ES2368285T3 - NEW GENES THAT CODIFY NEW PROTEOLITHIC ENZYMES. - Google Patents

Abstract

An isolated polynucleotide hybridizable under conditions very restrictive to polynucleotide SEQ ID NO: 10 or to the sequence SEQ ID NO: 67, wherein said polynucleotide encodes a protein that has protease activity.

Description

New genes encoding new proteolytic enzymes

Field of the Invention

The invention relates to newly identified polynucleotide sequences comprising genes that

5 encode new proteases isolated from Aspergillus niger. The invention relates to the full-length nucleotide sequence of the new genes, to the cDNA sequences comprising the full-length coding sequences of the new proteases, as well as to the amino acid sequences of the full-length functional proteins and fragments. and variants thereof. The invention also relates to methods for using these enzymes in industrial processes, and to methods for diagnosing fungal infections. Also included in the invention are cells transformed with a polynucleotide according to the invention, and cells in which a protease according to the invention is genetically modified to enhance or reduce its activity and / or level of expression.

Background of the invention

Proteolytic enzymes

Proteins can be considered heteropolymers that consist of amino acid building blocks

15 connected via a peptide link. The repetitive unit in proteins is the central alpha carbon atom with an amino group and a carboxyl group. Except for glycine, a so-called amino acid side chain replaces one of the two hydrogen atoms in the remaining alpha carbon. The amino acid side chain makes the central alpha carbon asymmetric. In general, the L-enantiomer of the amino acid is found in the proteins. The following terms describe the various types of polymerized amino acids. Peptides are short chains of amino acid residues with a defined sequence. Although there really is not a maximum to the number of residues, the term usually indicates a chain whose properties are mainly determined by its amino acid composition and which does not have a fixed three-dimensional conformation. The term polypeptide is usually used for longer chains, usually of defined sequence and length, and in principle of the appropriate length to fold into a three-dimensional structure. Protein is reserved for polypeptides that appear from

25 naturally and showing a defined three-dimensional structure. In the case where the main function of proteins is to catalyze a chemical reaction, it is usually called an enzyme. Proteases are the enzymes that catalyze the hydrolysis of the peptide bond in (poly) peptides and proteins.

Under physiological conditions, proteases catalyze the hydrolysis of the peptide bond. The International Union of Biochemistry and Molecular Biology (1984) has recommended using the term peptidase for the hydrolase set of the peptide bond (Subclass E.C 3.4.). The terms protease and peptide hydrolase are synonymous with peptidase, and can also be used here. Proteases comprise two classes of enzymes: endopeptidases and exopeptidases, which cleave peptide bonds at points within the protein, and remove amino acids sequentially from the term N or C respectively. Proteinase is used as a synonym for endopeptidase. The peptide bond can be produced in the context of di-, tri-, tetrapeptides, peptides, polypeptides or proteins. In general, the

The amino acid composition of natural peptides and polypeptides comprises 20 different amino acids, which show the L configuration (except for glycine, which has no chiral center). However, the proteolytic activity of proteases is not limited to peptides containing only the 20 natural amino acids. Peptide bonds can also be cleaved between so-called unnatural amino acids, as well as peptide bonds between modified amino acids or amino acid analogs. Some proteases do not accept the D enantiomers of amino acids in certain positions. In general, the remarkable stereoselectivity of proteases makes them very useful in the chemical resolution process. Many proteases show interesting secondary activities, such as esterase activity, thiol esterase activity and (des) amidase activity. These secondary activities are usually not limited to amino acids only, and can be very useful in bioconversions in the area of fine chemicals.

45 There are a number of reasons why filamentous fungal proteases, eukaryotic microorganisms, are of particular interest. The basic process of hydrolytic cleavage of peptide bonds in proteins seems costly and potentially harmful to an organism if not properly controlled. The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates in the cell, through the modification of the substrates that allow recognition by the respective proteases, by regulation via activation of cytogens , and the presence or absence of specific inhibitors, as well as through the regulation of protease gene expression. In fungi, proteases are also involved in other fundamental cellular processes, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation. In fact, Aspergillus nidulans and Neurospora crassa have been used as model organisms to analyze the molecular basis of a range of

55 physiological and developmental processes. Its genetics allow direct access to biochemical and genetic studies, under defined nutrient and culture conditions. In addition, a large group of pathogenic fungi for humans, livestock and crops have been isolated, and it has been suggested that proteolysis plays a role in their pathogenicity (host penetration, opposing host defense mechanisms and / or nutrition during infection ). Proteases are also frequently used in laboratory, clinical and industrial processes; the proteases

Both microbial and non-microbial are widely used in the food industry (cooking, brewing, cheese making, meat softening), in the tanning industry, and in the manufacture of biological detergents (Aunstrup, 1980). Commercial interest in exploiting certain filamentous fungi, especially Aspergilli, as hosts for the production of both homologous and heterologous proteins has also recently renewed the interests in fungal proteases (van Brunt, 1986ab). Proteases often cause problems in heterologous expression and homologous overexpression of protein in fungi. In particular, heterologous expression is prevented by proteolytic degradation of products expressed by homologous proteases. These commercial interests have resulted in detailed studies of proteolytic spectra and construction of protease deficient strains, and have improved knowledge about the expression and regulation of proteases in these organisms. Consequently, there is a great need to identify and eliminate new proteases in filamentous fungi.

Microorganisms such as, for example, fungi are particularly useful in large-scale protein production. In particular, when such proteins are secreted to the medium. Proteolytic enzymes play a role in these production processes. On the other hand, in particular particular proteolytic enzymes are needed for the proper processing of the target protein and the metabolic well-being of the production host. On the other hand, proteolytic degradation can significantly decrease the yield of secreted proteins. Misfolding in the secretion pathway can lead to degradation by intracellular proteases. This can be a particular problem when it comes to producing heterologous proteins. The details of proteolytic processes, which are responsible for the degradation of proteins that deviate from the fungal secretory process, are not known exactly. In eukaryotes, the degradation of cellular proteins is achieved by a proteasome, and usually involves ubiquitin labeling of the proteins to be degraded. In fungi, proteasomal and vascular proteases are also likely candidates for proteolytic degradation of poorly folded secretory proteins. Proteolytic degradation is probably cytoplasmic, but proteases resident in the endoplasmic reticulum cannot be excluded. From the point of view of improving the strain of the production host, the proteolytic system can be an interesting target for genetic manipulation and improvement of the production strain. Additional copies of protease genes, overexpression of certain proteases, modification of transcriptional control, as well as elimination procedures for suppression of protease genes can provide a more detailed idea of the function of a given protease. The suppression of genes encoding proteases can be a valuable strategy for the improvement of the host strain, in order to improve the production yield for homologous as well as heterologous proteins.

Eukaryotic microbial proteases have been reviewed by North (1982). More recently, Suarez Rendueles and Wolf (1988) have reviewed S. cerevisiae proteases and their function.

Apart from the hydrolytic cleavage of the bonds, proteases can also be applied in bond formation. Links, in this aspect, comprise not only peptide and amide links, but also ester type links. The fact that a protease catalyzes the cleavage or the formation of a particular bond depends primarily on the thermodynamics of the reaction. An enzyme such as a protease does not affect the equilibrium of the reaction. The balance depends on the particular conditions under which the reaction occurs. Under physiological conditions, the thermodynamics of the reactions favors the hydrolysis of the peptide, due to the thermodynamically very stable structure of the zwitterionic product. By applying physical-chemical principles to the influence of equilibrium, or by manipulating the concentrations or the nature of the reactants and products, or by exploiting the kinetic parameters of the enzymatic reaction, it is possible to apply proteases in order to synthesize peptide bonds. The addition of water-miscible organic solvents decreases the degree of ionization of the carboxylic component, thereby increasing the concentration of substrate available for the reaction. Biphasic systems, aqueous mimetics, reverse micelles, anhydrous media, or modified amino and carboxyl groups are often employed to invoke the precipitation of products to improve yields. When proteases with the correct properties are available, the application of proteases for synthesis offers substantial advantages. Since proteases are stereoselective as well as regioselective, sensitive groups in the reactants do not usually need protection, and the reactants do not need to be optically pure. Since the conditions of enzymatic synthesis are mild, racemization and decomposition of reactants or labile products can be avoided. Apart from the linkages between amino acids, also other compounds that have a primary amino group, a thiol group or a carboxyl group can be linked by appropriately selected proteases. In addition, esters, thiol esters, and amides can be synthesized by certain proteases. The protease has been shown to show regioselectivity in the acylation of mono-, di- and trisaccharides, nucleosides, and riboflavin. By proper formulation, problems with stability in the sometimes harsh reaction conditions can be avoided. Encapsulation and immobilization not only stabilize enzymes, but also allow easy recovery and separation from the reaction medium. Extensive crosslinking, treatment with aldehydes, or surface coating with certain polymers, such as dextrans, polyethylene glycol, polyimines, can substantially prolong the lifetime of the biocatalyst.

The natural roles of proteases

Traditionally, proteases have been considered as degrading enzymes, capable of cleaving proteins into small peptides and / or amino acids, and whose role is to digest the nutrient protein or participate in the turnover of cellular proteins. In addition, it has been shown that proteases also play important roles in a

wide variety of cellular processes, via mechanisms of selective modification by limited proteolysis, and thus can have essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980). It is assumed that the specificity of a proteinase is closely related to its physiological function and its mode of expression. With regard to the function of a particular protease, its locations are often very important; for example, a large number of vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988). The different roles of proteases in many cellular processes can be divided into four main protease functions: 1) protein degradation, 2) post-translational processing and (in) activation of specific proteins, 3) morphogenesis, and 4) pathogenesis.

An obvious role for proteases in organisms that use protein as a nutrient source is in the hydrolysis of nutrients. In fungi, this would involve degradation outside the cells by means of proteases of broad extracellular specificity. Protein degradation is also important for the rapid turnover of cellular proteins, and allows cells to eliminate abnormal proteins and adapt their protein complement to changing physiological conditions. Generally, rather broad specificity proteases should be extremely well controlled in order to protect the cell from random degradation of target proteins other than the correct ones.

Contrary to hydrolysis, polypeptide synthesis occurs in vivo by an ATP driven process in the ribosome. After all, the sequence in which the amino acids are linked is dictated by the information derived from the genome. This process is known as transcription. The primary translation products are often longer than the final functional products, and after transcription, additional processing of such precursor proteins by proteases is usually required. Proteases play a key role in the maturation of such precursor proteins to obtain the final functional protein. In contrast to the highly controlled clipping and remodeling of proteins, proteases can also be very destructive, and can completely degrade polypeptides into peptides and amino acids. In order to prevent proteolytic activity from firing earlier than necessary, proteases are subject to intense regulation. Many proteases are synthesized as larger precursors, known as cytogens, which will be activated when needed. Notably, this activation always occurs by proteolysis. Apart from direct involvement in processing, selective activation and inactivation of individual proteins are well known phenomena catalyzed by specific proteases.

The limited proteolysis selectivity seems to reside more directly in the proteinase-substrate interaction. The specificity can be derived from the proteolytic enzyme, which only recognizes specific amino acid target sequences. On the other hand, they may be the result of selective exposure of the "processing site" under certain conditions, such as pH, ionic strength or secondary modifications, thus allowing a non-specific protease to catalyze a very specific event. An example of the latter type is the activation of vascular cymogens by limited proteolysis.

Morphogenesis or differentiation can be defined as a regulated series of events that lead to changes from one state to another in an organism. Although in many cases direct relationships between proteases and morphological effects could not be established, the signs present suggest a significant implication of proteases in fungal morphogenesis; Apart from the wide protein turnover observed during the differentiation, sporulation, and germination of spores, it is thought that proteases are directly involved in normal processes such as branching of the water tip and formation of the septum (Deshpande, 1992).

Aspergillus species, in particular A. fumigatus and A. flavus, have been implicated as etiologic agents of a number of diseases in humans and animals, called aspergillosis (Bodey and Vartivarian, 1989). It has been repeatedly suggested that proteases are involved in the virulence of A. fumigatus and A. flavus, since there are many studies that relate segregated proteases and the virulence of bacteria. In fact, most human infections due to the Aspergillus species are characterized by extensive degradation of the lung parenchyma, which is mainly composed of collagen and elastin (Campbell et al., 1994). Research has focused on the putative role of segregated proteases in the virulence of A. fumigatus and A. flavus, which are the main human pathogens and are known to possess elastinolytic and collagenic activities (Kolattukudy et al., 1993). It was shown that these elastinolytic activities correlate in vitro with infectivity in mice (Kothary et al., 1984). It is known that two secreted proteases are produced by A. fumigatus and A. flavus, an alkaline serine protease (ALP) and a neutral metalloprotease (MEP). In, A. fumigatus, the two genes encoding these proteases, were isolated, characterized and broken (Reicherd et al, 1990; Tang et al, 1992, 1993; Jaton-Ogay et al., 1994). However, double alp mep mutants showed no differences in pathogenicity when compared to wild type strains. Therefore, it must be concluded that the secreted A. fumigatus proteases identified in vitro are not essential factors for tissue invasion (Jaton Ogay et al., 1994). Although A. fumigatus accounts for only a small proportion of airborne mold spores, it is the fungus most frequently isolated from the lung and sputum (Schmitt et al., 1991). Other explanations for the virulence of the fungus could be that the conditions in the bronchi (temperature and nutrients) are favorable for the parasitic growth of A. fumigatus. As a consequence, invasive aspergillosis could be a circumstantial event, when the host's pathogenic defenses have been weakened by immunosuppressive treatments or diseases such as AIDS.

Four important classes of proteases are known, and they are designated by the main functional groups in their active site: the "serine", the "thiol" or "cysteine", the "aspartic" or "carboxyl" and the "metallo" proteases. In Methods in Enzymology part 244 and 248 (A.J. Barrett ed., 1994 and 1995) a detailed review of the state of the art of these main classes of proteases, minor classes and unclassified proteases can be found.

Protease specificity

Apart from the catalytic machinery of proteases, another important aspect of proteolytic enzymes is the specificity of proteases. The specificity of a protease indicates which substrates are likely to hydrolyse the protease. The twenty natural amino acids offer a large number of possibilities to build peptides. For example, with twenty amino acids, about 400 dipeptides and 800 different tripeptides, etc. can be constructed. With longer peptides, the number of possibilities will be almost unlimited. Certain proteases hydrolyse only particular sequences in a very specific position. The interaction of the protease with the peptide substrate can encompass one to ten amino acid residues of the peptide substrate. With large protein substrates, there may be even more substrate residues that interact with proteases. However, this probably implies less specific interactions with protease residues outside the binding slit of the active site. In general, specific recognition is restricted to the linear peptide, which binds to the active site of the protease.

The nomenclature to describe the interaction of a substrate with a protease has been introduced in 1967 by Schechter and Berger (Biochem. Biophys. Res. Com., 1967, 27, 157-162), and is now widely used in the literature. In this system, the amino acid residues of the polypeptide substrate are considered to bind to the so-called active site subsites. By convention, these protease subsites are called S (for subsites), and the corresponding amino acid residues are called P (per peptide). The amino acid residues on the N-terminal side of the cleavable link are numbered P3, P2, P1, and those residues on the C-terminal side are numbered P1 ’, P2’, P3 ’. The P1 or P1 ’residues are the amino acid residues located near the cleavable link. The remains of the substrate around the cleavage site can then be numbered up to P8. The corresponding subsites in the protease that complement the substrate binding residues are numbered S3, S2, S1, S1 ’, S2’, S3 ’, etc. The preferences of the subsites at the peptide binding site determine the preference of the protease to cleave certain specific amino acid sequences at a particular point. The amino acid sequence of the substrate should conform to the preferences shown by the subsites. The specificity for a certain substrate clearly depends both on the binding affinity for the substrate and on the rate at which the cleavable link is subsequently hydrolyzed. Therefore, the specificity of a protease by a certain substrate is usually indicated by its ratio kcat / Km, better known as the specificity constant. In this specificity constant, kcat represents the replacement speed, and Km is the dissociation constant.

Apart from the amino acid residues involved in catalysis and binding, proteases contain many other essential amino acid residues. Some residues are critical in folding, some residues maintain the overall three-dimensional architecture of the protease, some residues may be involved in the regulation of proteolytic activity, and some remainder may direct the protease to a particular location. Many proteases contain, outside the active site, one or more binding sites for metal ions. These metal ions often play a role in the stabilization of the structure. In addition, segregated eukaryotic microbial proteases can be widely glycosylated. It occurs both in N-linked and O-linked glycosylation. Glycosylation can help protein folding, increase solubility, prevent aggregation and, as such, stabilize the mature protein. In addition, the degree of glycosylation can influence secretion as well as water binding by the protein.

