4, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 4, and/or

ii. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 95% of identity to SEQ ID NO: 5

(b) cultivating the cell of step (a) in a culture medium under conditions suitable for producing the enzyme composition; and

recovering the enzyme composition.

In another embodiment, the invention provides a method for producing the enzyme composition of the invention, wherein said method comprises

(a) introducing into a host cell a nucleic acid encoding for

ii. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5,

(c) recovering the enzyme composition.

In a particular embodiment, the invention provides a method for producing the enzyme composition of the invention, wherein said method comprises

(a) introducing into a host cell a nucleic acid encoding for

i. a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 4 (b) cultivating the cell of step (a) in a culture medium under conditions suitable for producing the enzyme composition; and

(c) recovering the enzyme composition.

In a further particular embodiment, the invention provides a method for producing the enzyme composition of the invention, wherein said method comprises

(a) introducing into a host cell a nucleic acid encoding for

i. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5

(b) cultivating the cell of step (a) in a culture medium under conditions suitable for producing the enzyme composition; and (c) recovering the enzyme composition.

In some preferred embodiments, the host cell is Aspergillus oryzae, Aspergillus sojae, Saccharomyces cerevisiae, and/or Kluveromyces lactis.

SEQ ID NO: 4 (AoS28A)

ATGCGGACTGCTGCTGCTTCACTGACGCTTGCTGCGACTTGTCTCTTTGAGTTGGC

ATCTGCTCGAGCGATTCATCCGCCCAGGCCGGTTCCTCCGCCGGTCTCTCGACCG

GTCTCGACGCAATCCTCGGTGGTTGAAGGAAACGCAACCTTCGAGCAACTCTTAG ATCATCATGACTCCTCAAAGGGGACTTTCTCGCAGCGTTACTGGTGGAGTACGGA GTACTGGGGTGGCCCCGGCTCACCGGTGCGTTTGTTGCCTTGCAGCGATACCTCG CCAGGTATCACCATGGTCGCTAACTTGGCGTCGTAGGTAGTATTATTCACCCCTG GTGAAGCTTCCGCCGATGGGTACGAGGGTTACCTCACTAATAACACATTGACTGG TCTATACGCGCAAGAAATTCAAGGTGCTGTGATTTTGATCGAGCGTGAGTGGGCC GTTTCCCTGGGAAAGAGTCGAACATTGATCTAAAGATGATCTAGACCGTTACTGG GGTGACTCCTCTCCTTATGAAGAACTCACAGCGGAGACGTTGCAGTATCTTACGC TGGAGCAGTCCATTCTGGACTTAACCCATTTTGCCGAAACGGTCCAGTTGGAATT TGATACTTCCAATAGCAGCAATGCACCAAAGGCGGTAAGATCGAGTCCTCATCC AGACGATACGATTGATAAAAGTCATATCCCTGACGTTAATATTATCAGCCATGGG TGTTGGTCGGTGGATCATACAGTGGTGCTTTGGCAGCTTGGACCGCCGCCGTCGC TCCAGAGACGTTCTGGGCCTATCATGCCACAAGCGCTCCTGTTCAAGCTATTGAC GATTTTGTGAGTGCCTCACACTCACTATGGGTGTTGGTGTAACGGCTTACAGATA TTGTAGTGGCAATATTTCGATCCCATCCGTCATGGCATGGCACCTAACTGTAGCA GAG AT GT C T C T C TGGT GGC C A AT C AC ATT GAT AC C GT C GGG A AG A AT GG AT C TGC CGCGGATCAACTTGCGCTAAAAGAACTCTTCGGCCTGGAAGCTCTTGAACACTAC GATGACTTTGCCGCGTAAGTACAAATAATCCGCAAAAGCAAGGTTAGACCTAAC TTCTACAGTGCCCTTCCGACGGGGCCCTATCTGTGGCAGTCCAATACTTTTGTGA CTGGGTACTCCAATTTCTTTGCCTTTTGCGACGCTGTTGAAGTACAGTTCTTCCCA TCACCAGCTTTGTCTGACGCATATATTGACTCATTTGGTCCAGAATGTCGAGGCC GGC GC C GC T GT T GT T C C AGG AC C AG AGGGT GT T GGC TT GC AG A AGGC T C T T AC GG GCTACGCGAATTGGTTCAATTCGACTATCATTCCAGGTTGTAAGTATCCTGCATC AAAATTATATCGATCAATCTGACTGTGTGGTTAGATTGTGCCAGCTACGGATATT GGACGGACAACCGGACTGTCGCCTGCTTCGACACCCACAATCCATCCAGTGCCAT ATTCACAGATACTTCGGTTGATAACGCTGTGGATCGACAATGGCAGTGGTTTCTG TGCAATGAACCTTTCTTCTGGTGGCAAGAGTAAGTTTCCTAGAACAATGTGCCGA

GTATACACTTACATTTGAATCTCAAGTGGTGCTCCGGAAGGTGTCCCAACAATTG TCCCTCGTACGATCAATGCAGAGTACTGGCAGCGTCAGTGCTCGTTATACTTCCC TGAAGTGAATGGTTACACCTACGGCAGTGCAAAGGGGAAGACTGCCGCGACAGT CAATACTTGGACAGGCGGATGGTCCGATTCCAAGAACACCAGTCGCTTGCTCTGG GTAAATGGGTAAGTGGAACCTTAGCTTGTAATCCACTGAGGGGTCGCAAAACAG CTTTACTAATTTATTCCCAGGCAATACGATCCCTGGCGCGATTCGGGCGTCTCTTC GACCCACCGTCCTGGTGGCCCATTGACGAGCACAGCTGATGAGCCAGTACAGGT TATCCCTGGTGGATTTCACTGCTCGGACTTGTATCTCAAGGATTATTTTGCAAATG CAGGCGTGAAGCAGGTGGTTGACAATGCAGTAGCTCAGATCAAGTCATGGGTTG CTGAATACTACAAATAG

SEQ ID NO: 5 (AoS28B)

ATGCGGACTGCTGCTGCTTCACTGACGCTTGCTGCGACTTGTCTCTTTGAGTTGGC ATCTGCTCGAGCCCTAAGCTTTCTCCCCGGCATCAAGGCCAATAATCTCCAACTC GCCTCGGTATTAGGTATCGATGGCCATACCGCCAGGTTCAATCCTGAGAAGATCG CAGAGACCGCTATCTCGCGCGGTTCTGGCTCAGAAGTCCCTGCCCGGCGGATATC GGTATGTCTTTACCAGTCAAGCTTTCTAGTATATGAGGTAAAATCTAACTCGGCG TTCAGATCCCCATTGACCATGAGGATCCATCTATGGGCACCTATCAGAACCGCTA CTGGGTTTCAGCAGACTTTTACAAGCCCGGTGGTCCCGTCTTTGTACTAGATGCC GGT G A AGGC A AT GC C T AC T C C GT GGC GC A AT C GT AT C T C GGC GG AT C GG AT A AC TTCTTCGCGGAGTACCTCAAGGAATTCAATGGGCTGGGTCTTGTGTGGGAGCATC GGTGAGCCACCTACCCTAGTCATCATTGTCATGATTGACCGCTAACCTCCGGTCC GATTGAAGTTACTATGGTGACTCTCTGCCCTTTCCTGTCAACACTAGCACCCCCA ACGAGCATTTCAAGTACCTCACCAACAGCCAGGCACTGGCTGACCTCCCTTACTT CGCTGAGAAGTTCACTCTCAACGGGACAGACTTGAGCCCCAAGTCCAGTCCCTG GATCATGCTCGGTGGCTCATACCCGGGCATGCGCGCGGCCTTCACCCGCAACGA GTACCCGGACACCATTTTCGCCTCGTTCGCAATGTCTGCGCCCGTCGAAGCCCGG GTCAACATGACCATCTACTTCGAGCAAGTCTACCGCGGCATGGTCGCGAACGGA CTGGGCGGCTGTGCCAAGGACCTCAAGGCCATCAACGACTACATCGACAGCCAA CTCGACAAGAAGGGCCAAGCCGCCGACGCCATCAAGACACTCTTCCTCGGTAAG GAAGGCATCCACAACTCCAACGGCGACTTCACCGCCGCGCTCGGAAGCATCTAC AACCTCTTCCAGAGCTACGGCGTCGACGGCGGCGAAGAAAGTCTCTCCCAGCTCT GCAGCTACCTCGACAAAGGCGCCAGCCCCAACGGCATCGCCCGGAAAATCGGAG TCAAGGAACTGACCGAGAAGTTCGCCGCCTGGCCCCCGCTTCTGTACCTCATCAA CCAGTGGGGCAGCCAGGTCGGTAACGGCGACTCCAACTGCAAGGGCCAGAACAA TTCCACCGAGACCGTCTGTGAGCTGGGCGGGCAGTTCACCGACCCCGACACCATC AGCTGGACCTGGCAGTACTGCACCGAATGGGGCTATCTCCAGGCCGACAACGTG GGCCCTCACTCCCTACTCTCCAAGTACCAGTCCCTGGAGTACCAGCAGTCCCTTT GCTACCGACAGTTCCCCGGCGCAAAGGAGAGTGGCCTGCTCCCCGAGCACCCGG AGGCGAACGAGACGAACGCCGAAACAGGCGGATGGACCATCCGTCCTTCCAATG TCTTCTGGAGCGCGGGCGAGTTCGATCCCTGGCGGACGTTGACGCCCTTGTCGAA TGAGACATTCGCGCCGAAGGGCGTGCAGATCTCCACCAATATCCCCAAGTGTGG TGTCGAGACACCTGAGAATGTGCTCTTCGGCTATGTCATTCCGAGGGCGGAGCAT TGCTTTGACTATGACTTGAGTTACAAGCCGGCTGATAAGTCGCGGAAGTTGTTCA GT C TT GC C T TG A AG A AGT GGC T C C C GT GC T GGC GGTC GG AGC AT GC T C C T A AGGG TGTACAGAGGAAGTGGATGTAA The nucleic acids encoding the endopeptidases of the enzyme composition of the invention include the nucleic acids whose sequences are provided herein or fragments thereof. The invention also includes mutant or variant nucleic acids any of whose bases may be changed from the corresponding base shown herein, while still encoding a endopeptidase that maintains activities of the endopeptidases of the invention, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those described herein, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. Also included in the invention are fragments of nucleic acids sufficient for use as hybridization probes to identify peptidases-encoding nucleic acids (for example AfuS28 mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of endopeptidase nucleic acid molecules. A nucleic acid molecule of the invention, e.g. , a nucleic acid molecule having the nucleic acid sequence comprising SEQ ID NOs: 4 and 5 a complement of this aforementioned nucleic acid sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOs: 4 and 5 as a hybridization probe, nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g. , as described in Sambrook et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded.

The term "probes", as used herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g. , 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double- stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules, which are present in the natural source of these nucleic acid molecules. Preferably, an "isolated" nucleic acid is free of sequences, which naturally flank the nucleic acid (e.g. , sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. Particularly, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85%) pure, and most preferably at least about 99%> pure. A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to endopeptidase nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. Oligonucleotides may be chemically synthesized and may also be used as probes.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleic acid sequence shown in SEQ ID NOs: 4 and 5, or a portion of this nucleic acid sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active fragment of a endopeptidase of the invention). A nucleic acid molecule that is complementary to the nucleic acid sequence shown in SEQ ID NOs: 4 and 5 is one that is sufficiently complementary to the nucleic acid sequence shown in SEQ ID NOs: 4 and 5 that it can hydrogen bond with little or no mismatches to the nucleic acid sequence shown in SEQ ID NOs: 4 and 5, thereby forming a stable duplex.

As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule.

Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differ from it with respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs or orthologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.

Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, 90% or 95% identity over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See, e.g. , Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993 , and below.

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level. Homologous nucleotide sequences encode those sequences coding for isoforms of endopeptidases of the invention. Isoforms can be expressed in the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences can include nucleotide sequences encoding a endopeptidase of the invention of species other than fungi. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions in SEQ ID NOs: 4 and 5, as well as a polypeptide possessing biological activity of the endopeptidase of the invention. The nucleic acid sequence identity may be determined as the degree of identity between two sequences. The identity may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See Needleman & Wunsch, J. Mol. Biol. 48:443-453 1970. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the nucleic acid sequence shown in SEQ ID NOs: 4 and 5. An endopeptidase of the invention is encoded by the open reading frame ("ORF") of a nucleic acid of said endopeptidase. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more. A nucleic acid fragment encoding a "biologically-active fragment of endopeptidase" can be prepared by isolating a fragment SEQ ID NOs: 4 and 5 that encodes an endopeptidase having a biological activity of the endopeptidases of the invention (the biological activities of the endopeptidases of the invention are described above), expressing the encoded portion of endopeptidase (for example, by recombinant expression in vitro) and assessing the activity of the encoded fragment of endopeptidase.

The invention further encompasses nucleic acid molecules that differ from the nucleic acid sequences shown in SEQ ID NOs: 4 and 5 due to degeneracy of the genetic code and thus encode the same endopeptidases that are encoded by the nucleic acid sequences shown in SEQ ID NOs: 4 and 5.

In addition to the fungal endopeptidase nucleic acid sequences shown in SEQ ID NOs: 4 and 5, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the endopeptidase polypeptides may exist within a population of various species. Such genetic polymorphisms in the endopeptidase genes may exist among individual fungal species within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding a endopeptidase, preferably a fungal endopeptidase. Such natural allelic variations can typically result in 1-5% variance in the nucleic acid sequence of the endopeptidase genes. Any and all such nucleic acid variations and resulting amino acid polymorphisms in the endopeptidase polypeptides, which are the result of natural allelic variation and that do not alter the biological activity of the endopeptidase polypeptides, are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding endopeptidases of the invention from other species, and, thus, that have a nucleic acid sequence that differs from the sequence SEQ ID NOs: 4 and 5 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the endopeptidase cDNAs of the invention can be isolated based on their homology to the fungal endopeptidase nucleic acids disclosed herein using the fungal cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein (an enzyme) encoded by an allelic variant of a gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NOs: 4 and 5.

In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Homologs or other related sequences (e.g., orthologs, paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular fungal sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning. Stringent conditions are known to those skilled in the art and can be found in Ausubel et al., (eds.), 1993 , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. ( 1989), 6.3. 1 -6.3.6 and and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY and Shilo & Weinberg, Proc Natl Acad Sci USA 78 : 6789-6792 ( 198 1 ).

For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOs: 4 and 5. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the endopeptidases of the invention without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. As used herein, the term "biological activity" or "functional activity" refers to the natural or normal function of the endopeptidases of the invention, for example the ability to degrade other proteins. Amino acid residues that are conserved among the endopeptidases of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known within the art. The person skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Furthermore, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1 %) in an encoded sequence are "conservative mutations" where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

Another aspect of the invention pertains to nucleic acid molecules encoding the endopeptidases of the invention that contain changes in amino acid residues that are not essential for activity. Such endopeptidases of the invention differ in amino acid sequence from SEQ ID NOs: 1 and 2 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a endopeptidase, wherein the endopeptidase comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOs: 1 and 2. Preferably, the endopeptidase encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOs: 1 and 2; more preferably at least about 70% homologous to SEQ ID NOs: 1 and 2; still more preferably at least about 80% homologous to SEQ ID NOS: 1 and 2; even more preferably at least about 90%) homologous to SEQ ID NOs: 1 and 2; and most preferably at least about 95% homologous to SEQ ID NOs: 1 and 2.

An isolated nucleic acid molecule encoding an endopeptidase of the invention homologous to the protein of SEQ ID NOs: 1 and 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleic acid sequence of SEQ ID NOs: 4 and 5 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded endopeptidase.

Mutations can be introduced into SEQ ID NOs: 4 and 5 by standard techniques, such as site-directed mutagenesis, PCR-mediated mutagenesis and DNA shuffling. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is a new amino acid that has similar properties and is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Non-conservative substitutions refer to a new amino acid, which has different properties. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, for a conservative substitution, a predicted non-essential amino acid residue in the endopeptidase of the invention is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a coding sequence of the endopeptidase of the invention, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity of the endopeptidase of the invention to identify mutants that retain activity. Following mutagenesis of SEQ ID NOs: 4 and 5, the encoded endopeptidase can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.

In preferred embodiments, the host cell for producing the enzyme composition of the invention in large amounts are recombinant fungus Aspergillus oryzae strains deposited on 19 December 2014 under accession numbers IHEM 26503 and IHEM 26504. This recombinant Aspergillus oryzae strains are engineered to produce prolyl endopeptidase of the invention AoS28A and AoS28B with high yields.

An advantage of gene overexpression in the native organism is that the production of recombinant protein can be obtained from genomic DNA and not obligatory from cDNA since the introns are naturally spliced from transcribed RNA. Yield of 28 μg AoS28A/ml and 23 μg AoS28B/ml were obtained in shaking liquid culture supernatants. Transfer to the industrial scale and optimization of culture conditions could further improve the production of secreted enzymes. In further preferred embodiment, the enzyme composition of invention is produced by

Aspergillus oryzae strain having accession number IHEM 26504 and/ 'or Aspergillus oryzae strain having accession number IHEM 26503. Thus according to an embodiment, the invention provides a method for producing the enzyme composition of the invention, wherein the method comprises:

(b) recovering the enzyme composition. In a particular embodiment, the invention provides a method for producing the enzyme composition of the invention, wherein the method comprises:

(a) cultivating Aspergillus oryzae strain having accession number IHEM 26504 and Aspergillus oryzae strain having accession number IHEM 26503 in a culture medium under conditions suitable for producing the enzyme composition; and (b) recovering the enzyme composition.