Regulation of proteolytic activity

A substantial number of proteases are subject to broad regulation of proteolytic activity in order to avoid unwanted proteolytic damage. To some extent, this regulation takes place at the transcriptional level. For example, in fungi, it seems that the transcription of secreted protease genes is sensitive to external sources of carbon and nitrogen, while genes encoding intracellular proteases are insensitive. Extracellular pH is felt by fungi, and some genes are regulated by pH. In this process, transcriptional regulatory proteins play a crucial role. Often, proteolytic processing of such regulatory proteins is the switch that turns the regulatory proteins on or off.

Proteases are subject to intra-as well as intermolecular regulation. This implies certain amino acids in the proteolytic enzyme molecule that are essential for such regulation. Proteases are typically synthesized as larger precursors, known as cytogens, that are catalytically inactive. Usually, the extension of the peptide chain that inactivates the precursor protease is located in the amino terminus of the protease. The precursor is better known as pro-protein. Since many of the proteases processed in this way are secreted from the cells, they also contain a signal sequence (pre-sequence), so that the entire precursor is synthesized as a pre-pro-protein. Apart from making the protease inactive, the propeptide is often essential to mediate productive folding. Examples of proteases include serine proteases (lytic protease alpha, subtilisin, acualisin, prohormone convertase), thiol proteases (cathepsin L and crucian), aspartic proteases (proteinase A and cathepsin D), and metalloproteases. In addition, the pro-peptide can

play a role in cell transport, either alone or in conjunction with signal peptides. This may facilitate interaction with cell chaperones, or may facilitate transport along the membrane. The size of the extension in the pre-pro-precursor protein can vary substantially, ranging from a short peptide fragment to a polypeptide, which can exist as an autonomous folding unit. In particular, it is often observed that these larger extensions are potent protease inhibitors, even after cleavage from the protease. It was noted that, even after cleavage, such pro-peptides could help in the proper folding of proteases. As such, it could be considered that pro-peptides function as molecular chaperones, and separate or additional co-expression of such pro-peptides could be advantageous for the production of proteases.

There is a substantial difference in the level of regulation between proteases that are secreted in the medium and proteases that remain intracellularly. Segregated proteases in the medium are usually no longer subject, after activation, to control, and therefore are usually relatively simple in their molecular architecture, which consists of a globular module. Intracellular proteases are necessarily subject to continuous control, in order to avoid damage to the cells. In contrast to segregated protease cymogens, in very complex regulatory proteases very large polypeptide segments can be inserted between the signal and the cytogen activation domain of the proteolytic module. Structure-function studies indicate that such non-proteasic parts may be involved in interactions with macroscopic structures, membranes, cofactors, substrates, effectors, inhibitors, ions, which regulate the activity and activation of the proteolytic module or modules or their (its) cytogen ( s). The non-proteolytic modules have a notable variation in size and structure. Many of the modules may exist as such, regardless of the proteolytic module. Therefore, it can be considered that such modules correspond to independent structural and functional units that are autonomous with respect to folding. The value of such a modular organization is that the acquisition of new modules can provide the recipient protease with recent novel binding specificities, and can lead to drastic changes in its activity, regulation and target selection. The principle of modular organized proteolytic enzymes can also be exploited by applying molecular biology tools to create new interactions, regulation, specificity, and / or target selection by shuffling modules. Although in general such additional modules are observed as terminal N or C extension, large insertions have also been observed within the outer loops of the catalytic domain. It is believed that, also in this case, the main folding of the protease still represents the essential topology for forming a functional proteolytic entity, and that the insertion can be considered as a folded substructure on the surface of the proteolytic module.

Molecular structure

In principle, the modular organization of larger proteins is a general theme in nature. In particular within larger multimodular networks, typical proteolytic modules show sizes of 100 to 400 amino acids on average. This corresponds to the average size of most globular proteolytic enzymes that are secreted into the medium. As explained above, the polypeptide modules are polypeptide fragments, which can be folded and operated as independent entities. Another term for such modules is domains. However, the domain is used in a wider context than the module. The term domain, as used herein, usually refers to a part of the polypeptide chain that represents in the three-dimensional structure a typical folding topology. In a protein, domains interact in varying degrees, but less frequently than structural elements do within domains. Other terms such as subdomain and folding unit are also used in the literature. As such, it is noted that many proteins that share a particular functionality can share the same domains. Such domains can be recognized from the primary structure, which can show certain sequence patterns, which are typical for a particular domain. Typical examples are mononucleotide binding folding, cellulose binding domains, helix-turn-helix DNA binding motif, zinc fingers, EF hands, membrane anchors. The modules refer to those domains that are expected to be able to fold and operate autonomously. The person skilled in the art knows how to identify particular domains in a primary structure by applying commonly available computer software to said structure and homologous sequences from other organisms or species.

Although multimodular or multidomain proteins may seem like a string of pearls, assemblies of more complex substantial architecture have been observed. In the case where the different beads reside in the same polypeptide chain, the beads are generally referred to as modules or domains. When the pearls do not reside in the same polypeptide chain, but form assemblies via non-covalent interactions, then the term subunit is used to designate the pearl. Subunits can be transcribed by the same gene, or by different genes. The multimodular protein can be proteolytically processed after transcription, leading to multiple subunits. Individual subunits can consist of multiple domains. Typically, globular proteins smaller than 100-300 amino acids usually consist of only one domain.

Molecular classification of proteolytic enzymes

In general, proteases are classified according to their molecular properties, or according to their functional properties. The molecular classification is based on the primary structure of the protease. The primary structure of a protein

represents its amino acid sequence, which can be derived from the nucleotide sequence of the corresponding gene. Extensive tracking of similarities in primary structures can allow the observation of similarities in the catalytic mechanism and other properties, which can be extended even to functional properties. The term family is used to describe a group of proteases that show an evolutionary relationship based on the similarity between their primary structures. It is believed that members of such a family have emerged through divergent evolution from the same ancestor. Within a family, additional subgrouping of primary structures based on more detailed refinement of sequence comparisons results in subfamilies. The classification according to the three-dimensional folding of the proteases can comprise the secondary structure, the tertiary structure and the quaternary structure. In general, the classification in the secondary structure is limited to the content and the coarse orientation of elements of the secondary structure. Similarities in tertiary structure have led to the recognition of superfamilies or clans. A superfamily or a clan is a group of families that are thought to have a common ancestor, since they show a common three-dimensional folding. In general, the tertiary structure is more conserved than the primary structure. As a consequence, the similarity of the primary structure does not always reflect similar functional properties. In fact, the functional properties may have diverged substantially, resulting in new interesting properties. At present, the quaternary structure has not been applied to classify various proteases. This may be due to some bias of the structural databases with respect to simple globular proteases. It is likely that many proteolytic systems that are subject to activation, regulation, or cascades of complex reactions, consist of multiple domains or subunits. General issues in the structural organization of such protease systems can lead to new types of classification.

Classification according to specificity

In the absence of sequence information, proteases have undergone various types of functional classification. The classification and nomenclature of enzymes by reference to reactions that are catalyzed is a general principle in enzymatic nomenclature. This approach is also the underlying principle of EC numbering of enzymes (Enzyme Nomenclature 1992 Academic Press, Orlando). In the Enzyme Nomenclature 1992 two types of proteases can be recognized (EC 3.4): those of exopeptidases (EC 3.4.11-19), and those of endopeptidases (EC 3.4.21-24, 3.4.99). Endopeptidases cleave peptide bonds in the internal regions of the peptide chain, far from the terms. Exopeptidases cleave only remnants of the ends of the peptide chain. Exopeptidases that act in the free N term can release a single amino acid residue, a dipeptide or a tripeptide, and are called respectively aminopeptidases (EC 3.4.11), dipeptidyl peptidases (EC 3.4.14) and tripeptidyl peptidase (EC 3.3. 14). Proteases that begin peptide processing from the term carboxyl, which release a single amino acid, are called carboxypeptidases (EC 3.4.16-18). Peptidyl dipeptidases (EC 3.4.15) remove a dipeptide from the term carboxyl. The exo- and endopeptidase together are dipeptidases (EC 3.4.13), which specifically cleave only dipeptides into their two amino acid halves. Omega peptidases (EC 3.4.19) eliminate terminal residues that are substituted, cyclic, or linked by isopeptide bonds.

Apart from the position in which the protease cleaves a peptide chain, for each type of protease an additional division based on the nature of the preferred amino acid residues in the substrate is possible. In general, proteases with a broad, medium and narrow specificity can be distinguished. Some proteases are simply named after specific hydrolyzing proteins or polypeptides, for example keratinase, collagenase, elastase. A narrow specificity can be fixed to a particular amino acid or to a particular sequence, which is removed or cleaved respectively. When the protease shows a particular preference for an amino acid at the P1 or P1 ’position, the name of this amino acid can be a qualifier. For example, prolyl amino peptidase removes proline from the amino terminus of a peptide (proline is the P1 moiety). X-Pro or proline is used when the bond is cleaved on the imino side of the proline (the proline is the remainder P1 ’); for example, proline carboxypeptidase removes proline from the term carboxyl. The prolyl endopeptidase (or Pro-X) cleaves behind the proline, while the proline endopeptidase (X-Pro) cleaves before a proline. The amino acid residue in front of the cleavable peptide bond refers to the amino acid residue that the carboxyl group brings to the peptide bond. The amino acid residue behind the cleavable peptide bond refers to the amino acid residue that the amino group brings to the peptide bond. According to the general convention, an amino acid chain ranges from the term amino (the beginning) to the term carboxyl (the end), and is numbered accordingly. Endoproteases can also show a clear preference for a particular amino acid in the P1 or P1 ’position, for example glycyl endopeptidase, peptidillisin endopeptidase, glutamyl endopeptidase. In addition, proteases may show a preference for a certain group of amino acids that share a certain resemblance. Such a preferred group of amino acids may comprise hydrophobic amino acids, only bulky hydrophobic amino acids, small hydrophobic amino acids, or only small amino acids, positively charged large amino acids, etc. Apart from the preferences for the P1 and P1 ’residues, there may also be particular preferences or exclusions for preferred residues by other protease subsites. Such multiple preferences may result in proteases that are very specific for only those sequences that satisfy multiple binding requirements at the same time. In general, it should be noted that proteases are rather promiscuous enzymes. Even a very specific protease can cleave peptides that do not meet the generally observed preference of the protease. In addition, it should be noted that environmental conditions such as pH, temperature, ionic strength, aqueous activity, solvent preference, presence of competing substrates or inhibitors, can influence protease preferences.

The environmental condition can not only influence the protease, but also influence the way in which the protease substrate is presented to the protease.

Classification by catalytic mechanism

Proteases can be subdivided based on their catalytic mechanism. It should be understood that for each

5 Catalytic mechanism, the previous classification based on specificity leads to an additional subdivision for each type of mechanism. Four important classes of proteases are known, and they are designated by the main functional group in the active site: serine proteases (endopeptidase EC 3.4.21, carboxypeptidase EC 3.4.16), thiol or cysteine proteases (endopeptidase EC 3.4.22, carboxypeptidase EC 3.4.18), carboxyl or aspartic proteases (endopeptidase EC 3.4.23), and metalloproteases (endopeptidase EC 3.4.24, carboxypeptidase EC 3.4.18). There are characteristic inhibitors of the members of each catalytic type of protease. These small inhibitors irreversibly modify an amino acid residue of the active protease site. For example, serine proteases are inactivated by phenylmethanesulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), which react with active serine, while chloromethyl ketone derivatives react with histidine in the catalytic triad. Phosphoramidon and 1,10-phenanthroline typically inhibit metalloproteases. Inhibition by

Pepstatin generally indicates an aspartic protease. E64 specifically inhibits thiol protease. Amastatin and bestatin inhibit various aminopeptidases. Substantial variations in the susceptibility of proteases to inhibitors are observed, even within a catalytic class. To some extent, this may be related to the specificity of the protease. In the event that the binding site architecture prevents an inhibitor based on the mechanism from approaching the catalytic site, then such a protease escapes the inhibition, and identification of the type of mechanism based on the inhibition is prohibited. Chymostation, for example, is a potent inhibitor for serine protease, with specificity similar to chymotrypsin; Elastatinal inhibits serine proteases similar to elastase, and does not react with trypsin or chymotrypsin; 4-amido PMSF (APMSF) inhibits only serine proteases with specificity similar to trypsin. Extensive reports of the use of inhibitors in the classification of proteases include Barret and Salvesen, Proteinase Inhibitors, Elsevier Amstardam, 1986; Bond and Beynon (eds.), Proteolytic Enzymes, A Practical

25 Approach, IRL Press, Oxford, 1989; Methods in Enzymology, eds. E.J. Barret, volume 244, 1994 and volume 248, 1995; E. Shaw, Cysteinyl proteinases and their selective inactivation, Adv Enzymol. 63: 271-347 (1990).

Classification according to optimal behavioral conditions

The catalytic mechanism of proteases, and the requirement of their conformational integrity, mainly determine the conditions under which the protease can be used. Finding the protease that behaves optimally under application conditions is an important challenge. Often, the conditions under which proteases are to function are not optimal, and represent a compromise between the ideal conditions for a particular application and the conditions that would best suit the protease. Apart from the particular properties of the protease, it should be noted that also the presentation of protein substrates depends on the conditions, and as such also determines which conditions are the most effective for proteolysis. The specifications for the enzyme

35 which are relevant for the application include, for example, the pH dependence, temperature dependence, sensitivity to or dependence on metal ions, ionic strength, salt concentration, solvent compatibility. Another vitally important factor is the specific activity of a protease. The higher the specific activity of the enzyme, the less enzyme is needed for a specific conversion. Lower enzyme requirements imply lower costs and lower levels of protein contamination.

The pH is an important parameter that determines the behavior of the protease in an application. Therefore, pH dependence is an important parameter for grouping proteases. The main groups that are recognized are acid proteases, neutral proteases, alkaline proteases, and very alkaline proteases. The optimum pH equals only to a certain extent the proteolytic mechanism; for example, aspartic protease often shows an optimum at acidic pH; metalloproteases and thiol proteases often behave optimally around neutral to slightly alkaline pH; Serine peptidases are mainly active in the alkaline and very alkaline region. For each class, exceptions are known. In addition, the overall water activity of the system also plays a role. The optimal pH of a protease is defined as the pH range in which the protease shows an optimal rate of hydrolysis for most of its substrates in a particular environment under particular conditions. This range can be narrow, for example a unit of pH, as well as quite ample, 3-4 units of pH. In general, the optimum pH also depends on the nature of the protein substrate. Both the replacement rate and the specificity may vary depending on the pH. For some efficacy, it may be desirable to use the protease away from its optimum pH, because the production of less desired peptides is avoided. The less desired peptides may be, for example, very short peptides, or peptides that cause a bitter taste. In addition, a narrower specificity may be a reason for choosing conditions that deviate from

55 the optimal conditions with respect to the replacement speed. Depending on the pH, the specificity may be narrow, for example by cleaving only the peptide chain at a particular position, or before or after a particular amino acid, or it may be broader, for example by cleaving a chain at multiple positions, or by cleaving before or after more different types of amino acids. In fact, pH dependence can be an important tool to regulate proteolytic activity in an application. In the event that the pH moves during the process, the proteolysis can cease spontaneously without the need for additional treatment to inactivate the protease. In some cases, the proteolysis itself may be the driver of the pH shift.

Very crucial for the application of proteases is its stability during handling and operation. Since protease stability is strongly affected by working temperature, stability is also often referred to as thermostability. In general, the stability of a protease indicates how long a protease retains its proteolytic activity under particular conditions. Particular conditions may include fermentation conditions, conditions during isolation and processing downstream of the enzyme, storage conditions, formulation and operating or application conditions. In the case where the particular conditions encompass high temperatures, stability generally refers to thermostability. Apart from the general causes for enzymatic inactivation, such as chemical modification, lack of folding, aggregation, etc., a major problem with proteases is that they easily undergo self-degradation. Especially for the use of proteases, the optimum temperature is a relevant criterion for grouping proteases. Although there are different definitions, economically the most useful definition is the temperature or temperature range in which the protease is most productive in a certain application. Protease productivity is a function of both stability and turnover rate. When the elevated temperature in general will increase the replacement speed, the rapid inactivation will contract the increase in the replacement speed, and will eventually lead to low productivity. The conformational stability of the protease under a given process condition will determine its maximum operating temperature. The temperature at which the protease loses its active conformation, often indicated as loss of folding or melting point, can be determined according to various methods, for example NMR, circular dichroism spectroscopy, differential scanning calorimetry, etc. For the protease, the loss of folding is usually accompanied by a tremendous increase in the rate of self-degradation.