In another particular embodiment, the invention provides a method for producing the enzyme composition rich in the enzyme AoS28A of the invention, wherein the method comprises:

(a) cultivating Aspergillus oryzae strain having accession number IHEM 26504 in a culture medium under conditions suitable for producing the enzyme composition; and

(b) recovering the enzyme composition. In a further particular embodiment, the invention provides a method for producing the enzyme composition rich in the enzyme AoS28B of the invention, wherein the method comprises:

(a) cultivating Aspergillus oryzae strain having accession number IHEM 26503 in a culture medium under conditions suitable for producing the enzyme composition; and (b) recovering the enzyme composition.

The expressed enzyme composition can be recovered and purified from recombinant cell cultures by methods well known to the person skilled in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps. Aspergillus oryzae strain having accession number IHEM 26504 overexpressees the gene encoding the enzyme AoS28A of the invention and thereby this Aspergillus oryzae strain is providing at least 1050 mU of AoS28A enzyme activity per ml of supernatant when grown in GP medium. This Aspergillus oryzae strain produces at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 150 folds more AoS28A than the native strain.

Aspergillus oryzae strain having accession number IHEM 26503 overexpressees the gene encoding the enzyme AoS28B of the invention and thereby this Aspergillus oryzae strain is providing at least 2600 mU of AoS28B enzme activity per ml of supernatant when grown in GP medium. This Aspergillus oryzae strain produces at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 150 folds more AoS28B than the native strain.

The host cell may be any of the host cells familiar to the person skilled in the art, including prokaryotic cells, eukaryotic cells, mammalian cells, insect cells, fungal cells, yeast cells and/or plant cells. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces and Staphylococcus, fungal cells, such as Aspergillus, yeast such as any species of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces, including Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe, insect cells such as Drosophila S2 and Spodoptera 5/9, animal cells such as CHO, COS or Bowes melanoma and adenoviruses. In some preferred embodiments, host cells include Aspergillus oryzae, Aspergillus sojae, Saccharomyces cerevisiae, and/or Kluveromyces lactis. The selection of an appropriate host is within the abilities of the person skilled in the art.

The production of a functional protein is intimately related to the cellular machinery of the organism producing the protein. The eukaryotic yeast, the methanoltrophic Pichia pastoris is typically used as the "factory" of choice for the expression of many proteins. P. pastoris has been developed to be an outstanding host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned: The P. pastoris transformation was first reported in 1985. The P. pastoris heterologous protein expression system was developed by Phillips Petroleum, see, e.g. , U.S. Patent NOs. 4,855,231, 4,857,467, 4,879,23 1 and 4,929,555, each of which is incorporated herein by reference. Compared to other eukaryotic expression systems, Pichia offers many advantages, because it does not have the endotoxin problem associated with bacteria or the viral contamination problem of proteins produced in animal cell cultures. Furthermore, P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P. pastoris expression system uses the methanol-induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme that catalyzes the first step in the metabolism of methanol. This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems. Furthermore, P. pastoris has the ability to spontaneously glycosylate expressed proteins, which also is an advantage over E. coli.

In one aspect, the nucleic acid sequences or vectors of the invention are introduced into the host cells, thus, the nucleic acids enter the host cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaP0₄ precipitation, liposome fusion, lipofection (e.g., LIPOFECTIN™), electroporation, viral infection, etc. The candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a endopeptidase of the invention, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced {e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors {e.g. , non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors" . In general, expression vectors of used in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors {e.g. , replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, L, Basic Methods in Molecular Biology, (1986)). The expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The invention also encompasses a transformed host cell comprising nucleic acid sequences encoding the endopeptidases of the invention, e.g., SEQ ID NOs: 4 or 5.

Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the nucleic acids coding for the endopeptidases of the invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the person skilled in the art. The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity. Following transformation of a suitable host cell and growth of the host cell to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired enzyme composition.

AoS28A and AoS28B prolyl endopeptidases of the invention are secreted protein in the culture medium. Culture supernatant can be harvested by centrifugation, filtration or decantation of the mycelium.

Host cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to the person skilled in the art. The expressed enzyme composition can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.

In further embodiment, the invention provides also a method for overexpressing recombinant endopeptidases of the invention in a host cell comprising expressing a vector comprising a nucleic acid of the invention, e.g., an exemplary nucleic acid of the invention, including, e.g., SEQ ID NO: 4 or 5 and biologically active fragments thereof, naturally occurring allelic variants thereof, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of identity to SEQ ID NO: 4 or 5. The overexpression can be effected by any means, e.g., use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.

According to some embodiments, the nucleic acid molecules of the invention can be expressed, or overexpressed, in any in vitro or in vivo expression system. Any cell culture systems can be employed to express, or over-express, recombinant endopeptidase, including bacterial, insect, yeast, fungal or mammalian cultures. Over-expression can be effected by appropriate choice of promoters, enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors (see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8), media, culture systems and the like. In one aspect, gene amplification using selection markers, e.g., glutamine synthetase (see, e.g., Sanders (1987) Dev. Biol. Stand. 66:55-63), in cell systems are used to overexpress the endopeptidase of the invention. Additional details regarding this approach are in the public literature and/or are known to the person skilled in the art, e.g., EP 0659215 (WO 9403612 Al) (Nevalainen et al); Lapidot (1996) J. Biotechnol. Nov 51 :259-64; Luthi (1990) Appl. Environ. Microbiol. Sep 56:2677-83 (1990); Sung (1993) Protein Expr. Purif. Jun 4:200-6 (1993).

Alternatively, if it is desired to produce the endopeptidases with other microorganisms than Aspergillus oryzae, it is possible that the genetic information of Aspergillus oryzae, which has been found initially by extensive screening and which has been proven to be a suitable source of the endopeptidases of the invention, can be transferred to another microorganism which is normally used for the production of endopeptidase endopeptidase s, such as Pichia pastoris that overexpresses the endopeptidases of the invention, thereby providing the desired enzyme composition. Further alternative to recombinant expression, an endopeptidase of the invention can be synthesized chemically using standard peptide synthesis techniques and purified using standard peptide purification techniques known to the person skilled in the art. In other aspects, fragments or portions of the polypeptides may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.

A "purified" polypeptide or protein or biologically-active fragment thereof is substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of chemical precursors or other chemicals" includes preparations of the endopeptidases of the invention in which the endopeptidase is separated from chemical precursors or other chemicals that are involved in the synthesis of the endopeptidase. For example, the endopeptidases of the invention have less than about 30% (by dry weight) of chemical precursors or non- endopeptidase chemicals, more preferably less than about 20%, still more preferably less than about 10%, and most preferably less than about 5% chemical precursors or non- endopeptidase chemicals. Furthermore, "substantially free of chemical precursors or other chemicals" would include oxidation byproducts. The person skilled in the art would know how to prevent oxidation, for example, by keeping chemicals in an oxygen free environment.

In another embodiment, the enzyme composition of the invention can be derived from Aspergillus species, Penicillium species, Fusarium species, Saccharomyces species, and/or Kluveromyces species that were modified to overexpress the endopeptidases of the invention. Preferably the enzyme composition of the invention is derived from Aspergillus fumigatus, Aspergillus oryzae, Aspergillus sojae Aspergillus niger, Aspergillus clavatus, Aspergillus glaucus, Aspergillus ornatus, Aspergillus cervinus, Aspergillus restrictus, Aspergillus ochraceus, Aspergillus candidus, Aspergillus flavus ; Aspergillus wentii, Aspergillus cremeus, Aspergillus sparsus, Aspergillus versicolor, Aspergillus nidulans, Aspergillus ustus, Aspergillus flavipes, Aspergillus terreus, Penicillium roqueforti, Penicillium candidum, Penicillium notatum, Penicillium camemberti, Penicillium glaucus, Penicillium expansum, Penicillium digitatum, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, griseofulvum, Penicillium purpurogenum, Penicillium rugulosum, Penicillium verrucolosum, Fusarium venenatum, Saccharomyces cerevisiae, and/or Kluveromyces lactis. The endopeptidases of the enzyme composition of the invention can be isolated from cells, such as Aspergillus species, Penicillium species, Fusarium species, Saccharomyces species, and/or Kluveromyces species that were modified to overexpress endopeptidases of the invention or culture supernatants by an appropriate purification scheme using appropriate protein purification techniques known to the person skilled in the art.

An "isolated" or "purified" polypeptide or protein or biologically-active fragment thereof is substantially free of cellular material or other contaminating proteins from the cell from which the endopeptidase of the invention is derived.

The language "substantially free of cellular material" includes preparations of endopeptidases of the invention in which the endopeptidase is separated from cellular material of the cells from which it is isolated or recombinantly-produced. For example the endopeptidases of the invention have less than about 30% (by dry weight) of cellular material (or a contaminating protein), more preferably less than about 20%, still more preferably less than about 10%, and most preferably less than about 5% of cellular material (or a contaminating protein). When the endopeptidase of the invention or biologically-active fragment thereof is recombinantly-produced, it is also preferably substantially free of any constituent of the culture medium, e.g. , culture medium components may represent less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the endopeptidase preparation.

Usually, the industrial production of enzymes is performed in a technical fermentation way using suitable microorganisms (bacteria, moulds, fungi). Usually the strains are recovered from natural ecosystems according to a special screening protocol, isolated as pure cultures as well as improved in their properties with respect to the enzyme spectrum and biosynthesis performance (volume/time yield). Enzyme production may also be carried out by methods developed in the future.

In a further embodiment, the present invention also encompasses a fungal enzyme extract, which comprises the enzyme composition according to the invention. Thus the fungal enzyme extract, comprising the enzyme composition according to the invention, can have the same or similar uses as disclosed herein for the enzyme composition of the invention. The fungal enzyme extract of the invention can be derived from the following species modified to overexpress the endopeptidases of the invention: Aspergillus species, Penicillium species, Fusarium species, Saccharomyces species, and/or Kluveromyces species, and preferably from Aspergillus so/^'aeAspergillus fumigatus, Aspergillus oryzae, Aspergillus niger, Aspergillus clavatus, Aspergillus glaucus, Aspergillus ornatus, Aspergillus cervinus, Aspergillus restrictus, Aspergillus ochraceus, Aspergillus candidus, Aspergillus flavus ; Aspergillus wentii, Aspergillus cremeus, Aspergillus sparsus, Aspergillus versicolor, Aspergillus nidulans, Aspergillus ustus, Aspergillus flavipes, Aspergillus terreus, Penicillium roqueforti, Penicillium candidum, Penicillium notatum, Penicillium camemberti, Penicillium glaucus, Penicillium expansum, Penicillium digitatum, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, griseofulvum, Penicillium purpurogenum, Penicillium rugulosum, Penicillium verrucolosum, Fusarium venenatum, Saccharomyces cerevisiae, and/or Kluveromyces lactis.

In a further embodiment, the present invention also encompasses a fungal enzyme extract of Aspergillus oryzae species of the invention, which comprises the enzyme composition according to the invention. Thus the fungal enzyme extract, comprising the enzyme composition according to the invention, can have the same or similar uses as disclosed herein for the enzyme composition of the invention.

In an embodiment, a fungal enzyme extract is an extract comprising an extract rich in AoS28A of the invention from Aspergillus oryzae strain deposited under accession number IHEM 26504 and an extract rich in AoS28B of the invention from Aspergillus oryzae strain deposited under accession number IHEM 26503.

In a further embodiment, AoS28A and AoS28B endopeptidases are isolated from such as Aspergillus oryzae species of the invention by conventional protein purification methods known to those skilled in the art, for example as described in the Current Protocols in Molecular Biology and the Current Protocols in Protein Sciences. The protein fraction of an extract from a Rothia species bacterium can be concentrated by ammonium sulphate precipitation, and then purified by ion exchange chromatography on DEAE SEPHAROSE® CL-6B and gel filtration on SEPHADEX® G-100. Sample fractions are taken at each step and assayed for cleavage activity in order to follow the location of the enzymes. For example, the enzymes can be at least 20% pure, at least 35% pure, at least 45% pure, at least 55% pure, at least 65% pure, at least 75% pure, at least 85% pure, at least 95% pure, at least 95% pure, at least 99% pure, wherein all the percentages between 20 and 99 are explicitly included.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention.

Examples

1. Aspergillus oryzae strains and expression plasmid

All Aspergillus oryzae strains used in this work were used food fermentations (Table 1). Aspergillus oryzae NF 1 is a uridine auxotroph derived from the strain TK3 which was used in food fermentation. Aspergillus oryzae NBRC 100959 is the strain RIB40, which was used for A. oryzae genome sequencing. The ribosomal DNA internally transcribed spacer 2 (ITS2) of all strains was PCR-amplified using the homologous sense and antisense primers PI and P2 (Table 2), and 20 ng of A. oryzae genomic DNA. The generated amplicons were sequenced for strain characterization. Seven, three, and two of the 12 strains used in this study had and ITS2 sequence identical to AB008417, AB008414 and AB008419, respectively.

Plasmid pKS l was designed for the heterologous production of the Aspergillus fumigatus secreted prolyl peptidase AfuS28 in A. oryzae. The sequence of pKS l was deposited under the accession number LN866854. This plasmid was constructed by cloning in pUC57 an expression cassette with the A. oryzae PYRG gene as a selection marker and the A. fumigatus AfuS28 encoding gene under the A. oryzae TEF 1 promoter. The expression cassette was generated by gene synthesis (Genecust, Dunedange, Luxembourg). An Xhol site was inserted in pKS l just downstream of the sequence coding for the A. fumigatus AfuS28 signal sequence. A Bglll site and a NotI site were inserted just after the stop sequence TAG of the AfuS28 ORF.

Table 1. Aspergillus oryzae strains

Strain Species name in ITS DNA AoS28

reference collection sequence secreted

prolyl

peptidase

NF1 A. oryzae AB008417 B

NBRC 100959 A. oryzae RIB40 AB008417 B

NRRL 447 A. flavus var. oryzae AB008417 B

NRRL 458 A. flavus var. oryzae AB008417 B

NRRL 2220 A. oryzae var. oryzae AB008417 A + B

IHEM 4381 A. oryzae var. brunneus AB008417 B

IHEM 5782 A. oryzae AB008417 B

NRRL 32657 A. oryzae AB008414 A + B

IHEM 4382 A. oryzae var. oryzae AB008414 A + B

IHEM 5778 A. oryzae AB008414 A + B

IHEM 5218 A. oryzae var. oryzae AB008419 A + B

IHEM 5790 A. oryzae AB008419 A + B

NF1

Transformants

IHEM 25504 A. oryzae KS36.9 AB008417 A (+ B)

IHEM 25503 A. oryzae KS7.1 AB008417 B 2. Aspergillus oryzae growth media

Aspergillus oryzae strains were routinely grown on 2% (w/v) malt agar medium (Oxoid, Basingstoke Hampshire, UK) to favour sporulation. To promote proteolytic activity at acidic pH, the strains were grown in liquid medium containing 2% glucose, 1% soy meal protein (Supro 171 1, Protein Technologies International, St Louis, MO) or 0.2% gliadin (Sigma- Aldrich, Buchs, Switzerland), 0.5% KH2P04, 0.5% KCl, 0.1% NH4C1, 0.05% MgS04 and 68 mM citrate buffer (pH 4.0). Minimum agar medium (MM) used for transformant selections was prepared following Cove (Cove, 1966). For AoS28A and AoS28B production, A. oryzae transformants were grown in glucose/peptone (GP) medium (2% glucose, 1% polypeptone, 0.5% KH2P04, 0.5% KCl, 0.1% NH4C1, 0.05% MgS04) and 68 mM citrate buffer (pH 4.0).

Table 2. Primers

Primer Oligonucleotide sequence ^b Location PCR product size

(bp) with cloning si

PI 5 ' - TGTGAATTGCAGAATTCCGTG-3 ' (SEQ ID NO. 6) A. oryzae ITS sequence 290

P2 5 ' - TTCAGCGGGTATCCCTACCTG-3 ' (SEQ ID NO. 7) Complement of A. oryzae

ITS sequence

P3 5 ' -GTTCC^rCCGGACACCATTTTCGCCTCGTTCGCC-S' (SEQ ID NO. 8) AoS28B 1069

P4 5 ' -CTTCC^rC TTACATCCACTTCCTCTGTACACCC-S' (SEQ ID NO. 9) Complement of AoS28B (NcoI-BamHI)

P5 5 ' -GTTTC^rC^CCGGGCGGCTTTTGCCCGTAACAAG-S' (SEQ ID NO. 10) AoS28C 1 169

P6 5 ' -CTTT 4GL4rC7TCATCCACTCCACATCTGGTACCC-3 ' (SEQ ID NO. 1 1) Complement of AoS28C (Rcal-Bglll)

P7 5 ' -GTTCC^rCCGGCGCCTTTGTCGCCTTCCTGCGCA-S' (SEQ ID NO. 12) AoS28D 1 153

P8 5 ' -CTTT 7 4rC TTAGAGTGGCACCTGGCTACGCCC-3 ' (SEQ ID NO. 13) Complement of AoS28D (Ncol- -BamHI)