In applications where low temperatures are required, the protease can be selected with emphasis on high intrinsic activity at low to moderate temperature. Since under such conditions inactivation is relatively slow, under these conditions activity can greatly determine productivity. In processes where a protease activity is required only for a short period, the stability of the protease can be used as a plug to turn off the protease. In such a case, a more labile protease may be preferable instead of a very thermostable protease.

Other environmental parameters that may play a role in selecting the appropriate protease may be its salt sensitivity. Compatibility with metal ions that are frequently found in low concentrations in various natural materials can be crucial for certain applications. In particular with metalloproteases, certain ions can replace the catalytic metal ion and reduce or even completely cancel the activity. In some applications, metal ions have to be added on purpose in order to avoid the removal of coordinated metal ions to the protease by washing. It is well known that for the sake of enzymatic stability and lifetime, calcium ions have to be supplied in order to avoid the dissociation of calcium bound to protein.

Most microorganisms show a certain tolerance with respect to adaptation to changes in the environmental condition. As a consequence, it is likely that at least the proteolytic spectrum that the organism is capable of producing shows at least similar tolerances. Such a proteolytic spectrum can be covered by many proteases that cover the entire spectrum together, or by only a few broad-spectrum proteases. Given the full proteolytic spectrum of a microorganism, it can be very important to consider the location.

Cellular location and characterization of proteolytic degradation and processing

From an industrial point of view, proteases that are excreted from the cell have specific advantages over large-scale productivity and stress tolerance, since they have to survive without the protection of the cell. The large group of cellular protease can be further subdivided into soluble and membrane bound. Attached to the membrane can comprise a protease inside as well as outside the membrane. The soluble intracellular protease can be further subdivided according to specific compartments of the cell in which they appear. Since the cell protects proteases to a certain degree from the environment, and because the cell controls the conditions in the cell, the intracellular protease may be more sensitive to large environmental changes, and its optimum can be better correlated with intracellular conditions. specific. Knowing the conditions of the cell department in which the protease resides can indicate your preferences. Where extracellular protease in general no longer requires any other regulation once excreted from the cell, intracellular proteases often undergo more complicated control and regulation.

With regard to the function of a particular protease, its location is often very important; for example, a large number of vacuolar and periplasmic proteases are involved in protein degradation, while many of the membrane-bound proteases are important in protein processing (Suarez Rendueles and Wolf, 1988).

In van den Hombergh: Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, a comprehensive review of the biological properties and evolution of proteases has been published.

The protease problem

An important reason for the interest in microbial proteases is the problem of protease-related expression observed in several expression hosts used in the bioprocessing industry. The increasing use of heterologous hosts for protein production, using recombinant DNA technology, has recently put this problem in the spotlight, since it seems that heterologous proteins are more prone to proteolysis (Archer et al., 1992; van den Hombergh et al., 1996b).

In S. cerevisiae, at the beginning of the eighties the problem of proteases and the implication of several proteases were recognized, thus complicating the breakdown approaches of genes selected as targets to solve this problem. During secretion, a protein is exposed to several proteolytic activities that reside in the secretory pathway. Additionally, in a prototrophic microorganism such as Aspergillus, secreted proteins can be exposed to various extracellular proteolytic activities.

The problem of heterologously expressed protein degradation is well documented in Aspergillus (van den Hombergh Thesis Landbouwuniversiteit Wageningen: An analysis of the proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2), and it has been given to be known in the expression of cow prochymyosin, human D-2 interferon, tPA, GMCSF, IL6, lactoferrin, lysozyme from egg white, porcine PLA2, pectin lyase B from A. niger, enterotoxin B from E. coli and E-glucuronidase, and pectate lyase 3 from Erwinia carotovora.

The problem of proteolysis can be solved at various stages in protein production. Bioprocess engineers can solve the problem of proteolysis by processing downstream at low temperatures, by early separation of product and protease or proteases, or by use of protease inhibitors. All this can lead to the successful reduction of the problem. However, it is certainly not eliminated, because much of the degradation occurs in vivo during protein production.

When it comes to understanding how proteolysis is controlled in the cell, an important issue refers to the recognition mechanism by which proteolysis is started. To what extent are proteolytically susceptible (heterologous) proteins recognized as aberrant due to misfolding, or, if correctly folded, as "strange", because they do not possess essential stability traits that are specific to the host. Various types of stress can cause the overall proteolysis in a cell to increase significantly. Factors known to increase the speed of proteolysis include lack of nutrients and other various types of stress (i.e. temperature rise, osmotic stress, toxic substances, and expression of certain heterologous proteins). To handle expression problems related to proteolysis in vivo, several approaches have proven successful, as will be explained below. However, it must be borne in mind that true “non-proteolytic cells” cannot exist, since proteolysis by intracellular proteases is involved in many essential metabolic reactions and “domestic maintenance and conservation”. Therefore, the reduction of proteolysis will be a process in which the changed genetic background that results in a reduced proteolysis has to be analyzed, looking for potential side effects that could lead to reduced protein production (for example, reduced growth rate or reduced sporulation).

Rupture of proteases in filamentous fungal expression hosts

Berka et al. (1990) describe the cloning and rupture of the pepA gene from A. awamori. More recently, three broken aspartyl proteases have been described in A. niger. Destroyers were described for both the main extracellular aspartyl proteases and the main vacuolar aspartyl protease. Double and triple destroyers were generated via recombination, and tested for protease spectra and the expression and secretion of the PELB pectin lyase protein from A. niger, which is very susceptible to proteolytic degradation (van den Hombergh et al. , nineteen ninety five). The rupture of pepA and pepB resulted in the reduction in both cases of extracellular protease activities, 80% and 6%, respectively. In the 'pepE destroyer, other protease activities (vacuolar) were also significantly affected, caused by the inactivation of the proteolytic cascade for other vacuolar proteases. Reduced extracellular activities were correlated with reduced in vitro degradation of PELB and improved in vivo expression of pelB (van den Hombergh et al., 1996f).

Filamentous fungi with protease deficient mutants (prt)

Several mutants deficient in Aspergillus proteases have been studied to know if protein production is improved. Archer et al. Describe the reduced proteolysis of chicken egg white lysozyme in supernatants of a double prt mutant of A. niger generated by Mattern et al. (1992), and conclude that, although degradation is not absent, it is significantly reduced. Van den Hombergh et al., (1995) show that the in vitro degradation of PELB from A. niger is reduced in the seven prt complementation groups that they have isolated. Virtually no degradation is observed in the prtB, prtF and prtG mutants. Recently, it was shown that pelB gene expression was improved in six complementation groups tested (prtA-F), and the highest levels of expression were observed in the prtB, prtF and prtG mutants. In addition to the individual mutants, which contained residual extracellular proteolytic activities ranging from 2-80% compared to wild-type activity, double mutants were generated both by recombination and by rounds.

additional mutagenesis. Via this approach, several double prt mutants were selected and subsequently characterized, which showed an additional reduction in PELB degradation compared to their parental strains.

Instead of eliminating protease activities via disruption or mutagenesis, reduced proteolysis can also be achieved via decreased interfering proteolytic activities. This can be achieved by genetically altering the promoter or other regulatory sequences of the gene. As shown by Fraissinet-Tachet et al. (1996), the extracellular proteases in A. niger are all regulated by the repression of the carbon catabolite and the repression of the nitrogen metabolite. The lack of nutrients also causes the overall rate of proteolysis in a cell to increase dramatically, which makes sense for a cell that lacks nutrients but has proteins, which, under famine conditions, are not necessary or only necessary in amounts smaller. In expression strategies that allow high expression in media containing high concentrations of glucose and ammonia, reduced proteolysis has been disclosed. Several constitutive glycolytic promoters (gpd and pkiA) are highly expressed under these conditions, and can also be used to drive gene expression (heterologous) in continuous fermentations. The type of lack of nutrients imposed can influence varying degrees in different proteases, which means that the importance of nutrient conditions in a given process depends on the type of proteolysis that is involved. Therefore, specific proteolysis can be induced by substrate limiting conditions, which are frequently used in many large-scale fermentation processes.

The protease problem can now be partially resolved by one or more of the above strategies. However, the residual proteolytic activity of proteolytic enzymes not yet identified is still a major problem in the art. In order to further reduce the level of unwanted proteolysis, there is a great need in the art to identify new proteases responsible for the degradation of homologous and heterologously expressed proteins. This invention provides such new protease gene sequences that encode new proteases. Once the primary sequence of a new protease is known, one or more of the above recombinant DNA strategies can be employed to produce mutants (lacking a gene) with reduced proteolytic activity.

Despite the widespread applications of proteases in a large number of industrial processes, current enzymes also have significant weaknesses with respect to at least one of the following properties.

When added to animal feed, current proteases are not sufficiently resistant to the digestive enzymes present in the digestive tract (GI) of, for example, pigs and poultry.

With respect to another aspect, currently available enzymes are not sufficiently resistant to specific conditions of (high) temperatures and (high) pressures that are applied during extrusion operations

or pellet formation.

Also, current enzymes are not sufficiently active in a pH range of 3-7, conditions that prevail in many food products, beverages, as well as in the digestive tract of most animals.

According to yet another aspect, the specificity of currently available proteases is very limited, which results in the inability of existing enzymes to degrade or dissolve certain "protease resistant" proteins, thus resulting in low peptide or amino acid yields. In addition, proteases with new specificities allow the synthesis of new peptides.

Still another drawback of the enzymes currently available is their low specific activity.

Therefore, it is clear that, for a large number of applications, there is a strong desire for proteases that are more resistant to digestive enzymes, at elevated temperature and / or pressure, and that show new specificities with respect to their hydrolysis sites. The present invention provides such enzymes.

Object of the invention

It is an object of the invention to provide new polynucleotides encoding new proteases. A further object is to provide naturally and recombinantly produced proteases, as well as recombinant strains that produce them. Such strains can also be used to produce classic fermentation products faster or with higher yields. Still another object of the invention is to provide a filamentous fungus strain that is defective in producing a protease according to the invention. Such strains can be used for more efficient production of heterologous or homologous proteins. Also the antibodies and fusion polypeptides are part of the invention, as well as methods for obtaining and using the polynucleotides and polypeptides according to the invention.

Summary of the invention

The invention provides new polynucleotides encoding new proteases.

More particularly, the invention provides polynucleotides having a nucleotide sequence that hybridizes (preferably under very restrictive conditions) to a sequence according to a sequence selected from the group consisting of SEQ ID NO: 10 or 67. Accordingly, the invention provides acids Nuclei having about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the sequences according to a sequence selected from the group consisting of SEQ ID NO: 10 or 67.

In a more preferred embodiment, the invention provides such an isolated polynucleotide obtainable from a filamentous fungus, preferably Aspergilli, in particular A. niger is preferred.

In one embodiment, the invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide with an amino acid sequence of SEQ ID NO: 124 or its functional equivalents.

In a further preferred embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide according to a sequence of SEQ ID NO: 124 or its functional equivalents.

In a preferred embodiment, the invention provides a protease gene according to a sequence of SEQ ID NO: 10. In another aspect, the invention provides a polynucleotide, preferably a cDNA encoding a protease of

A. niger of SEQ ID NO: 67, or variants or fragments of that polypeptide. In a preferred embodiment, the cDNA has a sequence selected from the group consisting of SEQ ID NO: 67 or its functional equivalents.

A genomic clone encoding a polypeptide according to the invention can also be obtained by selecting suitable probes to specifically amplify a genomic region corresponding to the sequences according to SEQ ID NO: 10 or its fragments, hybridizing that probe under conditions suitable to genomic DNA obtained from a suitable organism, such as Aspergillus, for example A. niger, amplifying the desired fragment, for example by PCR (polymerase chain reaction), followed by purification and cloning of the amplified fragment.

In even a further preferred embodiment, the invention provides a polynucleotide comprising the coding sequence of the genomic polynucleotides according to the invention; a polynucleotide sequence of SEQ ID NO: 10 is preferred.

In another preferred embodiment, the invention provides a cDNA obtainable by cloning and expressing a sequence of SEQ ID NO: 67 in a suitable host organism, such as A. niger.

A polypeptide according to the invention can also be obtained by cloning and expressing a sequence of SEQ ID NO: 10 in a suitable host organism, such as A. niger.

The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that can be used to amplify or detect the DNA according to the invention.

In a further preferred embodiment, a vector is provided in which the polynucleotide sequence according to the invention is functionally linked with regulatory sequences suitable for the expression of the amino acid sequence encoded in a suitable host cell, such as A. niger or A. oryzea . The invention also provides methods for preparing polynucleotides and vectors according to the invention.

The invention also relates to recombinantly produced host cells containing heterologous or homologous polynucleotides according to the invention.

In one embodiment, the invention provides recombinant host cells in which the expression of a protease according to the invention is significantly reduced, or in which the protease activity is reduced,

or in which the protease is even inactivated. Such recombinants are especially useful for the expression of homologous or heterologous proteins.

In another embodiment, the invention provides recombinant host cells in which the expression of a protease according to the invention is significantly increased, or in which the protease activity is increased. Such recombinants are especially useful for the expression of homologous or heterologous proteins in which maturation is seriously impeded in the case where the required proteolytic cleavage becomes the speed limiting stage.

In another embodiment, the invention provides a recombinantly produced host cell containing heterologous or homologous DNA according to the invention, preferably DNA encoding proteins that possess signal sequences, and in which the cell is capable of producing a functional protease according to the invention, preferably a cell capable of overexpressing the protease according to the invention, for example an Aspergillus strain comprising an increasing number of copies of a gene or cDNA according to the invention.

In another embodiment, the invention provides a recombinantly produced host cell containing heterologous or homologous DNA according to the invention, and wherein the cell is capable of secreting a functional protease according to the invention, preferably a cell capable of overexpressing and secreting the protease according the invention, for example an Aspergillus strain comprising an increasing number of copies of a gene or cDNA according to the invention.

In yet another aspect of the invention, a purified polypeptide is provided. Polypeptides according to the invention include polypeptides encoded by polynucleotides according to the invention. A polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171 or its functional equivalents is especially preferred.

The invention also provides antibodies that react with a polypeptide according to the invention. These antibodies may be polyclonal, although it is especially preferred that they are monoclonal antibodies. Such antibodies are particularly useful for purifying the polypeptides according to the invention.

Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention. The invention also provides methods for obtaining the polypeptides according to the invention.

The invention further relates to a method for diagnosing aspergillosis by detecting the presence of a polypeptide according to the invention or its functional equivalents, or by detecting the presence of a DNA according to the invention or its fragments or functional equivalents.

The invention also relates to the use of the protease according to the invention in an industrial process as described herein.

Detailed description of the invention

Polynucleotides

The present invention provides polynucleotides encoding proteases that have an amino acid sequence of SEQ ID NO: 124 or its functional equivalents. The sequence of these genes was determined by sequencing a genomic clone obtained from Aspergillus niger. The invention provides polynucleotide sequences comprising the gene that encodes these proteases, as well as their complete cDNA sequence and their coding sequence. Accordingly, the invention relates to an isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 10, or a sequence of SEQ ID NO: 67, or its functional equivalents.

More particularly, the invention relates to an isolated polynucleotide hybridizable under restrictive conditions to a polynucleotide of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, preferably under very restrictive conditions. Advantageously, such polynucleotides can be obtained from filamentous fungi, in particular from Aspergillus niger. More specifically, the invention relates to an isolated polynucleotide having a nucleotide sequence according to a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67.

The invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide according to SEQ ID NO: 124 or its functional equivalents.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that can be isolated from chromosomal DNA, which include an open reading frame encoding a protein, for example a protease of A niger A gene can include coding sequences, non-coding sequences, introns and regulatory sequences. In addition, a gene refers to an isolated nucleic acid molecule as defined herein.