P9 5 ' -GTTTCJCG^GCGATTCATCCGCCCAGGCCGGTTCC-S' (SEQ ID NO. 14) AoS28A 2058

PIO 5 ' -CTTTj(^4rCCCTATTTGTAGTATTCAGCAACCCATG-3 ' (SEQ ID NO. 15) Complement of AoS28A (Xhol- -BamHI)

Pll 5 ' -GTTTCrCGL4G XCTAAGCTTTCTCCCCGGCATC-3 ' (SEQ ID NO. 16) AoS28B 1780

P12 5 ' -CTTTCCCCCCC TTACATCCACTTCCTCTGTACACCC-S' (SEQ ID NO. 17) Complement of AoS28B (Xhol- -Notl)

P13 ₅ , ._{GXXXCrCGL4 G}-_{CGAXXCAXCCGCCCAGGCCGGXXCC} _₃ > (SEQ ID NO. 18) AoS28A 2066

P14 5 '- (Xhol- -BamHI)

CTTTGG^JCCCTAGTGATGGTGATGGTGATGTTTGTAGTATTCAGCAACCC Complement of AoS28A

ATG-3 ' (SEQ ID NO. 19)

₅ , _GTTTCrc^_{GCCCTAAGCTTTCTCCCCGGCATC 3} , _{(SEQ ID N0 20)}

P15 AoS28B 1798

P16 5 '- (Xhol- -Notl)

CTTTCCCCCCC TTAGTGATGGTGATGGTGATGCATCCACTTCCTCTGTACA Complement of AoS28B

CCC-3 ' (SEQ ID NO. 21) ^a Primers P I and P2 were used for PCR amplification of the ribosomal DNA internally transcribed spacer 2 (ITS2) of A. oryzae strains. Primers P3 t P8 were used for antigen production in E. coli. Primers P9 to P 16 were used for AoS28 gene expression in A. oryzae.

b Italicized nucleotides represent cloning sites

3. Antigen preparations and rabbit antiserum productions

Large antigen peptides (200-300 aa) corresponding to partial amino acid sequences of A. oryzae and Aspergillus flavus putative proteases (endopeptidases) of the S28 family were produced using plasmid pET-1 laH6, a derivative of pET-1 la made for His₆-tagged peptide production. For the synthesis of peptides corresponding to partial amino acid sequences of three A. oryzae proteases (S28B, S28C and S28D), sense and antisense primer pairs P3/P4, P5/P6 and P7/P8 (Table 2) were used to amplify A. oryzae RIB40 (NBRC 100959) genomic DNA. A 1059 bp DNA fragment encoding part (amino acid 175 to 527) of a fourth putative protease of the S28 family in A. flavus (MER 161 166) but not identified in the A. oryzae genome was synthesized by Genecust. This protein, called S28A, was 64% identical to the previously characterized AfuS28, and appeared to be a putative orthologue of this prolyl peptidase. The PCR products and synthesized DNA were digested with either Ncol or Real, and ΒαηϊΆΙ to be subsequently cloned into the Ncol and ΒαηϊΆΙ sites of pET-l laH6. The resulting plasmids were used to transform E. coli BL21 for heterologous His₆-tagged peptides production. Cells were grown at 37°C to an Οϋ₆οο of 0.6 and His₆-tagged peptide expression was induced by adding IPTG to a 0.1 mM final concentration. Incubation was continued for an additional 4 h at 37°C. Cells were collected by centrifugation (4,500 ^χ g, 4°C, 10 min), and the His₆-tagged peptides were extracted by lysis with guanidine hydrochloride buffer and Ni-NTA resin affinity (Life Technologies, Carlsbad, CA, USA) columns according to the manufacturer. The column was washed with 0.1 M sodium phosphate buffer (pH 5.9) containing 8 M urea. Thereafter, antigen was eluted with the same buffer adjusted at pH 4.0. Rabbit antisera were made by Eurogentec (Liege, Belgium) by using the purified polypeptides as antigens.

4. Overexpression of the genes encoding AoS28A and AoS28B in A. oryzae

Aspergillus oryzae genomic DNA was isolated from freshly growing mycelium using a Qiagen DNAeasy Plant mini Kit (Qiagen, Hilden, Germany). DNA encoding AoS28A and AoS28B with and without a C-terminal H₆-tag sequence was amplified by PCR with a standard protocol using homologous sense and antisense primers (P9/P10 and P13/P14 for AoS28A, and P1 1/P12 and P15/P16 for AoS28B, Table 2) and 200 ng of A. oryzae genomic DNA. The PCR product was digested with Xhol and either BgUI, or BamHI, or Notl for which a site was previously designed at the 5' end of the primers, and fused to the large 6.5 kb fragment of pKSl digested with zoI/Bglll or XhollNotl to generate expression plasmids. Aspergillus oryzae NF1 transformation with either Smal or Sacl/Spel linearized plasmid DNA was performed by electoporation and transformants were selected on MM. For recombinant enzyme production, one litre flasks containing 200 ml of acidic soy meal protein medium (pH 4.0) were inoculated with approximately 10⁸ spores of selected transformants, and incubated for 48 h at 30°C on an orbital shaker at 200 rpm.

5. Purification of AoS28A and AoS28B from A. oryzae transformants

For enzyme characterization, His₆-tagged AoS28 and AoS28B without an His₆-tag were produced in A. oryzae selected transformants. Ala-Ala-Pro-p-nitroanilide (AAP-pNA) (Genecust, Dunedange, Luxembourg) was used as a substrate to test enzyme activity in culture supernatant and to select active fractions. Protein concentrations were measured by the method of Bradford using a commercial kit (Bio-Rad, Hercules, CA), and using different amounts of bovine serum albumin as standards. The secreted proteins from 200 ml of A. oryzae culture supernatant were concentrated by ultrafiltration to 2.5 ml using a Centricon Plus-70 (30 kDa cut-off) (Millipore, Volketswil, Switzerland). Thereafter, the concentrate was desalted with PD10 column (Amersham Pharmacia, Diibendorf, Switzerland). After the concentration and desalting steps, His6-tagged AoS28A was extracted with a Ni-NTA resin (Life Technologies) column with histidine elution buffer (50 mM histidine in PBS) as known in the prior art. For AoS28B purification, the active fractions eluted from the PD10 column were pooled and applied to a hydroxyl apatite (HPT) (Bio-Rad) column which had previously been equilibrated with a 10 mM Na phosphate buffer (pH 7.0). After washing the column with the same buffer, the recombinant protein was eluted with a 100 mM sodium phophate buffer (pH 7.0). 6. Proteolytic activities

Exoproteolytic activities were tested with synthetic substrates supplied by Genecust. Stock solutions were prepared at 100 mM concentration and stored at -20°C. AP-pNA, AAP-pNA, APP-pNA, AAAP-pNA and Suc-AAAP-pNA were dissolved in Ethanol/DMSO (50%/50%, v/v). The reaction mixture contained a concentration of 10 mM substrate and the enzyme preparation (between 0.1 to 1.0 μg per assay) in 100 μΐ of 50 mM citrate buffer (pH values from 2.0 to 7.0) or in 50 mM of Tris buffer (pH values from 7.0 to 9.0). After incubation at 37°C for 10-240 min (depending on the activity of the enzyme preparation), the reaction was terminated by adding 5 μΐ of glacial acetic acid and then 0.9 ml of water. The released pNA was measured by spectrophotometry at λ= 405 nm. A control with substrate but without enzyme was carried out in parallel. The AoS28 activities were expressed in mU (μηιοΐεβ of released pNA/min) using AAP-pNA as a substrate.

7. Protein extract analysis

SDS-PAGE of the different protein extracts was performed on a 10% separating gel. The gels were stained with Coomassie brilliant blue R-250 (Bio-Rad, Hercules, CA, USA). N- glycosidase F digestion of protein extracts was performed as knonw in the prior art. Western blots were revealed using rabbit antisera and alkaline phosphatase conjugated goat anti-rabbit IgG (Bio-Rad).

8. Gene intron exon structures

The cDNA encoding AoS28A and AoS28B was obtained by RT-PCR with a Qiagen OneStep RT-PCR kit following protocol provided by the supplier. RNA extracted from selected A. oryzae transformants and homologous sense and antisense primers (Table 2) were used. Aspergillus oryzae total RNA was isolated using the filamentous fungi protocol of a Qiagen RNeasy total RNApurification kit. The PCR product was digested with Xhol and either Bglll or Not! for which a site was previously designed at the 5' end of the primers, and cloned into pKJl 13 digested with XhoI/BamHI for subsequent sequencing. 9. Enzymatic degradation of the 33-mer

Enzymatic degradation was tested with the 33-mer

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:22) obtained from Altergen (Schiltigheim, France). Stock solutions were prepared at 10 mg/ml concentration in water. Concentrations of AoS28A and AoS28B in the enzyme preparations were measured by BCA assay (Pierce Biotechnology, Rockford, IL) prior to their dilution in water to get 80 μg/ml solutions. The reaction mixture contained 4 μΐ of substrate solution (40 μg) and 12.5 μΐ of both enzyme solutions (1 μg) in 71 μΐ of pH 3.8 buffer (20 mM citrate buffer) or of pH 7.8 buffer (20 mM Tris buffer). After incubation at 37°C under agitation for 0, 2.5, 5, 7.5, 10, 15, 20, 30, and 60 min, the reaction was terminated by adding 990 μΐ of stopping solution containing ACN 50% and FA 0.1%. The peptides in solution were analyzed by mass spectrometry (MS, see herein). Reaction mixtures with only one of the two enzymes, as well as controls without substrate and/or without enzyme, were prepared in parallel. Water and solvents were obtained from Biosolve (Valkenswaard, The Netherlands). 10. Gliadin enzymatic degradation - comparison AfuS28 + SedB and AoS28A + AoS28B

The degradation of the 33-mer of gliadin was studied at two different pH, either in a 20 mM citrate buffer at pH 3.8 or in a 20 mM Tris buffer at pH 7.8. Two pairs of enzymes (AfuS28 + SedB and AoS28A + AoS28B) ensured the degradation. 33-mer of gliadin was obtained from Altergen (Schiltigheim, France). A 10 mg/mL solution of the 33-mer of gliadin was prepared in water. For all four conditions, an Eppendorf was prepared containing buffer in an appropriate volume to get a final volume of 100 μΙ_, when the 33- mer of gliadin and the enzymes are added; 4 μΙ_, of the 33-mer of gliadin solution (40 μg) were added. The solution was placed in an incubator at 37°C with agitation. At time = 0 min, the enzymes were added (1.88 + 2.30 μg for Afus28 and SedB, 2.85 + 2.13 μg for AoS28A and B). Every 2.5 or 5 minutes, 10 μΙ_, of the solution were taken and immediately diluted in 990 μΙ_, of a solution containing ACN 50% and FA 0.1%. The solution at time = 0 min was taken before the addition of enzymes.

11. MS analysis and data processing

Kinetic MS analyses were performed on a Waters Xevo TQ-S triple quadrupole (Waters corp, Milford, MA, USA) equipped with its standard electrospray interface (ESI). The reaction mixtures diluted with stopping solution were infused using the built-in system at a rate of 25 μΐ/min in combination with an LC flow of ACN 50% + FA 0.1% at 300 μΐ/min from a Waters Acquity UPLC i-Class. MeOH was loaded between every analysis to wash the system. Analyses were operated in the MS I scan mode using positive ionization during one minute, after an initial minute of infusion for equilibration. Data were acquired in continuum mode over the 800- 1500 m/z range with a scan time of 1 s. The capillary and cone voltages were set to 3.80 kV and 62 V, respectively. The source temperature, desolvation temperature, cone gas flow and desolvation gas flow were set to 150°C, 300°C, 150 1/h and 800 1/h, respectively. MeOH 50% was used as purge solution for the fluidics. Spectra 7 to 60 were combined and smoothed (mean, 2x2). Kinetic plots were based on the areas of the peaks. Raw data were acquired and processed with MassLynx 4.1 (SCN 901) software from Waters. High-resolution MS analyses were performed on a Thermo Fisher Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using positive ionization mode in Full MS mode, with a scan range set at 150-1500 m/z in the ESI positive mode with a heated ESI probe (H-ESI). The m/z resolution was set at 35000, AGC Target = l e6, maximum IT = 100 ms, microscans = 1, sheath gas = 10 (arb), aux gas = 0 (arb), spare gas = 0 (arb), spray voltage = 3000 V, capillary temperature = 320°C and probe heater temperature = 30°C.

12. Optimal pH activity of AoS28A and Ao S28B

The optimal pH for enzymatic activities was determined using the Ellis and Morrison buffer system. The buffer contained three components with different pKa values while the ionic strength of buffer remained constant throughout the entire pH range examined. The pH of the buffer was adjusted from 2 to 8 in half-pH unit increments with 1M HC1 or 1M NaOH. 0.2 μg of purified AoS28A and AoS28B were incubated with Ala-Ala-Ala-Pro- pNA at 1 mM concentration for 30 min at 37C at pH 2.0 to 8.0 with increment of 0.5 units in a total volume of 0.1 ml. The released pNA was measured in an ELISA reader at 405 nm. A control with substrate but without enzyme was carried out in parallel and the absorbances were substracted from the values obtained in presence of the enzymes. Finally, a combination of AoS28A and AoS28B (each 0.2 μg) was also performed to assess that all the pH interval was covered for activity when the two enzymes were associated.

AoS28A is at least 3 fold more active than AoS28B (based on a similar molar ratio) to cleave AAAP-pNA and exhibits a wide range of activity from pH 2.0 to pH 8.0 with an optimal pH ranging from pH 4 to 5 and a significant activity at pH 2.0 representing 37 % of the maximum activity reached at pH 4.5 (OD at 0.218 at pH 2.0 vs 0.588 at pH 4.5). AoS28A is therefore very active in the gastric fluid. AoS28B pH optimal activity is rightward shifted with an optimal activity at pH 6.5. AoS28B surpasses the activity of AoS28A at pH 6.5 and pH 7.0. This result indicates that AoS28B is the best enzyme to digest substrates at pH encountered in the intestinal fluid. See Table 3 for results.

Finally, the combination of both enzymes confirms that they represent the best choice to ensure full digestion of AAAP-pNa from pH 2.0 to 8.0. Table 3

pH Optimum for AoS28A and B

pH Optimum for AoS28A and B (Blank substracted)

Results are expressed in absorbance at 405 nm

RESULTS

Major secreted prolyl peptidases secreted by various A. oryz e strains

Aspergillus oryzae strains were grown for 48 h in acidic soy meal protein and gliadin medium buffered at pH 4.0. Secreted prolyl peptidase activity using AAP-pNA as a substrate varied from 13 to 150 U/ml depending on the strain. NRRL 2220 was the most active strain. Less than 1.0 U/ml was measured when A. oryzae strains were grown in MM and GP medium. It was first tried to identify which prolyl peptidase was secreted by A. oryzae RIB40 (Table 1). Culture supernatant was concentrated by ultrafiltration, desalted and applied to a hydroxylapatite (HPT) column which had previously been equilibrated with a 10 mM Na phosphate buffer (pH 7.0) as described in Example section. All prolyl peptidase activity was retained on HPT, but could be eluted from the column with a 100 mM sodium phosphate buffer (pH 7.0). The active fractions eluted from the HPT column were pooled, concentrated by ultrafiltration and the proteins were separated by SDS-PAGE electrophoresis. No protein band could be detected in SDS-PAGE gels stained with Coomasie blue. However, western blot analysis using a prepared antiAoS28B antiserum revealed a smearing signal for a putative protein of approximately 60-100 kDa. A 60 kDa protein band was detected after endoglycosidase treatment of samples from the same active eluted fractions. No signal was detected using antisera raised against AoS28A, AoS28C and AoS28D large peptides. It was concluded that the major prolyl peptidase secreted by A. oryzae RIB40 was AoS28B. When concentrated culture supernatant of strains NRLL2220 was applied to a HPT column, 25% of the total prolyl peptidase activity was found to be not retained. Western blot analysis of the HPT column filtrate revealed a smearing band for a protein of approximately 70 kDa using prepared anti-AoS28A antiserum. A 60 kDa protein band was detected after endoglycosidase treatment of the same samples. In addition, prolyl peptidase activity retained on HPT (75%) could be eluted from the column with 100 mM sodium phosphate buffer (pH 6.0) and was found to be AoS28B like in RIB40 strain. No signal was detected using antisera raised against AoS28C and AoS28D large peptides in either HPT column filtrate containing AoS28A or eluted fractions containing AoS28B. It was concluded that two major prolyl peptidases, AoS28A and AoS28B, were secreted by NRRL220.