A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, or a functional equivalent thereof, is You can isolate using standard molecular biology techniques and the sequence information provided here. For example, using all or part of the nucleic acid sequence of a sequence of SEQ ID NO: 10, or the nucleotide sequence of a sequence of SEQ ID NO: 67, as a hybridization probe, nucleic acid molecules can be isolated according to the invention using standard hybridization and cloning techniques (for example, as described in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

In addition, a nucleic acid molecule that encompasses all or a portion of a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67 can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67.

A nucleic acid 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. He

Nucleic acid thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.

In addition, oligonucleotides that correspond to or are hybridizable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, for example using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in a sequence of SEQ ID NO: 67. The sequence of a sequence of SEQ ID NO: 67 corresponds to the coding region of the protease cDNA. of A. niger. This cDNA comprises sequences encoding the A. niger protease polypeptide according to a sequence of SEQ ID NO: 124.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement to the nucleotide sequence shown in a sequence of SEQ ID NO: 10

or a sequence of SEQ ID NO: 57, or a functional equivalent of these nucleotide sequences.

A nucleic acid molecule that is complementary to another nucleic acid sequence is one that is sufficiently complementary to the other nucleotide sequence, so that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.

One aspect of the invention relates to isolated nucleic acid molecules encoding a polypeptide of the invention or a functional equivalent thereof, such as a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as a hybridization probe. to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for amplification or mutation of nucleic acid molecules.

An "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately adjacent to both coding sequences with which it is immediately contiguous (one at the 5 'end and one at the 3' end) in the genome of natural origin of the organism from which it derives. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5 ’non-coding sequences (e.g., promoters) that are immediately adjacent to the coding sequence. Therefore, the term includes, for example, a DNA

A recombinant that is incorporated into a vector, in a plasmid or virus that replicates autonomously, or in the genomic DNA of a prokaryotic or eukaryotic, or that exists as a separate molecule (for example, a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene that encodes an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or viral precursors or other chemicals. (when chemically synthesized). In addition, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment, and would not be found in the natural state.

As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), and DNA analogs or

35 RNAs generated using nucleotide analogs. The nucleic acid molecule may be single stranded or double stranded, but preferably it is double stranded DNA. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (for example, inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base matching capabilities, or that have increased resistance to nucleases.

Another embodiment of the invention provides an isolated nucleic acid molecule that is antisense to a protease nucleic acid molecule, for example the coding strand of a protease nucleic acid molecule. Also complementary to the nucleic acid molecules described herein are included within the scope of the invention.

Sequencing errors

45 Sequence information, as provided here, should not be interpreted so closely as to require the inclusion of erroneously identified bases. The specific sequences described herein can be easily used to isolate the entire gene from filamentous fungi, in particular A. niger, which in turn can easily be subjected to additional sequence analysis, thereby identifying sequencing errors.

Except as otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule here were determined using an automated DNA sequencer, and all amino acid sequences of the polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as before. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be determined more precisely by other approaches, including methods of

Manual DNA sequencing, well known in the art. As is also known in the art, an individual insertion or deletion in a particular nucleotide sequence compared to the actual sequence will cause a shift of the frame in the translation of the nucleotide sequence, so that the predicted amino acid sequence encoded by a particular nucleotide sequence it will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, starting at the point of such insertion or deletion.

The person skilled in the art is able to identify such bases, erroneously identified, and knows how to correct such errors.

Fragments of nucleic acids, probes and primers

A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114, for example a fragment that can be used as a probe or primer, or a fragment encoding a portion of a protease-like protein. The nucleotide sequence determined from the cloning of the protease and cDNA gene allows the generation of probes and primers designed for use in identifying and / or cloning other members of the protease family, as well as protease homologs from other species. The probe / primer typically comprises substantially purified oligonucleotide, which typically comprises a region of nucleotide sequence that preferentially hybridizes under very restrictive conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 to SEQ ID NO: 114, or a functional equivalent thereof.

Probes based on the nucleotide sequences of the proteases can be used to detect transcripts or genomic protease sequences encoding the same homologous proteins or proteins, for example in other organisms. In preferred embodiments, the probe further comprises a labile group attached thereto, for example the labile group may be a radioisotope, a fluorescent compound, an enzyme, or an enzymatic cofactor. Such probes can be used as part of a diagnostic assay kit to identify cells that express a protease-like protein.

Identity and homology

The terms "homology" or "percent identity" are used interchangeably here. For the purposes of this invention, it is defined herein that, in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., jumps may 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 or nucleotide residues are then compared at the corresponding amino acid positions or nucleotide positions. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the position corresponding to the second sequence, then the molecules are identical in that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (ie,% identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100).

Preferably, the two sequences have the same length.

The skilled person will be aware of the fact that there are several different computer programs to determine the homology between two sequences. For example, a sequence comparison and the determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix, and a jump weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will produce slightly different results, but that the overall percentage identity of two sequences is not significantly altered when They use different algorithms.

In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using an NWSgapdna.CMP matrix and a weight of jump of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity of two amino acid or nucleotide sequences is determined using the algorithm by E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0) (available at http: // vega (igh.cnrs.fr/bin/align -guess.cgi), using a weight remnants table of PAM120, a jump length penalty of 12, and a jump penalty of 4.

The nucleic and protein acid sequences of the present invention can also be used as an "interrogation sequence" to conduct a search against public databases to, for example, identify other family members or related sequences. Such searches can be carried out using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be carried out with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to protease nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protease protein molecules of the invention. To obtain alignment with jumps for comparison purposes, Gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When the BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Hybridization

As used herein, the term "hybridize" is intended to describe conditions for hybridization and washing, in which the nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least around 80%, even more preferably at least about 85% to 90%, more preferably at least 95% homologous to each other, typically remain hybridized to each other.

A preferred non-limiting example of such hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 1 X SSC, 0.1% SDS at 50 ° C, preferably at 55 ° C, preferably at 60 ° C, and even more preferably at 65 ° C.

Very restrictive conditions include, for example, hybridizing at 68 ° C in 5x SSC / 5x Denhardt solution / 1.0% SDS, and washing in 0.2x SSC / 0.1% SDS at room temperature. Alternatively, washing can be carried out at 42 ° C.

The expert will know what conditions to apply for the restrictive and very restrictive hybridization conditions. In the art there is easily additional guidance in relation to such conditions, for example in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y .; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Of course, a polynucleotide that hybridizes only to a poly A sequence (such as the 3 'terminal poly (A) stretch of mRNAs), or to a complementary stretch of T (or U) moieties, will not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule that contains a stretch of poly (A) or its complement (eg, virtually any clone of Double stranded cDNA).

Obtaining full-length DNA from other organisms

In a typical approach, cDNA libraries constructed from other organisms will be identified, for example filamentous fungi, in particular of the Aspergillus species.

For example, Aspergillus strains can be identified for homologous protease polynucleotides by Northern blot analysis. With the detection of polynucleotide homologous transcripts according to the invention, cDNA libraries can be constructed from RNA isolated from the appropriate strain, using standard techniques well known to those skilled in the art. Alternatively, a library of total genomic DNA can be identified using a probe that can hybridize to a protease polynucleotide according to the invention.

Homologous gene sequences can be isolated, for example by carrying out PCR using two oligonucleotide primers or sets of two degenerate oligonucleotide primers designed based on the nucleotide sequences taught herein.

The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new protease nucleic acid sequence, or a functional equivalent thereof.

The PCR fragment can then be used to isolate a full length cDNA clone by a variety of known methods. For example, the amplified fragment can be labeled and used to identify a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment can be used to identify a genomic library.

PCR technology can also be used to isolate full-length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction on the RNA can be carried out using a

oligonucleotide primer specific for most of the 5 ′ end of the amplified fragment to prime the synthesis of the first strand.

The resulting RNA / DNA hybrid can be "colear" (for example, with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and then the synthesis of the second strand can be primed (for example , with a poly-C primer). In this way, cDNA sequences can be easily isolated in the 5 'direction of the amplified fragment. For a review of useful cloning strategies, see for example Sambrook et al., Above; and Ausubel et al., above.

Vectors

Another aspect of the invention relates to vectors, preferably expression vectors, which contain a nucleic acid encoding a protease protein or a functional equivalent thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop, in which additional segments of DNA can be ligated. Another type of vector is a viral vector, in which additional segments of DNA can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, bacterial vectors having an origin of bacterial replication, and episomic mammalian vectors). Other vectors (eg, non-episomic mammalian vectors) are integrated into the genome of a host cell with introduction into the host cell, and thereby replicate together with the host genome. In addition, 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 useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein, since 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 (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which have equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected based on the Host cells to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence or sequences in a manner that allows the expression of the nucleotide sequence (eg, in a transcription / translation system in vitro, or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells, and those that direct the expression of the nucleotide sequence only in a certain host cell (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. Expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (for example protease proteins, mutant forms of protease proteins, fragments, variants or functional equivalents. thereof, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed for the expression of protease proteins in prokaryotic or eukaryotic cells. For example, protease proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further explained in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 and T7 polymerase promoter regulatory sequences.

Useful expression vectors of the present invention include vectors derived from chromosomes, episomes, and viruses, for example vectors derived from bacterial plasmids, bacteriophages, yeast episoma, chromosomal elements of yeasts, viruses such as baculovirus, papova virus, vaccine virus, adenovirus, avian smallpox virus, pseudorabies virus, and retroviruses, and vectors derived from their combinations, such as those derived from genetic elements of plasmids and bacteriophages, such as cosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and retroviral LTR promoters, to cite a few. Other suitable promoters will be known by the skilled person. In a specific embodiment, promoters that are capable of directing a high level of protease expression in filamentous fungi are preferred. Such promoters are known in the art. The expression constructs

they may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning, and a termination codon properly located at the end of the polypeptide to be translated.

Vector DNA can be introduced into prokaryotic or eukaryotic cells, via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to a variety of techniques recognized in the art for introducing foreign nucleic acid (eg, DNA) into a host cell, including coprecipitation with calcium phosphate or calcium chloride. calcium, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986), and other manuals laboratory.

For stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these members, a gene encoding a selectable marker (for example, antibiotic resistance) is generally introduced into the host cells, together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. The nucleic acid encoding a selectable marker can be introduced into a host cell in the same vector as that encoding a protease protein, or it can be introduced into a separate vector. Stably transfected cells with the introduced nucleic acid can be identified by drug selection (for example, cells that have incorporated the selectable marker gene will survive, while the other cells will die).

Protein expression in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters that direct the expression of fusion proteins or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded there, for example the amino acid term of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein, to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.

As indicated, the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or resistance to neomycin for the culture of eukaryotic cells, and resistance to tetracycline or ampicillin for culture in E. coli and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture media and conditions for the host cells described above are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE60 and PQE9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among the preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pZT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the expert.

Among the bacterial promoters known for use in the present invention are the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the PR, PL phage promoters of the lambda phage, and the trp promoter, the promoter of HSV thymidine kinase, the SV40 early and late promoters, retroviral LTR promoters, such as those of Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein I promoter.

The transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are elements that act in cis of DNA, usually around 10 to 300 bp, that act to increase the transcriptional activity of a promoter in a given type of host cell. Examples of enhancers include the SV40 enhancer, which is located on the late side of the 100 to 270 bp origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancers.

For secretion of the translated protein in the lumen of the endoplasmic reticulum, in the periplasmic space or in the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide, or they may be heterologous signals.

The polypeptide can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals but also additional heterologous functional regions. Thus, for example, a region of additional amino acids, particularly charged amino acids, can be added to the N terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide, to facilitate purification.

Polypeptides according to the invention

The invention provides an isolated polypeptide having an amino acid sequence of SEQ ID NO: 124, an amino acid sequence obtainable by expressing a polynucleotide according to the invention or in a preferred embodiment of a sequence of SEQ ID NO: 10 in an appropriate host, thus as an amino acid sequence obtainable by expressing a polynucleotide sequence of SEQ ID NO: 67 in an appropriate host. Also, within the present invention a peptide or polypeptide comprising a functional equivalent of the above polypeptides is comprised. The above polypeptides are collectively included in the expression "polypeptides according to the invention".

The terms "peptide" and "oligopeptide" are considered synonymous (as is normally recognized), and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptide bonds. The word "polypeptide" is used here for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right, and in the direction from the amino term to the carboxy term. The one-letter amino acid code used herein is commonly known in the art, and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

By "isolated" polypeptide or protein is meant a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins, expressed in host cells, are considered isolated for the purposes of the invention such as native or recombinant polypeptides that have been substantially purified by any suitable technique, such as, for example, the method. single stage purification described in Smith and Johnson, Gene 67: 31-40 (1988).

The protease according to the invention can be recovered and purified from recombinant cell cultures by well known methods, including precipitation with ammonium or ethanol sulfate, acid extraction, anionic or cationic exchange chromatography, phosphocellulose chromatography, interaction chromatography hydrophobic, affinity chromatography, hydroxylapatite chromatography, and lecithin chromatography. For analytical purposes, high performance liquid chromatography ("HPLC") is most preferably used for purification.

The polypeptides of the present invention include naturally purified products, products of chemical synthetic processes, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant cells, of Insects and mammals. Depending on the host employed in a recombinant production process, the polypeptides of the present invention may be glycosylated, or they may be non-glycosylated. In addition, the polypeptides of the invention may also include an initial modified methionine moiety, in some cases as a result of host-mediated processes.

Furthermore, a protein according to the invention can be a precursor protein such as a cymogen, a hybrid protein, a protein obtained as a prosequence or a pre-prosequence, or any other type of immature form.

Protein fragments

The invention also relates to biologically active fragments of the polypeptides according to the invention.

Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protease protein (eg, the amino acid sequence of a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171), which includes a smaller number of amino acids than the full length protein, and shows at least one biological activity of the corresponding full length protein. Typically, biologically active fragments comprise a domain or motif with at least one protease protein activity. A biologically active fragment of a protein of the invention can be a polypeptide having, for example, a length of 10, 25, 50, 100 or more amino acids. In addition, other biologically active portions can be prepared by recombinant techniques, in which other suppressed

regions of the protein, and can be evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.

The invention also relates to nucleic acid fragments encoding the above biologically active fragments of the protease protein.

5 Fusion Proteins

The proteins of the present invention or their functional equivalents, for example their biologically active portions, can be operably linked to a non-protease polypeptide (eg, heterologous amino acid sequences), to form fusion proteins. As used herein, a "chimeric protein" or "fusion protein" of protease comprises a protease polypeptide operably linked to a non-protease polypeptide. A "protease polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a polypeptide sequence according to the invention, while a "non-protease polypeptide" refers to a polypeptide having an amino acid sequence that corresponds to a protein that is not substantially homologous to a protein according to the invention, for example a protein that is different from the protease protein and that is derived from the same organism or from a different organism. Within a protein of

In protease fusion, the protease polypeptide may correspond to all or a portion of a protein according to the invention. In a preferred embodiment, a protease fusion protein comprises at least one biologically active fragment of a protein according to the invention. In another preferred embodiment, a protease fusion protein comprises at least two biologically active portions of a protein according to the invention. Within the fusion protein, the expression "operably linked" is intended to indicate that the protease polypeptide and the non-protease polypeptide are fused within each other. The non-protease polypeptide can be fused to the N-terminus or the C-terminus of the protease polypeptide.

For example, in one embodiment, the fusion protein is a GST-protease fusion protein in which the protease sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant protease. In another embodiment, the fusion protein is a protein of

25 protease containing a heterologous signal sequence at its N terminus. In certain host cells (eg, mammalian and yeast host cells), protease expression and / or secretion can be increased through the use of a signal sequence heterologous

In another example, as the heterologous signal sequence, the gp67 secretory sequence of the baculovirus envelope protein (Current Protocols in Molecular Biology, Ausubel et al., Eds., John Wiley & Sons, 1992) can be used. Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melitin and alkaline phosphatase of the human placenta (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al., Above) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

A signal sequence can be used to facilitate the secretion and isolation of a protein or polypeptide from the

Invention. Signal sequences are typically characterized by a nucleus of hydrophobic amino acids that are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from mature proteins as they pass through the secretory pathway. The signal sequence directs the secretion of the protein, such as from a eukaryotic host in which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be easily purified from the extracellular medium by methods recognized in the art. Alternatively, the signal sequence can be ligated to the protein of interest using a sequence that facilitates purification, such as with a GST domain. Thus, for example, the sequence encoding the polypeptide can be fused to a marker sequence, such as a sequence encoding a polypeptide, which facilitates purification of the fused polypeptide. In certain embodiments

Preferred of this aspect of the invention, the marker sequence is a hexahistidine peptide, such as the label provided in the pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl Acad. Sci. USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. The HA tag is another peptide useful for purification that corresponds to an epitope derived from influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984), for example.