Further immunoblotting analysis revealed that AoS28B was secreted at pH 4.0 in a protein medium by all 12 A. oryzae strains used in this study. In the same growth conditions, AoS28A was secreted by 3 and 2 A. oryzae reference strains with an ITS2 sequence identical to AB008414 and AB008419, respectively (Table 1). Noteworthy, BLAST analysis revealed that a lot of strains with the AB008414 sequence are named A. flavus in reference collections, while strains with AB008419 sequences are named A. sojae or A. parasiticus. AoS28A was not detected in culture supernatant in 5 strains, all with an ITS2 sequence identical to AB008417. The defect of a gene coding for a S28A prolyl peptidase appears to be a feature of most A. oryzae strains with the AB008417 ITS2 sequence, and strain NRRL2220 which secreted AoS28A appeared as an exception among these strains. Overexpression of the genes encoding AoS28A and AoS28B in A. oryzae

In order to produce large amounts of prolyl peptidase for further purification and characterization, the genes encoding AoS28A and AoS28B were overexpressed in A. oryzae NFl. The genomic DNA encoding AoS28B could be amplified using specific 5'-sense and 3'- antisense primers (Table 2) using A. oryzae NFl, RIB40 and NRLL2220 genomic DNA as the template. AoS28A encoding DNA could be amplified using genomic DNA of strain NRLL2220 but not using that of strains RIB40 and NFl . The A. oryzae expression plasmids pAoS28A and pAoS28B carrying genomic DNA encoding NRRL2220 AoS28A and RIB40 AoS28B, respectively, were subsequently constructed as described in Material and Method section. More than 100 URA+ A. oryzae NFl transformants were obtained with pAoS28A and pAoS28B. In a first round of selection, fifty URA+ transformants were tested for prolyl peptidase activity by a quick test consisting in measuring the activity obtained from a carrot of agar taken from the edge of the colony. Activity varied in a 1 to 20 ratio from one clone to another. For each transformation assay with pAoS28A and pAoS28B, the three clones appearing the more active were purified by growing fungal colonies from single spores on MM agar. Each transformant was subsequently grown in acidic soy meal protein liquid medium for 48 h to select the most active one producing either AoS28A or AoS28B for further investigations. The finally selected transformants, KS36.9 (IHEM 26504) producing AoS28A and KS7.1 (IHEM 26503) producing AoS28B, were found to produce 1050 and 2600 U/ml of prolyl peptidase activity, respectively, using AAP-PNA as a substrate. Prolyl peptidase activity of both transformants was found to be approximately 50% lower in GP medium than in SP medium and in gliadin medium. For comparison, the activity of the non-transformed NF1 strain in GP medium was below 1.0 U/ml, and 10 U/ml in SP and gliadin medium. In further experiments, AoS28A and aoS28B were tentatively produced with a His₆-tag sequence at their C-terminus. Recombinant His₆-tagged AoS28A could be purified from A. oryzae culture supernatant by affinity chromatography using Ni-NTA resin for further characterization. His₆-tagged AoS28B did not bind to Ni-NTA resin, and purification of this second enzyme was performed using HPT as described in Material and Method section.

To promote proteolytic activity at acidic pH, the strains were grown in liquid medium containing 2% glucose, 1% soy meal protein (Supro 171 1, Protein Technologies International, St Louis, MO) or 0.2% gliadin (Sigma- Aldrich, Buchs, Switzerland), 0.5% KH2P04, 0.5% KC1, 0.1% NH4C1, 0.05% MgS04 and 68 mM citrate buffer (pH 4.0).

The yield was about 40 μg/ ml in A. oryzae culture supernatant (Figure 2). Intron-exon structures of the genes encoding AoS28

The cDNA fragments encoding AoS28A and AoS28B were obtained by RT-PCR using RNA extracted from selected A. oryzae transformants. The intron-exon structure of AoS28A and that of AoS28B were determined by comparing the cDNA sequences with the A. oryzae and A. flavus genome sequences. Nine introns in positions similar to those of the genes encoding AfuS28 and AN PEP were revealed in the gene encoding AoS28A. Two introns were found in the gene encoding AoS28B.

Properties of AoS28A and AoS28B

AoS28A was found to be active at pH values from 2.0 to 6.0 with an optimal value at pH 4.0 at 37°C with AAP-pNA as a substrate. AoS28B was found to be active at pH values from 3.0 to 7.0 with an optimal value at pH 4.5. At optimal pH, specific AoS28A activity was 37 mU^g protein, while that of AoS28B was 1 12 mU^g protein. From these values, the amount of secreted AoS28A and AoS28B in soy meal protein medium was assessed as 28 and 23 (1050 and 2600 U/ml, respectively). Recombinant AoS28A and AoS28B also very efficiently released pNA when APP-pNA, AAAP-pNA and Suc-AAAP-pNA were used as substrates. At optimal pH, AoS28A (AoS28B) activity was 2 (37), 16 (240), and 33 (38) mmol/min/mg, respectively. Both enzymes showed no activity on the DppIV substrate AP-pNA, and on the sedolisin substrate APF-pNA. AoS28A and AoS28B are glycoproteins as attested by a reduction in their molecular weights following treatment with N-glycosidase F (Figure 3). The apparent molecular mass of each deglycosylated protein (approximately 60 kDa) was close to that of the calculated molecular mass of the polypeptide chain deduced from the nucleotide sequence encoding both proteases. These enzymes belong to the same family as the human lysosomal Pro-Xaa carboxypeptidase (MEROPS, Accession MER000446) which is a physiological activator of plasma prekallikrein. This enzyme is the prototype of the S28 family for which the Ser, Asp, His catalytic triad of this enzyme has been determined from its crystal structure. Using comparative alignments, the putative catalytic triad of AoS28A and AoS28B could be deduced. Figure 4 shows AoS28A (a) and AoS28B (b) extracted from culture supernatants of A. oryzae overproducing transformants before (1) and after (2) deglycosylation. SDS-PAGE gels (10%) were stained with Coomasie Blue. Figure 14 shows determination of specific activities of Aos28A (yellow circles) and Aos28B (blue triangles) at different pH values using AAP-pNA as a substrate. Absorbance was measured at 405 nm at 37°C.

Enzymatic degradation of the 33-mer

Degradation of the 33-mer peptide was monitored using either AoS28A, or AoS28B, or both enzymes. The reaction was completely stopped by a l OOx dilution in stopping solution containing 50% of ACN and 0.1 % of formic acid. Samples were analyzed by MS in random order, and the intensity of all detected peptidic peaks was measured as described in Examples section. None of these peptides were detected in any control sample containing no enzyme or no substrate. The 33-mer was found to be stable for one hour in our experimental conditions without enzyme. In contrast, the peak intensities of the 33-mer rapidly decreased in the presence of enzyme(s). At pH 3.8 (Figure 5(a)) the peptide was not detected after 2.5 min with AoS28B or AoS28A+AoS28B, or after 15 min in the presence of AoS28A. At pH 7.8, the digestion was almost completed after 30 min of incubation with the combination of both enzymes (AoS28A being the most efficient) (Figure 5(b)).

The search for the degradation products of the 33-mer produced by either AoS28A, or AoS28B or both enzymes resulted in the neoformation of 12 peptides. The size of the detected peptides ranged from 7 (lower limit set to the instrument) to 28 amino acids. Some detected peptides corresponded to several fragments of the 33-mer since its structure is made of 3 repetitions of the same sequence of 7 amino acids (QPQLPYP, SEQ ID NO: 25). Most peptides were detected in all experimental conditions (at both pH and using both enzymes), except at pH 7.8 with AoS28B. In this case, only peptides 1-33 and 6-33 were detected, the latter with stable intensity over time, indicating that it was the unique product of degradation.

All over time peak intensities of the peptides that were produced by digestion of the 33- mer using either AoS28A, or AoS28B, or both enzymes were similar except at pH 7.8 with AoS28B. These peptides were not detected at 0 min, showed maximal peak intensities between 2.5 and 30 minutes before decreasing, probably because of their further degradation in smaller fragments. The peak intensities of the smallest detected peptides also decreased with time, suggesting that they were also further digested. Indeed, tetramers and pentamers such as peptide 6-9 and fragments QPQLP (SEQ ID NO: 26), QLPYP (SEQ ID NO: 27), and YPQP (SEQ ID NO: 28) were detected by Orbitrap MS, but not by the triple quadrupole MS because of the lower m/z limit set to 800.

The following results were obtained by a careful examination of the products of degradation of the 33-mer: First, both AoS28A and AoS28B were strict prolylendopeptidases with the 33-mer as a substrate, as all detected peptides without exception are produced by cleavage of the peptide bonds at the C-terminal side of Proline residues. Second, the digestion of the 33-mer with both enzymes was found to occur sequentially from its N-terminus as all detected peptides of > 10 amino acids contained the C-terminus of the 33-mer and no N-terminal fragments such as 1-19 or 1-12 were detected (Figure 6). An analytical bias was excluded since smaller N-terminal peptides were detected (e.g. peptide 1-9). Third, the digestion of the 33-mer and its fragments by the combination of both enzymes was always faster or at least equivalent to the digestion by the sole enzymes. This result is explained by the difference in activities of both enzymes rather than by the higher amount of enzyme in solution (2x 1 μg). Indeed, the simultaneous use of both enzymes with slightly different activities provides an interesting synergic effect by reducing the number of limiting steps of degradation. For example, 15 min was needed for AoS28A to completely digest the 33-mer while Aos28B had the same effect in 2.5 min (Figure 5(a)). Conversely, AoS28A could efficiently cleave the QP//QLP bond that AoS28B could not, e.g. to produce the 10-33 peptide (Figure 6). Finally, the intensities measured for all immunotoxic peptides, i.e. peptides with nine of more amino acids and all non-antigenic peptides were searched and summed. Figure 1 shows that all peptides equal or larger than nine amino acids in size were completely digested at acidic pH by the combination of both enzymes after 20 min, while 30 to 60 min were necessary when only one of the enzymes was used in the same experimental conditions (Figure 1(a)). In contrast, high quantities of these peptides larger than 9 amino acids in size were found at pH 7.8, in particular when the sole Aos28B is used (Figure 1(b)). Conversely, increasing quantities of peptides smaller than nine amino acids in size were detected in all conditions (Figure 1(c) and (d)).

Comparative data

The the digestion of a 33-mer of gliadin was also tested by the two different combination of enzymes: AoS28A + B of the present invention and AfuS28 + SedB of the prior art at two different pH (3.8 and 7.8). The combination of AoS28A + B enzymes digests the 33-mer of gliadin and produces new peptides of smaller size. After few minutes, only fragments of peptides containing 7 of less amino acids are detected (Figure 7) and see Table 4 for detailed results. This result is similar to the one obtained for the degradation by the combination of AfuS28 + SedB. The main difference between the degradation of the 33-mer of gliadin by the two combination of enzymes is the kinetic of digestion. At pH 3.8, the combination of AoS28A + B totally digested the 33-mer of gliadin in 5 minutes, while AfuS28 + SedB fully digested the same peptide in 15 minutes only (Figure 8). At pH 7.8, the combination of AoS28A + B digested the 33-mer of gliadin in 10 minutes; the combination of AfuS28 + SedB did not completely digest the same peptide after 25 minutes since 10 % of the 33-mer of gliadin is still present (Figure 8). The fast generation of a fragment of 10 +/- 1 amino acids (m/z 11 12.05, fragment of unknown composition) is observed. However, it decreases after t = 5 min with all enzymes. The peptide is most probably digested in smaller fragments. Its degradation is clearly faster and went to completion by the combination of enzymes AoS28A + B whereas some residual peptide remains intact (i.e, 10 %) with AfuS28 + SedB at pH 3.8 and even was merely not digested by this combination at pH 7.8 (Figure 9). The kinetic of two 7-mers was studied: YPQPQPF (SEQ ID NO: 29) (Figure 10) and YPQPQLP (SEQ ID NO: 30) (Figure 11). The concentration of both peptides is zero or almost zero and rapidly increases. Contrasting with the concentration of the 10 +/- 1 amino acids peptides mentioned above, the concentrations of the 7-mers do not decrease in most of the conditions during the 20 or 30 minutes of the experiment. This may indicate that the peptides are not further digested, or that the kinetic of their production is similar or higher than the one of their degradation. In conclusion, the digestion of the 33-mer of gliadin by the combination of AoS28A + B of the present invention is faster than by the combination of AfuS28 + SedB of the prior art. This faster kinetic may be advantageous for the degradation of gliadin in the frame of celiac disease. Indeed, the degradation of the 33-mer of gliadin by this combination of AoS28A + B at pH 3.8, similar to the gastric pH, was complete after 5 minutes and should allow to prevent the passage in the intestinal tract of residual cytotoxic 33 mer of the gliadine.

Table 4 - intensities of 4 ions measured in the infusions of the reactional medium. See the experimental conditions section for more details.

20.0 7.88E+05

25.0 7.00E+05

30.0 7.67E+05 m/z 876.8 Afiis28 + TPP Afiis28 + TPP Aos28 A + B Aos28 A + B

Time (min) pH 3.8 pH 7.8 pH 3.8 pH 7.8 time

0.0 0 1.55E+05 8.59E+04 1.08E+05

2.5 6.86E+05 2.19E+05 4.96E+05 4.93E+05

5.0 1.41E+06 2.65E+05 6.81E+05 1.28E+06

7.5 1.70E+06 3.01E+05 1.10E+06 1.58E+06

10.0 2.14E+06 4.34E+05 1.57E+06 1.74E+05

15.0 3.08E+06 8.35E+05

20.0 1.24E+06

25.0 1.38E+06

30.0 1.87E+06 m/z 842.98 Afiis28 + TPP Afiis28 + TPP Aos28 A + B Aos28 A + B

Time (min) pH 3.8 pH 7.8 pH 3.8 pH 7.8

0.0 1.44E+05 1.51E+05 1.65E+05 3.19E+05

2.5 1.83E+06 1.11E+06 4.83E+06 6.00E+06

5.0 2.45E+06 1.42E+06 7.76E+06 3.38E+06

7.5 3.32E+06 1.53E+06 8.64E+06 3.54E+06

10.0 3.02E+06 1.79E+06 9.00E+06 1.09E+05

15.0 2.81E+06 1.78E+06

20.0 1.99E+06

25.0 1.77E+06

30.0 2.11E+06

Claims

1. An enzyme composition comprising a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and/or a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

2. The enzyme composition of claim 1 comprising a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

3. The enzyme composition of claim 1 or 2, wherein the prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 is derived from Aspergillus oryzae deposited under accession number IHEM 26504, and the prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2 is derived from Aspergillus oryzae deposited under accession number IHEM 26503.

4. The enzyme composition of claim 1 consisting of an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and/or an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

5. The enzyme composition of claim 4 consisting of an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 and an extract derived from a host cell overexpressing a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2.

6. The enzyme composition of claim 4 or claim 5, wherein the host cell

overexpressing the prolyl endopeptidase AoS28A contains a suitable promoter and a nucleic acid encoding for a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 4 and wherein the host cell overexpressing the prolyl endopeptidase AoS28B contains a suitable promoter and a nucleic acid encoding for a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5.

7. The enzyme composition of claim 4 or claim 5, wherein the host cell overexpressing the prolyl endopeptidase AoS28A is Aspergillus oryzae strain deposited under accession number IHEM 26504, and the host cell overexpressing the prolyl endopeptidase AoS28B is Aspergillus oryzae strain deposited under accession number IHEM 26503.

8. The enzyme composition of any one of claims 1 -7, wherein a prolyl endopeptidase AoS28A comprising or consisting of SEQ ID NO: 1 is fused to the signal sequence SEQ ID NO: 3, and/or a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 2 is fused to the signal sequence SEQ ID NO: 3.

9. A pharmaceutical composition comprising an effective amount of the enzyme composition of any one of claims 1-8 and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

10. A food supplement comprising the enzyme composition of any one of claims 1-8.

1 1. The enzyme composition of any one of claims 1 -8 for use in a method for treating and/or preventing a condition selected from the group comprising celiac disease, digestive tract bad absorption, an allergic reaction, an enzyme deficiency, a fungal infection, mycoses, Crohn disease, sprue and wound healing.

12. The enzyme composition for use according to claim 1 1, wherein the allergic reaction is a reaction to gluten.

13. A method of degrading a polypeptide substrate, wherein said method comprises contacting the polypeptide substrate with the enzyme composition of any one of claims 1-8.

14. The method of degrading a polypeptide substrate according to claim 13, wherein said enzyme composition sequentially digests a full-length polypeptide substrate or a full-length protein and wherein the polypeptide substrate is selected from the group comprising casein, gluten, bovine serum albumin or fragments thereof .

15. A method of detoxifying gliadin, wherein said method comprising contacting gliadin containing food product with an effective dose of the enzyme composition of any one of claims 1-8.

16. A method for improving food digestion in a mammal, wherein said method comprising oral administration to the said mammal of the enzyme composition of any one of claims 1-8.

17. The method for improving food digestion of claim 16, wherein the food contains proline rich nutriments such as gluten.

18. A method for producing the enzyme composition of any one of claims 1-8, wherein said method comprises

(a) introducing into a host cell a nucleic acid encoding for

ii. a prolyl endopeptidase AoS28B comprising or consisting of SEQ ID NO: 5,

(c) recovering the enzyme composition.

19. The method for producing the enzyme composition according to claim 18, wherein the host cell is Aspergillus oryzae, Aspergillus sojae, Saccharomyces cerevisiae, and/or Kluveromyces lactis.

20. A method for producing the enzyme composition of any one of claims 1-8, wherein said method comprises

(b) recovering the enzyme composition.

21. A recombinant Aspergillus oryzae strain, wherein the strain is fungus Aspergillus oryzae deposited under accession number IHEM 26503.

22. A recombinant Aspergillus oryzae, wherein the strain is fungus Aspergillus oryzae deposited under accession number IHEM 26504.