Preferably, a chimeric or protease fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments encoding the different polypeptide sequences are linked together in the frame according to conventional techniques, for example using terms with blunt ends or stepped ends for ligation, digestion by restriction enzymes to provide appropriate terms, filled with the cohesive ends as appropriate, treatment with alkaline phosphatase to avoid undesirable binding, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary protrusions between two consecutive gene fragments that can subsequently be hybridized and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al.

John Wiley & Sons: 1992). In addition, many expression vectors that already encode a fusion moiety (for example, a GST polypeptide) are commercially available. A nucleic acid encoding the protease can be cloned into such an expression vector, so that the fusion moiety is linked in the frame to the protease protein.

Functional equivalents

5 The terms "functional equivalents" and "functional variants" are used interchangeably here. The functional equivalents of a DNA according to the invention are isolated DNA fragments encoding a polypeptide that shows a particular function of an A. niger protease as defined herein. A functional equivalent of a polypeptide according to the invention is a polypeptide that shows at least one function of an A. niger protease as defined herein.

Functional protein or polypeptide equivalents may contain only conservative substitutions of one or more amino acids of a sequence of SEQ ID NO: 124 or substitutions, insertions or deletions of nonessential amino acids. Consequently, a non-essential amino acid is a moiety that can be altered in a sequence of SEQ ID NO: 124 without substantially altering the biological function. For example, it is predicted that amino acid residues that are conserved among the protease proteins of the present invention are particularly

15 not susceptible to alteration. Furthermore, it is not likely that the amino acids conserved between the protease proteins according to the present invention and other proteases are susceptible to alteration.

The term "conservative substitution" means that substitution in which the amino acid residue is replaced by an amino acid residue having a similar side chain. These families are known in the art, and include amino acids with basic side chains (for example lysine, arginine and histidine), acid side chains (for example aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine , glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine).

The equivalents of functional nucleic acids may typically contain silent mutations or mutations that do not alter the biological function of the encoded polypeptide. Accordingly, the invention provides nucleic acid molecules that encode protease proteins that contain changes in amino acid residues that are not essential for a particular biological activity. Such protease proteins differ in the amino acid sequence of a sequence of SEQ ID NO: 124 although they retain at least one biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises a substantially homologous amino acid sequence of at least about 80%, 85%, 90%, 95%, 96%. , 97%, 98%, 99% or more homologous to the amino acid sequence shown in a sequence of SEQ ID NO: 124.

For example, in Bowie, J.U. et al., Science 247: 1306-1310 (1990) provides a guide that refers to how

35 obtain phenotypically silent amino acid substitutions, a reference in which the authors indicate that there are two main approaches to study the tolerance to change of an amino acid sequence. The first method is based on the evolution process, in which mutations are accepted or rejected by natural selection. The second approach uses genetic manipulation to introduce amino acid changes at specific positions of a cloned gene, and selects or identifies sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors also indicate what changes are probably permissive in a certain position of the protein. For example, more buried amino acid residues require non-polar side chains, while few features of the surface side chains are generally preserved. Other such phenotypically silent substitutions are described in Bowie et al., Above, and the references cited therein.

An isolated nucleic acid molecule encoding a protease protein homologous to the protein of SEQ ID NO: 124 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequences encoding according to SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, so that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein. Such mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

The term "functional equivalents" also encompasses orthologs of the A. niger protease protein. The orthologs of the A. niger protease protein are proteins that can be isolated from other strains or species, and possess a similar or identical biological activity. Such orthologs can be easily identified as they comprise an amino acid sequence that is substantially homologous to a sequence of SEQ ID NO: 124.

As defined herein, the term "substantially homologous" refers to a first amino acid or nucleotide sequence containing a sufficient or minimum number of identical or equivalent amino acids or nucleotides (eg, with similar side chain) to a second sequence of nucleic or nucleotide acids, so that the first and second amino acid or nucleotide sequences have a common domain. For example, the

amino acid or nucleotide sequences containing a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or more, are defined here as sufficiently identical.

Also, nucleic acids encoding other members of the protease family, thus having a nucleotide sequence that differ from a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, are within the scope of the invention. In addition, nucleic acids encoding protease proteins from different species, which thus have a nucleotide sequence that differs from a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, are within the scope of the invention. .

Nucleic acid molecules corresponding to variants (for example natural allelic variants) and homologs of the protease DNA of the invention can be isolated based on their homology with the protease nucleic acids described herein using the cDNAs described herein or a suitable fragment of they, as a hybridization probe according to standard hybridization techniques, preferably under very restrictive hybridization conditions.

In addition to the naturally occurring allelic variants of the protease sequence, the skilled person will recognize that changes can be made by mutation in the nucleotide sequences of a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, leading from that way to changes in the amino acid sequence of the protease protein without substantially altering the function of the protease protein.

In another aspect of the invention, improved protease proteins are provided. Enhanced protease proteins are proteins in which at least one biological activity is improved. Such proteins can be obtained by randomly introducing mutations throughout all or part of the protease coding sequence, such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and can be identified for biological activity. For example, the technique provides standard assays to measure the enzymatic activity of proteases, and thus improved proteins can be easily selected.

In a preferred embodiment, the protease protein has an amino acid sequence according to a sequence of SEQ ID NO: 124. In another embodiment, the protease polypeptide is substantially homologous to the amino acid sequence according to a sequence of SEQ ID NO: 124, and retains at least one biological activity of a polypeptide according to a sequence of SEQ ID NO: 124, although it differs in the amino acid sequence due to natural variation

or mutagenesis as described above.

In a further preferred embodiment, the protease protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridizing to a nucleic acid according to a sequence of SEQ ID NO: 10 or a sequence of SEQ ID NO: 67, preferably in very restrictive hybridization conditions.

Consequently, the protease protein is a protein that comprises an amino acid sequence of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence shown in a sequence of SEQ ID NO: 124, and retains at least one functional activity of the polypeptide according to a sequence selected from the group consisting of SEQ ID NO: 124.

The functional equivalents of a protein according to the invention can also be identified, for example, by detecting combinatorial libraries of mutants, for example truncation mutants, of the protein of the invention in search of protease activity. In one embodiment, a varied library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A varied library of variants can be produced, for example, by enzymatically linking a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or, alternatively, as a set of fusion proteins. larger (for example, for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477).

In addition, fragment libraries of the coding sequence of a polypeptide of the invention can be used to generate a varied population of polypeptides to identify a subsequent selection of variants. For example, a library of fragments of coding sequences can be generated by treating a double-stranded PCR fragment of the coding sequence of interest with a nuclease under conditions where the nicking occurs only about once per molecule, denaturing the double-stranded DNA, by renaturing the DNA to form double-stranded DNA, which may include sense / antisense pairs of different indented products, removing the single-stranded portions of the reformed duplexes by treatment with S1 nuclease, and ligating the library of the resulting fragment into an expression vector. By this method, an expression library encoding N-terminal and internal fragments of various sizes of the protein of interest can be derived.

Several techniques are known in the art to identify gene products from combinatorial libraries obtained by truncation point mutations, and to identify cDNA libraries in search of gene products.

that have a selected property. The most widely used techniques, which are susceptible to high throughput analysis, to identify large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expressing combinatorial genes under conditions in which which the detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recurrent assembly mutagenesis (REM), a technique that enhances the frequency of functional mutants in libraries, can be used in combination with identification assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl Acad. Sci. USA 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

In addition to the protease gene sequence shown in a sequence of SEQ ID NO: 10, it will be apparent to the person skilled in the art that polymorphisms of the DNA sequence may exist in a given population that can lead to changes in the sequence of Protease protein amino acids. Such genetic polymorphisms may exist in cells from different populations, or in a population, due to natural allelic variation. Allelic variants can also include functional equivalents.

Fragments of a polynucleotide according to the invention may also comprise polynucleotides that do not encode functional polypeptides. Such polynucleotides can function as probes or primers for a PCR reaction. Such polynucleotides can also be useful when it is desired to abolish the functional activity of a protease in a particular organism (gene lacking mutants).

The nucleic acids according to the invention, regardless of whether they encode functional or non-functional polypeptides, can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a protease activity include, among others, (1) isolating the gene encoding the protease protein, or its allelic variants, from a cDNA library, for example from organisms other than A. niger; (2) in situ hybridization (eg FISH) to metaphase chromosomal extensions, to provide a precise chromosomal location of the protease gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis, to detect the expression of protease mRNA in specific tissues and / or cells, and (4) probes and primers that can be used as a diagnostic tool to analyze the presence of a hybridizable nucleic acid to the protease probe in a given biological sample (for example, tissue).

A method for obtaining a functional equivalent of a protease gene or cDNA is also encompassed by the invention. Such a method involves obtaining a labeled probe that includes an isolated nucleic acid encoding all or a portion of the sequence according to a sequence of SEQ ID NO: 124 or a variant thereof; identify a library of nucleic acid fragments with the labeled probe, under conditions that allow hybridization of the probe to the nucleic acid fragments in the library, thereby forming duplexes of nucleic acids, and preparing a full length gene sequence to from the nucleic acid fragments in any labeled duplex, to obtain a gene related to the protease gene.

In one embodiment, a protease nucleic acid of the invention is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to a nucleic acid sequence shown in a sequence of SEQ ID NO: 10, a sequence of SEQ ID NO: 67, or the complement thereof.

In another preferred embodiment, a protease polypeptide of the invention is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, homologous to the amino acid sequence shown in a sequence of SEQ ID NO: 124.

Host cells

In another embodiment, the invention relates to cells, for example transformed host cells or recombinant host cells, which contain a nucleic acid encompassed by the invention. A "transformed cell"

or "recombinant cell" is a cell in which (or an ancestor in which) a nucleic acid according to the invention has been introduced by means of recombinant DNA techniques. Both prokaryotic and eukaryotic cells are included, for example bacteria, fungi, yeasts, and the like; filamentous fungal cells, in particular Aspergillus niger, are especially preferred.

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific and desired manner. Such modifications (for example, glycosylation) and processing (for example, cleavage) of protein products can facilitate the optimal functioning of the protein.

Various host cells have characteristic and specific mechanisms for post-translational processing and the modification of proteins and gene products. Appropriate cell lines or host systems, familiar to those skilled in the art of molecular biology and / or microbiology, may be chosen to ensure the desired and correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for proper processing can be used

of the primary transcript, glycosylation, and phosphorylation of the gene product. Such host cells are well known in the art.

Host cells also include, but are not limited to, mammalian cell lines, such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroidal plexus cell lines.

5 If desired, the polypeptides according to the invention can be produced by a stably transfected cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public; methods for constructing such cells are also publicly known, for example in Ausubel et al. (higher).

Antibodies

The invention further relates to antibodies, such as monoclonal or polyclonal antibodies, which specifically bind to protease proteins according to the invention.

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) includes intact molecules as well as antibody fragments (such as, for example, Fab and F (ab ') 2 fragments) that are capable of specifically binding to protease protein. Fab and F (ab ’) 2 fragments lack the Fc fragment of the antibody

15 intact, they clear up more rapidly from the circulation, and may have less non-specific binding to tissues of an intact antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Thus, these fragments are preferred.

The antibodies of the present invention can be prepared by any of a variety of methods. For example, cells expressing the protease protein or an antigen fragment thereof can be administered to a mammal, in order to induce the production of sera containing polyclonal antibodies. In a method

20 preferred, a protease protein preparation is prepared and purified to make it substantially free of natural contaminants. Such preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or their protease protein binding fragments). Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, p. 563-681 (1981)). In general, such procedures involve immunizing an animal (preferably a mouse) with a protease protein antigen, or with a cell that expresses the protease protein. Splenocytes from such mice are removed and fused with a suitable myeloma cell line. Any suitable myeloma cell line 30 according to the present invention can be used; however, it is preferable to use the progenitor myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastro-enterology 80: 225-232 (1981)). The hybridoma cells obtained through such selection are then evaluated to identify clones that secrete antibodies capable of binding to the

35 protease protein antigen. In general, the polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al., Above, mixed with an adjuvant, and can be injected into a host mammal.

In particular, various host animals can be immunized by injection of a polypeptide of interest. Examples of suitable host animals include rabbits, mice, guinea pigs, and rats. 40 various adjuvants can be used to increase the immune response, depending on the host species, including, but not limited to, Freund's adjuvant (complete and incomplete), adjuvant mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, polyols of the Pluronic type, polyanions, peptides, oily emulsions, hemocyanin of California limpe, dinitrophenol, BCG (bacillus Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules

45 derived from the sera of immunized animals.

Such antibodies can be any kind of immunoglobulin, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridomas that produce the mAbs of this invention can be cultured in vitro or in vivo.

Once produced, polyclonal or monoclonal antibodies are tested for recognition

A specific protease polypeptide or functional equivalent thereof in an immunoassay, such as a Western blot analysis or an immunoprecipitation analysis, using standard techniques, for example as described in Ausubel et al., Above. Antibodies that specifically bind protease proteins or their functional equivalents are useful in the invention. For example, such antibodies can be used in an immunoassay to detect protease in pathogenic or non-pathogenic strains of Aspergillus (for example, in extracts

Aspergillus 55).

Preferably, the antibodies of the invention are produced using fragments of protease polypeptides that probably appear to be antigenic, by criteria such as high frequency of charged moieties. By

For example, such fragments can be generated by standard PCR techniques, and then can be cloned into the pGEX expression vector (Ausubel et al., above). Then, the fusion proteins can be expressed in E. coli and can be purified using an affinity matrix in agarose gel with glutathione, as described in Ausubel et al., Above. If desired, several (for example, two or three) fusions can be generated for each protein, and each fusion can be injected into at least two rabbits. Antibodies can be produced by serial injections, typically including at least three revaccination. Typically, antibodies are tested for their ability to immunoprecipitate a recombinant protease polypeptide or its functional equivalents, while unrelated proteins can serve as a control for the specificity of the immune reaction.

Alternatively, the techniques described for the production of single chain antibodies (U.S. Patents 4,946,778 and 4,704,692) can be adapted to produce single chain antibodies against a protease polypeptide or its functional equivalents. Kits for generating and identifying phage display libraries are commercially available, for example, from Pharmacia.

Additionally, examples of methods and reagents particularly susceptible for use in generating and identifying the antibody presentation library can be found, for example, in U.S. Pat. No. 5,223,409; in PCT Publication No. WO 92/18619; in PCT Publication No. WO 91/17271; in PCT Publication No. WO 20791; in PCT Publication No. WO 92/20791; in PCT Publication No. WO 92/15679; in PCT Publication No. WO 93/01288; in PCT Publication No. WO 92/01047; in PCT Publication No. WO 92/09690; in PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; There et al. (1992) Hum. Antibod Hybridomas 3: 8185; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J. 12: 725-734.

Polyclonal and monoclonal antibodies that specifically bind to protease polypeptides or their functional equivalents can be used, for example, to detect the expression of a protease gene or a functional equivalent thereof, for example, in another strain of Aspergillus. For example, the protease polypeptide can be readily detected in conventional immunoassays of Aspergillus cells or extracts. Examples of suitable assays include, without limitation, Western blotting, ELISAs, radioimmunoassays, and the like.

By "specifically binds" is meant that an antibody recognizes and binds to a particular antigen, for example a protease polypeptide, but does not recognize or bind substantially to other unrelated molecules in a sample.

The antibodies can be purified, for example, by affinity chromatography methods, in which the polypeptide antigen is immobilized on a resin.

An antibody directed against a polypeptide of the invention (eg, monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. In addition, such an antibody can be used to detect the protein (for example, in a cell lysate or a cell supernatant) in order to evaluate the abundance and expression pattern of the polypeptide. Antibodies can also be used diagnosticly to monitor protein levels in cells or tissue as part of a clinical trial procedure, for example to determine the efficacy of a given treatment regimen, or in the diagnosis of aspergillosis.

Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, E-galactosidase or acetylcholinesterase; Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamin fluorescein, dansyl chloride or phycoerythrin; An example of a luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin, and aecuorin; and examples of suitable radioactive materials include 125I, 131I, 35S or 3H.

Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, for example hydrophilic regions. Hydrophobic plots of the proteins of the invention can be used to identify hydrophilic regions.

The antigenic peptide of a protein of the invention comprises at least 7 (preferably 10, 15, 20, or 30) contiguous amino acid residues of the amino acid sequence of a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, and encompasses an epitope of the protein so that an antibody raised against the peptide forms a specific immune complex with the protein.

Preferred epitopes encompassed by the antigenic peptide are protease regions that are located on the surface of the protein, for example hydrophilic regions, hydrophobic regions, alpha regions, beta regions, winding regions, back regions and flexible regions.

Immunoassays

The qualitative or quantitative determination of a polypeptide according to the present invention in a biological sample can be performed using any method known in the art. Antibody-based techniques provide special advantages to test specific polypeptide levels in a biological sample.

In these, specific recognition is provided by the primary antibody (polyclonal or monoclonal), but the secondary detection system can use fluorescent materials, enzymes, or other conjugated secondary antibodies. As a result, an immunocomplex is obtained.

Accordingly, the invention provides a method for diagnosing whether a certain organism is infected with Aspergillus, which comprises the steps of:

10 x isolate a biological sample from said organism suspected of being infected with Aspergillus,

x reacting said biological sample with an antibody according to the invention,

x determine if immunocomplexes are formed.

The tissues can also be extracted, for example, with urea and neutral detergent, for protein release for the Western blot or dot / groove assay. This technique can also be applied to body fluids.

Other antibody-based methods, useful for detecting protease gene expression, include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). For example, protease-specific monoclonal antibodies can be used both as an immunoabsorbent and as an enzymatically labeled probe, to detect and quantify the protease protein. The amount of protease protein present in the sample can be calculated by referring to the amount present in a sample.

20 standard preparation using a linear regression computer algorithm. In another ELISA assay, two different specific monoclonal antibodies can be used to detect the protease protein in a biological fluid. In this assay, one of the antibodies is used as the immunoabsorbent, and the other as the enzymatically labeled probe.

The above techniques can be carried out essentially as a "one stage" or "two stage" assay. He

"One step" assay involves contacting the protease protein with immobilized antibody, and, without washing, contacting the mixture with the labeled antibody. The "two stage" assay involves washing before contacting the mixture with the labeled antibody. Other conventional methods may also be employed as appropriate. It is usually desirable to immobilize a component of the test system on a support, thereby allowing other components of the system to contact the component and to be

30 easily removed from the sample.

Suitable enzyme markers include, for example, those of the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate. The activity of an oxidase label can be evaluated by measuring the concentration of hydrogen peroxide formed by the reaction of antibody labeled with the enzyme / substrate.

In addition to enzymes, other suitable markers include radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent markers , such as fluorescein and rhodamine, and biotin.

Specific binding of a test compound to a protease polypeptide can be detected, for example, in vitro by reversibly or irreversibly immobilizing the protease polypeptide on a substrate, for example the surface 40 of a well of a polystyrene microtiter plate of 96 wells Methods for immobilizing polypeptides and other small molecules are known in the art. For example, microtiter plates can be coated with a protease polypeptide by adding the polypeptide in a solution (typically, at a concentration of 0.05 to 1 mg / ml in a volume of 1-100 ul) to each well, and incubating the plates at room temperature up to 37 ° C for 0.1 to 36 hours. Polypeptides that do not bind to the plate can be removed by stirring the excess solution of the plate, and then washing the plate (once, or repeatedly) with water or with a buffer. Typically, the polypeptide is contained in water or in a buffer. The plate is then washed with a buffer lacking the bound polypeptide. To block free protein binding sites in the plates, the plates are blocked with a protein that is not related to the bound polypeptide. For example, 300 ul of bovine serum albumin (BSA) at a concentration of 2 mg / ml in Tris-HCl is suitable. Suitable substrates include

50 those substrates containing a defined cross-linking chemistry (for example, plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Corning Costar Corp. (Cambridge, MA), for example). If desired, as a substrate, a particle in the form of a pearl can be used, for example agarose in pearls or sepharose in pearls.

The binding of the test compound to the polypeptides according to the invention can be detected by any of a variety of methods known in the art. For example, a specific antibody can be used in a

immunoassay If desired, the antibody can be labeled (for example, fluorescently or with a radioisotope) and detected directly (see, for example, West and McMahon, J. Cell Biol. 74: 264, 1977). Alternatively, a second antibody can be used for detection (for example, a labeled antibody that binds to the Fc portion of an anti-AN97 antibody). In an alternative detection method, the protease polypeptide is labeled, and the label is detected (for example, by labeling a protease polypeptide with a radioisotope, fluorophore, chromophore, or the like). In yet another method, the protease polypeptide is produced as a fusion protein with a protein that can be detected optically, for example the green fluorescent protein (which can be detected under UV light). In an alternative method, the protease polypeptide can be covalently linked to or fused with an enzyme that has a detectable enzymatic activity, such as horseradish peroxidase, alkaline phosphatase, a-galactosidase, or glucose oxidase. The genes encoding all these enzymes have been cloned and are readily available for use by those skilled in the art. If desired, the fusion protein can include an antigen, and such antigen can be detected and measured with a polyclonal or monoclonal antibody using conventional methods. Suitable antigens include enzymes (for example, horseradish peroxidase, alkaline phosphatase, and a-galactosidase) and non-enzymatic polypeptides (eg, serum proteins, such as BSA and globulins, and milk proteins, such as caseins).

Epitopes, antigens and immunogens

In another aspect, the invention provides a peptide or polypeptide comprising a portion that possesses an epitope of a polypeptide of the invention. The epitope of this portion of the polypeptide is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the complete protein is the immunogen. It is believed that these immunogenic epitopes are confined to a few loci in the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen, H. M. et al., Proc. Natl Acad, Sci. USA 81: 3998-4002 (1984).

As for the selection of peptides or polypeptides that possess an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that technique that the relatively short synthetic peptides that mimic Part of a protein sequence are usually able to elicit an antiserum that reacts with partially imitated protein. See, for example, Sutcliffe, J. G. et al., Science 219: 660-666 (1984). Peptides capable of causing sera reactive with the protein are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are not confined in immunodominant regions of intact proteins (i.e., immunogenic epitopes) or at the amino or carboxyl terminals. Peptides that are extremely hydrophobic, and those of six residues or less, are generally ineffective in inducing antibodies that bind to the imitated protein; usually the longest, most soluble peptides are effective, especially those that contain proline residues. Sutcliffe et al., Above ?. For example, 18 to 20 peptides designed according to these guidelines, which contain 8-39 residues that cover 75% of the sequence of the HAI polypeptide chain of influenza virus hemagglutinin, induced antibodies that reacted with the HA1 protein or virus intact; and 12/12 peptides from MuLV polymerase and 18/18 from rabies glycoprotein induced antibodies that precipitated the respective proteins.

Peptides and polypeptides of the invention that possess antigenic epitopes are therefore useful for eliciting antibodies, including monoclonal antibodies, that specifically bind to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of splenocytes from donors immunized with a peptide that possesses an antigenic epitope generally secretes antibody reactive with the native protein. Sutcliffe et al., Above, in 663. Antibodies caused by peptides or polypeptides possessing antigenic epitopes are useful for detecting mimicked protein, and antibodies directed against different peptides can be used to track the fate of various regions of a precursor. protein that undergoes post-translation processing. Peptides and antipeptide antibodies can be used in a variety of qualitative or quantitative assays for the mimicked protein, for example in competition assays, since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace larger peptides in immunoprecipitation assays. See, for example, Wilson, I.A. et al., Cell 37: 767778 in 777 (1984). The antipeptide antibodies of the invention are also useful for purification of the imitated protein, for example by adsorption chromatography, using methods well known in the art.

The peptides and polypeptides of the invention possessing antigenic epitopes designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 and about 30 amino acids contained in the amino acid sequence. of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the complete amino acid sequence of a polypeptide of the invention, they are also considered peptides or polypeptides of the invention that possess epitopes, and are also useful for inducing antibodies that react with the imitated protein. Preferably, the amino acid sequence of the epitope-possessing peptide is selected to provide substantial solubility in aqueous solvents (i.e.,

the sequence includes relatively hydrophilic moieties, and very hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.

Peptides and polypeptides of the invention that possess epitopes can be produced by any conventional means to obtain peptides or polypeptides, including recombinant means using nucleic acid molecules of the invention. For example, a short sequence of amino acids that possess epitopes can be fused to a larger polypeptide that acts as a carrier during recombinant production and purification, as well as during immunization to produce antipeptide antibodies.

Peptides that possess epitopes can also be synthesized using known methods of chemical synthesis. For example, Houghten has described a simple method for the synthesis of large number of peptides, such as 10-20 mg of 248 peptides from 13 different residues representing individual amino acid variants of a HAI polypeptide segment that were prepared and characterized (by ELISA type binding studies) in less than four weeks. Houghten, R. A., Proc. Nati Acad. Sci. USA 82: 5131-5135 (1985). This "simultaneous multiple peptide synthesis (SMPS)" process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure, the individual resins for solid phase synthesis of various peptides are contained in separate solvent-permeable packages, which allow the optimal use of the many identical repetitive stages involved in solid phase methods.

A manual procedure allows 500-1000 or more syntheses to be carried out simultaneously. Houghten et al., Above in 5134.

Peptides and polypeptides of the invention that possess epitopes are used to induce antibodies according to methods well known in the art. See, for example, Sutcliffe et al., Above; Wilson et al., Above; Chow, M. et al., Proc. Natl Acad. Sci. USA 82: 910-914; and Bittle, F.J. et al., J. Gen. Virol. 66: 2347-2354 (1985).

Generally, animals can be immunized with free peptide; however, the antipeptide antibody titer can be increased by coupling the peptide to a macromolecular carrier, such as Californian limpet hemocyanin (KLH) or tetanus toxoid. For example, cysteine-containing peptides can be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides can be coupled to a carrier using a more general binding agent, such as glutaraldehyde. .

Animals such as rabbits, rats and mice are immunized with free or carrier-coupled peptides, for example by intraperitoneal and / or intradermal injection of emulsions containing about 100 ug of Freund's carrier protein or peptide and adjuvant protein. Several revaccinations may be necessary, for example at intervals of about two weeks, to provide a useful antipeptide antibody titer that can be detected, for example, by ELISA using a free peptide adsorbed to a solid surface. The titer of antipeptide antibodies in the serum from an immunized animal can be increased by selecting antipeptide antibodies, for example by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

Peptides of the invention that possess immunogenic epitopes, that is, those parts of a protein that elicit an antibody response when the complete protein is the immunogen, are identified according to methods known in the art. For example, Geysen et al., 1984, above, describe a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. The interaction of the peptides synthesized with the antibodies is then easily detected without removing them from the support. In this manner, a peptide that possesses an immunogenic epitope of a desired protein can be routinely identified by any person skilled in the art. For example, the immunologically important epitope in the gloss protein coat protein was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides that cover the entire 213 amino acid sequence of the protein. Then, a complete peptide substitution set was synthesized in which the 20 amino acids were in turn substituted at each position within the epitope, and particular amino acids conferring specificity for the reaction with the antibody were determined. Thus, by this method, peptide analogs of the peptides of the invention possessing epitopes can be obtained routinely. U.S. Patent No. 4,708,781 to Geysen (1987) further describes this method for identifying a peptide that possesses an immunogenic epitope of a desired protein.

Even more, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method for detecting or determining the sequence of monomers (amino acids or other compounds) that is a topological equivalent of the epitope (ie, a "mimetope") that is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a method for detecting or determining a monomer sequence that is a topographic equivalent of a ligand that is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, R.A. et al. (1996) in Peralkylated Oligopeptide Mixtures describes linear C1-C7 alkyl oligopeptides, and sets and libraries of such peptides, as well as methods for using such sets and libraries of oligopeptides to determine the sequence of a peralkylated oligopeptide that preferentially binds

to an acceptor molecule of interest. Thus, by these methods, non-peptide analogs of the peptides of the invention possessing epitopes can be obtained routinely.

Elimination or reduction of protease activity

The present invention also relates to methods for producing a mutant cell of a progenitor cell, which comprises destroying or suppressing a nucleic acid sequence encoding the protease or a control sequence thereof, which results in the mutant cell produce less protease than the progenitor cell.

The construction of strains that have reduced protease activity can be conveniently achieved by modifying

or inactivating a nucleic acid sequence necessary for the expression of protease activity in the cell. The nucleic acid sequence to be modified or inactivated can be, for example, a nucleic acid sequence encoding the protease or a part thereof essential to show protease activity; or the nucleic acid sequence may have a regulatory function necessary for the expression of the protease from the coding sequence of the nucleic acid sequence. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, that is, a part that is sufficient to affect protease expression. Other control sequences for possible modification include, but are not limited to,

15 a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal sequence, and a termination site.

Modification or inactivation of the nucleic acid sequence can be performed by subjecting the cell to mutagenesis and selecting cells in which the ability to produce protease has been reduced or eliminated. Mutagenesis, which can be specific or random, can be carried out, for example, using a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR-generated mutagenesis. In addition, mutagenesis can be carried out by use of any combination of these mutagenizing agents. Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-Nnitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS) , sodium bisulfite,

25 formic acid, and nucleotide analogs. When such agents are used, mutagenesis is typically carried out by incubating the cell to be mutagenized in the presence of the mutagenizing agent of choice, under suitable conditions, and selecting cells that exhibit reduced expression or no expression of protease activity.

Modification or inactivation of the production of a protease of the present invention can be achieved by introduction, substitution, or elimination of one or more nucleotides in the nucleic acid sequence encoding the protease or a regulatory element required for transcription or translation of the same. For example, nucleotides can be inserted or removed to result in the introduction of a stop codon, the removal of the stop codon, or a change of the open reading frame. Such modification or inactivation can be achieved by site-directed mutagenesis or PCR-generated mutagenesis, according to methods known in the art.

Although, in principle, the modification can be carried out in vivo, that is, directly on the cell that

35 expressing the nucleic acid sequence to be modified, it is preferred that the modification be carried out in vitro, as exemplified below.

An example of a convenient way to inactivate or reduce production by a host cell of choice is based on gene replacement or gene disruption techniques. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous gene or gene fragment of interest is mutagenized in vitro to produce a defective nucleic acid sequence, which is then transformed into the host cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker that can be used for the selection of transformants in which the gene encoding the protease has been modified or destroyed.

Alternatively, the modification or inactivation of the nucleic acid sequence encoding a protease of the present invention can be carried out by consolidated antisense techniques, using a nucleotide sequence complementary to the sequence encoding the protease. More specifically, the production of protease by a cell can be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence encoding the protease, which can be transcribed into the cell and is capable of hybridizing to the protease mRNA produced in the cell. The amount of translated protease is thus reduced or eliminated, under conditions that allow the complementary antisense nucleotide sequence to hybridize to the protease mRNA.

It is preferred that the cell to be modified according to the methods of the present invention be of microbial origin, for example a fungal strain that is suitable for the production of desired protein products, whether homologous

55 or heterologous to the cell.

The present invention further relates to a mutant cell of a progenitor cell comprising an interruption or deletion of a nucleic acid sequence encoding the protease or a control sequence thereof, which results in the mutant cell producing less protease than the progenitor cell.

The mutant cells thus created, deficient in protease, are particularly useful as host cells for the expression of homologous and / or heterologous polypeptides. Therefore, the present invention further relates to methods for producing a homologous or heterologous polypeptide, comprising (a) culturing the mutant cell under conditions that lead to the production of the polypeptide; and (b) recover the polypeptide. In the present context, the expression "heterologous polypeptides" is defined herein as polypeptides that are not native to the host cell, a native protein in which modifications have been made that alter the native sequence, or a native protein whose expression is quantitatively altered. as a result of manipulation of the host cell by recombinant DNA techniques.

The methods of the present invention for producing an essentially protease-free product are of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes. Protease-deficient cells can also be used to express heterologous proteins of interest to the food industry, or of pharmaceutical interest.

Use of proteases in industrial processes

The invention also relates to the use of the protease according to the invention in a select number of industrial and pharmaceutical processes. Despite the long-term experience obtained with these processes, the protease according to the invention has a number of significant advantages over the enzymes currently used. Depending on the specific application, these advantages may include aspects such as lower production costs, greater specificity against the substrate, lower antigenicity, less undesirable secondary activities, higher yields when produced in a suitable organism, more adequate pH and temperature ranges, better flavors of the final product, as well as aspects of kosher and food grade.

In large-scale industrial applications aimed at food or feed production, proteolytic enzymes are commonly used to improve aspects such as protein solubility, yields of extraction, viscosity or taste, texture, nutritional value, minimization of antigenicity or antinutritional factors, color or functionality, as well as aspects of the processing such as filtering capacity of the protein raw material. In these applications, the protein raw material may be of animal or vegetable origin, and examples include vegetable proteins such as soy protein, wheat gluten, rapeseed protein, pea protein, alfalfa protein, sunflower protein, protein bean, cottonseed or sesame seed protein, corn protein, barley protein, sorghum protein, potato protein, rice protein, coffee protein, and animal derived protein, such as milk protein (e.g. casein, whey protein), egg white, fish protein, meat protein, including gelatin, collagen, blood protein (eg hemoglobin), hair, feathers and fishmeal.

An important aspect of the proteases according to the invention is that they cover a full range of pH and temperature optimum that are ideally suited for a variety of applications. For example, many large-scale processes benefit from relatively high processing temperatures, 50 degrees C or higher, to control the risks of microbial infections. Several proteases according to the invention meet this demand, but at the same time show non-extreme thermal stability, so that they resist attempts to inactivate the enzyme by an additional heat treatment. This last feature allows production routes that produce final products free of residual proteolytic activity. Similarly, many feed and food products have slightly acidic pH values, so that proteases with acidic or near neutral pH optimum are preferred for processing. A protease according to the invention also meets this requirement.

The specificity of endoproteases is usually defined in terms of preferential cleavage of bonds between the carboxyl of the amino acid residue at position P1 and the amino group of the residue at position P1 ', respectively. The preference may be predominantly conditioned by P1 (for example positively charged moieties in trypsin substrates), by P1 '(for example hydrophobic moieties in thermolysin cleavage), or therefore P1 and P2 (for example specific cleavages between two positively charged moieties by serine endoprotease of the adrenal medulla). In some cases, more distant residues can determine the preference of cleavage, for example P2 for streptococcal peptidase A. It is known that some remains have a negative influence on splits; It is well known that proline bonds in the P1 ’position are resistant to the action of many proteases. Most endoproteases preferentially cleave in a hydrophobic environment or in the vicinity of negatively charged residues. For example, industrially available endoproteases such as chymotrypsin (obtained from bovine pancreas) or subtilisin, neutral metalloprotease or thermolysin (all obtained from the Bacillus species) tend to favor excision “behind” hydrophobic amino acids such as –Phe, -Leu and –Tyr. Other industrially available endoproteases (obtained from bovine pancreas) preferentially cleave behind –Arg and –Lys, and papain (a complex mixture of various enzymes that include proteases obtained from papaya fruits) preferentially cleave behind –Arg.

In contrast, peptide bonds formed by small size residues such as Ala, Gly, Ser, Thre, as well as Ile and Pro, are poor substrates (Keil, B et al .; Protein Seq Data Anal (1993) 5; 401- 407). This situation has profound implications for the pharmaceutical industry, the beverage and food industry, agribusiness and even the chemical industry. A protease according to the invention has unusual cleavage preferences.

Exopeptidases act only near the ends of the polypeptide chains. Those that act in the free N term release an individual amino acid residue (called aminopeptidases) or a dipeptide or a tripeptide (called dipeptidyl peptidases and tripeptidyl peptidases). Those that act in the free C term release a single moiety (the so-called carboxypeptidases) or a dipeptide (the so-called peptidyl dipeptidases). Carboxypeptidases are intended for three groups based on the catalytic mechanism, that is, carboxypeptidases of the serine type, metallocarboxypeptidases, and carboxypeptidases of the cysteine type. Other exopeptidases are specific for dipeptides (so-called dipeptidases), or are capable of cleaving peptide bonds different from those of the alpha-carboxylic or alpha-amino groups (so-called omega peptidases). Examples of such new omega peptidases are pyroglutamyl peptidase and acylaminoacyl peptidase as identified in the present invention (see Table 1, genes 18 and 45, respectively).

Typical examples of industrial application that depend on the use of pure endoproteases and in which the protease according to the invention can be expected to provide superior performance include the processing of materials of plant or animal origin. These processing steps can be directed to modify a large set of characteristics of the raw material or the (partially) purified protein fraction. For example, these processing steps can be aimed at maximizing product solubilities, filtration capacities, separation capacities, yields of protein extraction and digestion capacities, or minimizing toxicities, bad flavors and viscosities. In addition, the treatment can be directed to alter physical-chemical characteristics of the raw material or of the purified (or partially purified) protein. These advantages apply not only if the endoprotease according to the invention is applied as a processing aid in industrial applications, but also if it is applied as an active enzyme component in animal feed. Specifically, the endoprotease according to the invention can be applied as a bread improver in the bakery industry, for example to retard the hardening of the bread, or to decrease the viscosity of the dough. Or the endoprotease can be used in the beer and wine industry, to prevent or minimize the formation of undesirable protein cloudiness. As an alternative, it can be used in the brewing industry to optimize the extraction yields of cereal protein used in the preparation of must. In addition, it can also be used advantageously in the dairy industry as a milk coagulation agent, with superior characteristics, or to optimize the texturing, foaming or hardening characteristics of various milk components. Another application in the dairy industry is the use of the new protease in the preparation of enzyme modified cheeses (EMC).

In addition, various protein substrates can be subjected to an endoprotease according to the invention, usually in combination with other proteolytic enzymes, to obtain hydrolysates for medical or non-medical applications. Here, the endoprotease according to the invention is surprisingly effective in achieving complete hydrolysis of the protein substrate so that even the protease resistant parts are completely hydrolyzed; The endoprotease is also surprisingly active minimizing the allergenicity of the final hydrolyzate, or suppressing the formation of unpleasant bitter flavors.

More specifically, the endoprotease according to the invention is characterized by its preference for cleaving proteins in unusual peptide bonds, especially with the small amino acid residues of Ala, Gly, Ser and Thr, or the Ile and Pro residues in the position P1 or P1 '(Keil, B et al .; Protein Seq Data Anal (1993) 5; 401-407). As a result, those fractions of the proteinaceous starting materials that resist hydrolysis by using endoproteases of the prior art can be dissolved and hydrolyzed using the endoprotease according to the invention. Non-limiting examples of such protease-resistant fractions include so-called extensins in plant materials and collagen, gelatin but also specific dairy components in material of animal origin.

Various food products, such as, for example, soy beans, contain trypsin inhibitors. These proteins inhibit the trypsin activity in the digestive tract of, for example, pigs and poultry. This trypsin inhibitory activity results in suboptimal protein digestibility in these animals, resulting in increased waste production and poor economy. This problem can be overcome in part by roasting soy beans at elevated temperatures. Two different types of trypsin inhibitors have been identified in soy beans, that is, trypsin inhibitors of the Bowman-Birk type, and trypsin inhibitors of the Kunitz type.

The invention now provides an alternative way to degrade trypsin inhibitory activity with respect to roasting, because it provides a cysteine protease (EC 3.4.22, table 1) capable of cleaving the leucine 176-aspartate 177 peptide bond near the carboxylic terminus of the Kunitz-type trypsin inhibitor (as reviewed in Wilson (1988) in CRC Critical Reviews in Biotechnology 8 (3): 197-216). This results in the inactivation of this trypsin inhibitor in soybeans. It was surprisingly found that cysteine proteases secreted by the fungus Aspergillus niger met these criteria much better than similar enzymes derived from other organisms.

Proteases are also widely used in the art of cheese making. In cheese production, it is necessary to coagulate the milk from the cheese to be able to separate the materials from the cheese, for example casein, from the whey of the milk. Several milk coagulating enzymes have been described, also referred to as coagulants, and include chemosin (bovine), bovine pepsin, porcine pepsin, as well as microbial enzymes such as Rhizomucor miehei protease, Rhizomucor pusillus protease, and parasitic Cryptonectria protease. The chemosin is

It can be obtained from calf stomachs, but it can also be produced microbially, for example, by Kluyveromyces lactis. All these enzymes are characterized by having a specificity for the peptide bond between residue 105 (phenylalanine) and residue 106 (methionine), or the bond adjacent to that in N-casein. This means that, using these enzymes to obtain cheeses, N-casein is divided into the junction between para-N-casein and the macropeptide moiety called glucomacropeptide (GMP) that has negative charges. When this occurs, the macropeptide diffuses into the whey, its stabilizing effect on the solubility of casein micelles is lost, and casein micelles can begin to be added once enough kappa-casein has been hydrolyzed. For further elaboration on the enzymatic coagulation of milk (for example D.G. Dalgleish in Advanced Dairy Chemistry vol. 1 edited by P.F. Fox, Elsevier, London, 1992).

The coagulants currently available allow a rather high yield of cheeses; however, it should be noted that, due to the enormous volumes of cheese produced, an increase in yield of the order of magnitude of tens of percentage points may constitute a substantial economic advantage. Consequently, there is a great need in the coagulant technique with an (even slightly) improved performance.

Coagulants are characterized by their high specificity for the substrate, which, however, depends on the pH and temperature. In a typical cheese making process, the pH will change from the initial pH of 6.3 to lower pH values, in the range of 4.5-5.5, depending on the final value of the conditions used during the process of cheese production. Some coagulants are more sensitive to pH changes than others. Rhizomucor pusillus protease, for example, is more sensitive to pH changes than chemosin. In addition to pH, other parameters, such as temperature and water content, can also affect the specificity of the protease. It is well known that most coagulants show a changing specificity for the substrate when the pH changes, resulting in an altered proteolytic activity in the final stages of the cheese making process. It is also well known that coagulants differ in the degree of proteolysis of casein; They may also show differences in peptide patterns produced during proteolysis. There are relevant factors during cheese maturation, and they can affect the properties of cheese such as taste, aroma and texture. In some cases, coagulants give rise to unwanted effects, such as the formation of bitter-tasting peptides, or bad flavors. In addition, changes in proteolytic specificity can lead to a reduction in yield. Pepsin, a well known component in many bovine chemosin preparations, is an example of a protease that results in lower yields and taste effects when compared to pure chemosin. There is still a need for coagulants that give rise to a new improved cheese texture and flavor. Such new coagulants result in the accelerated development of flavor and texture profiles related to cheese aging, thereby providing a substantial economic benefit.

It is well known that free amino acids are very important in the generation of flavor and aroma. Especially, the amino acids leucine, phenylalanine, methionine and valine play an important role in the generation of flavor and aroma components typical of cheese. Free amino acids are converted, via fermentation by microorganisms that are added during the cheese manufacturing process, into the real aroma and flavor generating compounds, such as methanediol, dimethyl disulfide, methylpropanoic acid and methylpropanal. Exopeptidases play an important role in the generation of free amino acids. However, they can only be effective when combined with an appropriate specificity endoprotease. Appropriate combinations of exo- and endopeptidases can be used in cheese making, resulting in the manufacture of cheeses with new and improved flavor profiles.

The enzymes according to the invention can be used to hydrolyze protein materials of animal origin, such as whole milk, skim milk, casein, whey protein, or mixtures of casein and whey protein. Such mixtures of casein and whey protein can be used, for example, in ratios similar to those found in human milk. In addition, the enzyme mixture according to the invention can be used to hydrolyze plant-derived protein materials, such as, for example, wheat gluten, malted or malted barley or other cereals used to obtain beer, soy milk, its concentrates or isolates. , corn protein concentrates and their isolates, and rice proteins.

Within the area of large-scale industrial processes, some applications are based only on the use of endoproteases, while in other applications combinations of endoproteases with exoproteases are essential. Typical examples that depend on the use of pure endoproteases and in which the protease according to the invention can provide superior performance include applications such as processing of soy or pea or cereal proteins aimed at minimizing viscosities or optimizing foaming or other characteristics. physicochemical, bread improvers in the bakery industry also aimed at reducing the viscosity of the dough, processing aids in the beer and wine industry aimed at preventing protein cloudiness or optimizing cereal extraction yields, food additives in the bioindustry aimed at enhancing intestinal absorption or modulating microbial activities in the intestine, auxiliary to processing in the dairy industry aimed at optimizing the coagulation, foaming or hardening characteristics of various milk components. In addition, for specific market segments, proteins derived from milk or soy or collagen are exposed to proteases to produce so-called protein hydrolysates. Although the main markets for these protein hydrolysates are infant formulas and food products for hospitalized people, products intended for people without

Medical needs, such as athletes, or people on a slimming diet, form a rapidly growing segment. In all these applications, protein hydrolysates offer attractive advantages, such as reduced allergenicities, facilitated gastrointestinal uptake, less chemical deterioration of desirable amino acids such as glutamine and cysteine, and, finally, absence of protein precipitation in acidic beverages during prolonged storage periods. All these advantages can be combined if the hydrolyzate is offered as a mixture of di-and tripeptides. However, currently all commercially available hydrolysates are produced by combining several endoproteases. This last approach implies a non-uniform and incomplete degradation of the protein. To obtain the desired mixture of di- and tripeptides, a hydrolysis process involving a combination of various di- and tripeptidyl peptidases would be ideal. Unfortunately, only a few of these enzymes from food grade and industrially acceptable microorganisms are known, not to mention industrially available. According to the invention, several of the enormously useful di- and tripeptidyl peptidases are economically obtainable in a relatively pure state. Those di- or tripeptidyl peptidases that have low selectivity for the substrate to be cleaved are preferred, that is, they have only minimal preferences for cleavage of amino acid residues. Combinations of those di- or tripeptidyl peptidases that hydrolyze a high percentage of naturally occurring peptide bonds are preferred. Despite this high activity towards naturally occurring peptide bonds, total hydrolysis to free amino acids is avoided due to the nature of di- and tripeptidyl peptidases. Also preferred are those di- or tripeptidyl peptidases that are optimally active between pH 4 and 8, and show adequate temperature stability. Proper temperature stability implies that at least 40%, preferably at least 60%, more preferably between 70 and 100% of the initial hydrolytic activity survives after heating the enzyme together with the substrate for 1 hour at 50 degrees C.

Although the process with respect to an efficient production of mixtures of di- or tripeptides or di-and tripeptides depends on the availability of the enzymes according to the invention, the first enzymatic incubation with the protein substrate will usually be an endoprotease. Preferably, an endoprotease with a broad-spectrum endopeptidase suitable for the situation, for example subtilisin (DSM Delvolase), neutral metalloprotease (NOVO Neutrase) or thermolysin (Daiwa Kasei Thermoase) for almost neutral conditions, and pepsin or aspergilopepsin ( for example Sumizyme AP of Shin Nihon, Japan) for acidic conditions. The objective of this first digestion is to improve the solubility, reduce the viscosity, and reduce the thermosetting characteristics of the water / protein mixture. In addition, this pretreatment with an endonuclease is essential to create sufficient starting points for di- and tripeptidyl peptidases, thereby accelerating the process of di- or tripeptide formation. Optionally, at this stage of the process, or later, a protease intended to detach the hydrolyzate may be included, together with the di- or tripeptidyl peptidases.

The main objective of the latter hydrolysates is to minimize the allergenicity of the product, or facilitate gastrointestinal uptake. In the production of such hydrolysates, the use of dipeptidyl- and tripeptidylpeptidases is especially important, since they offer an efficient way of producing hydrolysates.

Other applications in these food and feed industries rely entirely on combinations of one or more endoproteases with one or more exoproteases. Such combinations of an endoprotease with an exoprotease are typically used in industries to improve aspects such as the taste and color of the final product. The reason for this is that the development of taste and color depends largely on the presence of free amino acids. Free amino acids can be obtained not only by exoproteases such as carboxypeptidases and aminopeptidases, but also by peptidyl dipeptidases. If combined with endoproteases, or even with dipeptidyl- or tripeptidyl-peptidases, carboxypeptidases, aminopeptidases and peptidyl dipeptidases can create greater amounts of free amino acids in less time. However, in all these processes an uncontrolled release of amino acids or even non-protein components should be avoided, to minimize undesirable side reactions.

Although free amino acids as such can cause a number of flavor impressions, these flavor impressions are very basic (bitter, sweet, sour and "umami"), and the concentration of amino acids required to perceive these flavors is very high. Despite these high threshold values, free amino acids are capable of creating important sensory effects at much lower concentration intervals, through a number of mechanisms that enhance flavor. One of these mechanisms involves the combination of free amino acids with sugars in the so-called Maillard reactions. Compared to free amino acids, overwhelmingly complex taste and odor systems can be developed with these Maillard products, with threshold values that are several orders of magnitude smaller than those recorded for free amino acids. Maillard products are formed at elevated temperatures, usually during cooking, baking or roasting, when food or feed products are prepared. During these treatments, both color and a large set of aromas develop. In these reactions, the amino groups react with reducing compounds as the first stage, and finally lead to a complete family of reaction pathways. In foods or feedstuffs, the amino compounds involved are predominantly free amino acids that are released from the protein raw material by various proteases, and the required reducing compounds primarily represent reducing sugars. The implication is that, during the processing of the raw material, the unwanted release of free amino acids and sugars should be avoided, to minimize the bad flavors that could be generated during subsequent heating stages, such as during spray drying or during sterilization This last notion emphasizes once again the benefit of superior purity and low costs in use of the enzyme according to the invention.

Apart from Maillard reactions, amino acids can also undergo significant chemical transitions at room temperatures. The latter type of transitions depend on enzymes, and are quite common in fermented foods such as beer, yogurt, cheese maturation and meat and wine maturation processes. In these fermentation processes, free amino acids are released from the raw materials used by the added proteases, or by proteolytic enzymatic activity from the raw material or the microbial ferments used. During the maturation phase, the microbial metabolic activity then converts free amino acids into derivatives with increased sensory properties. For example, L-leucine, Lisoleucine and L-valine lead to the formation of valuable fusel alcohols such as amyl alcohols and isobutanol in the fermentation of beer. Similarly, cheese volatiles, such as methanethiol and dimethyl disulfide, have been traced to the appearance of methionine in cheese, as well as methylpropanoic acid and methylpropanal to valine. Finally, the free amino acid glutamate can create potent flavor enhancing effects, due to its synergy with RNA breakdown products, called 5’-ribonucleotides. If combined with the appropriate concentrations of 5'-ribonucleotides, such as 5’-IMP and 5’-GMP, it is known that the threshold of detection of the umami flavor generated by glutamate is reduced by almost two orders of magnitude.

In order to obtain pronounced and precise flavor effects in all these processes, protein substrates should be hydrolyzed using a combination of an endo- and an exoprotease, in which at least one of the endo- or exoprotease, preferably both the endo - like exoprotease, they are pure and preferably selective with respect to a specific set of amino acids or preferably release the preferred amino acid or amino acids. The proteases thus preferred are characterized by a high selectivity with respect to the amino acid sequence that can be cleaved, notion which makes the enzymatic category in Aspergillus known as "mature" particularly important.

Apart from the food and feed industries, proteases are also commonly applied by the chemical, pharmaceutical, diagnostic and personal care industries.

In the personal care industry, proteases are used to create peptides that are added to a variety of products, to improve aspects such as sensation, shine or skin protection. In addition, there is a new trend to the direct topical application of the protease. Very similar to the enzymatic use in the leather industry, the main objective in this last application is to clean, remove hair and soften the skin.

In the chemical and pharmaceutical industry, proteases are being developed as valuable tools to produce expensive ingredients or intermediates. In these industries, proteases are not only used because of their hydrolytic capacity, but also because of their ability to synthesize natural or non-natural amino acid peptides. This last option is clearly demonstrated by the possibility of synthesizing aspartame from its amino acid-based building blocks using an endoprotease such as thermolysin.

Unlike the situation in the food and feed industry, the stereo- and regioselectivity of proteases are also considered important advantages, although unusual reaction conditions may be required to achieve the desired chemical transformation. Typical examples of the application of proteases in this industry include the use of endoproteases, aminopeptidases, as well as carboxypeptidases, in the production of various intermediates for drugs such as insulin, antibiotics, renin, and ACE inhibitors. In Industrial Biotransformations, A. Liese, K. Seelbach, C. Wandrey, Wiley-VCH; ISBN 3-527-30094-5 presents a review of such uses.

In view of the desired specificities, stereo- and regioselectivities, the absence of secondary activities and resistance to unusual reaction conditions such as high concentrations of solvents, the improved behavior of the protease according to the invention offers substantial advantages.

From a pharmaceutical point of view, the role of proteases is illustrated by a substantial number of references in Martindale’s, “The Extra Pharmacopoeia” (Pharmaceutical Press, London, UK). In addition, the important role of very specific proteases in regulating all types of biological processes is illustrated by the fact that many hormones become active only after the processing of a precursor molecule, mostly inactive, by such a very specific protease. Active inhibitors against certain categories of such specific proteases have been implicated in the development of all kinds of new drugs. Therefore, new and effective protease inhibitors can now be identified using the sequences provided herein.

Table 1

SEQ ID Number

Function of the encoded protein EC number

Gen

CDNA  Protein

one

58 115 Pepsin A3 EC3.4.23.1

2

59 116 Metalloprotease EC3.4.24.56

3

60 117 Acylaminoacyl peptidase  EC3.4.19.1

4

61 118 Tripeptidylaminopeptidase EC3.4.14.

62

119 serine carboxypeptidase EC3.4.16.6

6

63 120 Endoprotease serine EC3.4.21.

7

64 121 Carboxypeptidase Y EC3.4.16.5

8

65 122 aspergilopepsin II - hom EC3.4.23.19

9

66 123 Tripeptidyl peptidase EC3.4.14.9

 67

124 Tripeptidyl peptidase EC3.4.14.9

eleven

68 125 aspergilopepsin II - hom EC3.4.23.19

12

 69 126 Tripeptidyl peptidase EC3.4.14.9

13

 70 127 Metalloprotease EC3.4.24.

14

 71 128 aspergilopepsin I EC3.4.23.18

 72

129 Pepsinogen E EC3.4.23.25

16

73 130 aspergilopepsin I - hom EC3.4.23.18

17

 74 131 aspergilopepsin II EC3.4.23.19

18

 75 132 Pyro-Glu peptidase EC3.4.19.3

19

 76 133 dipeptidyl peptidase EC3.4.14.2

 77

134 aminopeptidase secr. EC3.4.11.10

twenty-one

 78 135 Alkaline D-peptidase EC3.4.16.4

22

 79 136 Carboxypeptidase EC3.4.16.1

2. 3

 80 137 Carboxypeptidase EC3.4.16.1

24

81 138 Carboxypeptidase-II EC3.4.16.1

 82

139 aspartic proteinase EC3.4.23.

26

 83 140 Tripeptidyl peptidase EC3.4.14.9

27

 84 141 Carboxypeptidase EC3.4.16.1

28

 85 142 cysteine proteinase EC3.4.22.

29

 86 143 Metallocarboxypeptidase EC3.4.17.

87

144 Subtilisin hom. EC3.4.21.62

31

 88 145 Carboxypeptidase Y EC3.4.16.5

32

89 146 Metalloprotease EC3.4.24.

33

90 147 Carboxypeptidase Y EC3.4.16.5

3. 4

 91 148 Metalloprotease EC3.4.24.

92

149 Tripeptidyl peptidase EC3.4.14.9

36

 93 150 aspartic protease EC3.4.23.24

37

 94 151 aspartic protease EC3.4.23.24

38

 95 152 Pepsin A3 EC3.4.23.1

39

 96 153 aspartic protease EC3.4.23.24

40

 97 154 aspartic protease EC3.4.23.24

41

 98 155 Kex EC3.4.21.61

42

 99 156 Serine protease EC3.4.21.

43

 100 157 Glutamyl endoprotease EC3.4.21.82

44

 101 158 Aspergilopepsin II - hom EC3.4.23.19

Four. Five

 102 159 acylaminoacyl peptidase EC3.4.19.1

46

 103 160 Tripeptidylaminopeptidase EC3.4.14.

47

 104 161 Serine Carboxypeptidase EC3.4.16.6

48

 105 162 Gly-X carboxypeptidase EC3.4.17.4

49

106 163 aspartic proteinase EC3.4.23.

fifty

 107 164 Tripeptidyl peptidase EC3.4.14.9

51

 108 165 Carboxypeptidase-I EC3.4.16.1

52

 109 166 serine carboxypeptidase EC3.4.16.6

53

 110 167 serine carboxypeptidase EC3.4.16.6

54

 111 168 aminopeptidase secr. EC3.4.11.10

55

 112 169 Prolil endopeptidase EC3.4.21.26

56

 113 170 Aspergilopepsin I - hom EC3.4.23.18

57

 114 171 Aminopeptidase EC 3.4.11.

EXAMPLES

Example 1

Evaluation of proteolytic activity and specificity

5 Protease specificity can be explored using various peptide substrates. Synthetic substrates are widely used to detect proteolytic enzymes in identification, in fermentation, during isolation, to study enzymatic activity, to determine enzymatic concentrations, to investigate specificity, and to explore interactions with inhibitors. To study the activity of the protease, preferably peptide p-nitroanilides are used, since the activity can be followed continuously, and therefore allows the

10 kinetic measure. The cleavage of peptide p-nitroanilides can be followed by measuring the increase in adsorption at 410 nm by releasing 4-nitroanilide. Paranitroanilide substrates are generally used for serine and cysteine proteases. In addition, peptide thioesters and peptide derivatives of 7-amino-p-methylcoumarin are used. Peptide thioesters are very sensitive substrates for serine and metalloproteases, which have a relatively high turnover rate, since the thioester bond is easier to cleave than the amide bond. Cleavage

Thiol esters can be followed with a thiol reagent such as 4,4-dithiopyridine (324 nm) or 5,5-dithiobis2-nitrobenzoic acid (405 nm). Usually the same increased rate of turnover is observed for the cleavage of ester bonds relative to the amide bond. The best known substrates for studying esterase activity of proteases are those derived from p-nitrophenol. The release of p-nitrophenol can be monitored at different wavelengths, depending on the pH used, for example around neutral pH a

20 wavelength of 340 nm, while, above pH 9, monitoring is performed around 405 nm. In addition, the hydrolysis of the esters can also be followed by titration using pH measuring equipment. In case of qualitative measurement of esterase activity, pH sensitive dyes can be applied.

Alternatively, the peptides can be attached to a fluorescent leaving group. Proteolysis is accompanied by an increase in fluorescence when monitored at the appropriate wavelengths. Usually 25 peptidyl 2-naphthylamides and peptidyl 4-methyl-7-coumarylamides are used. The release of, for example, 7-amino-4-methylcoumarin is

measured using an excitation wavelength of 350 nm, and an emission wavelength of 460 nm. The use of 7-amino-4-trifluoromethylcoumarin has the advantage that the leaving group is both chromogen (380 nm absorption) and fluorogen (400 nm excitation, 505 nm emission). When it is essential that an amino acid is present on both sides of the cleavable link, the introduction of a group that turns off the fluorescence may be useful. The general characteristics of such substrates are that the peptide sequence separates a fluorescent donor group from an acceptor group, which acts as the fluorescence quencher. The cleavage of the peptide bond between the quencher group and the fluorophore will lead to a substantial increase in fluorescence. Several donor-acceptor pairs have been disclosed, including o-aminobenzoic acid (Abz) as donor and 2,4-dinitrophenyl (Dnp) as acceptor, 5 - [(2'-aminoethyl) -amino] naphthalenesulfonic acid (EDANS) ) as a donor and 4 - [[4 '(dimethylamino] phenyl] azo] -benzoic acid (DABCYL) as an acceptor The Abz / EDDnp pair represents a very convenient donor-acceptor pair, since, after total hydrolysis, the Fluorescence increases by a factor of 7 to 100, and the absorption spectrum of EDDnp does not change with pH.In addition, the peptide sequence can contain up to 10 residues without loss of the quenching effect.As the size of the peptides increases connected, the position of the cleavable link can be made less specific, therefore, in addition to establishing whether proteolysis occurs, additional analysis of the products may be necessary, this can be done by analyzing and separating the produced peptides by HPLC, and determining amino acid sequence of the fragments In addition, the digested peptide composition can be directly analyzed using a combined HPLC / mass spectroscopy technique.

To study the specificity of proteases, apart from using peptides of defined sequence, synthetic peptide libraries can also be used. Peptides are synthesized by solid phase synthesis in a random or semi-random manner. For example, Meldal et al. (PNAS USA 91, 3314, 1994) disclose the preparation of a family of protease substrates starting from H-Lys (Abz) -resin, extending the resin with peptides to a length of six amino acids, and finally coupling Tyr (NO2 ) to the peptides. Each resin bead has a unique sequence, and, when treated with proteases, the most susceptible become fluorescent as the Tyr-containing peptide (NO2) is released. Sequence analysis of the peptides in the susceptible ones will give information on the protease information.

Protease activity is usually expressed in units. Generally, the international standard unit (IU) is defined as the amount of enzyme that, under defined conditions, transfers one micromol of substrate per minute. Specifically, with proteases, the UI would refer to the hydrolysis of one micromol peptide bond per minute. However, in the case of protease units, deviations from the international definition are more a rule than an exception. As with the model peptides, which are specifically cleaved in a bond, the calculation of the UIs is direct, for protein substrates, in which the protease can cleave at various positions in varying degrees, many definitions of deviating unit are used . Apart from a definition of the unit used, any hydrolysis experiment requires an adequate description of the conditions in which the units are measured. Such conditions comprise, for example, the substrate concentration, the enzyme-substrate ratio, the pH and the temperature. Typical assays to determine the specific activity of proteases comprise a protein substrate, such as, for example, denatured hemoglobin, insulin or casein. The polypeptide substrate is digested by a protease under fixed conditions for a fixed time interval. The undigested and large polypeptides are precipitated with TCA, and the TCA soluble product is determined by measuring the absorbance at 220 or 280 nm, or by assessing the soluble peptides with the folin reagent, ninhydrin, 2,4 fluro, dinitrobenzene / chloride dansyl, TNBS method, or fluorescein. Instead of marking the product after hydrolysis, polypeptide substrates that are already labeled by specific dyes or fluorophores, such as, for example, fluorescein, can also be used. In addition, standard amino acid analysis methods can be applied using standard laboratory analyzers. In order to get an idea of the size distribution of the peptides generated by a protease, gel chromatography experiments can be carried out. In addition to this, HPLC is applied using reverse phase techniques, in order to obtain a better resolution of the peptide patterns generated by the protease. The course of hydrolysis of protein substrates is usually expressed in degree of hydrolysis or DH. In the case where the pH figure is used to follow the course of hydrolysis, DH can be derived from the base consumption during hydrolysis (Enzymatic Hydrolysis of Food Protein, J. Adler-Nissen, 1986, Elsevier Apllied Science Publishers LTD). DH is related to various useful functional properties of the hydrolyzate, such as solubility, emulsifying capacity, foaming and foam stability, milkshake expansion, organoleptic quality. In addition, flavor is an important aspect of food grade hydrolysates. Bitterness can be a major problem in protein hydrolysates. The termination of the hydrolysis reaction can be accomplished by changing the pH, by thermal inactivation, denaturing agents such as SDS, acetonitrile, etc.

The polypeptides shown in Table 1 were expressed and purified at least partially according to standard procedures known in the art. They were analyzed according to at least one of the methods described above, and were found to have the activities listed in Table 1.

Example 2

Direct determination of the kcat / Km ratio for protease substrates

Synthetic substrates can be used to monitor enzyme activity during purification, to determine enzyme concentration, to determine inhibition constants, or to investigate substrate specificity. The determination of the kcat / Km ratio gives a measure of the substrate specificity. It allows comparing the specificity of different substrates by an enzyme, or comparing the hydrolysis rates with different enzymes that cleave the same substrate. This relationship has a unit of a second order velocity constant, and is then expressed as 1 / (concentration.time). Substrates that have a kcat / Km ratio in the range of 10.5-10.6 M-1.s-1 are considered to be very good substrates, that is, they have good affinity and rapid turnover. However, some substrates can be very specific with values of kcat / Km in the range of 10.4 M-1.s-1.

The ratio kcat / Km can be calculated after the determination of the individual parameters. In that case, Km and Vm can be obtained from various linear graphical representations (for example the Hanes or Cornish-Bowden method, or by a non-linear regression method. Knowing that Vm = kcat.Et (in which Et is the final active enzyme concentration), then kcat = Vm / Et. The determination of the Kcat / Km ratio by the above method can be avoided when product or substrate inhibition occurs, or when the substrate precipitates at concentration However, it is possible to obtain an exact value of the ratio kcat / Km working under first-order conditions, that is, at a substrate concentration well below the estimated km Under these conditions, the Michaelis-Menten equation : v = (Vm.S) / (Km + S) becomes:

v = (Vm.S) / Km since S «Km

or v = (Vm / Km) .S = kobs. S = -dS / dt

which is integrated as lnS = -kobs.t + lnSo, in which So is the starting substrate concentration and S is the substrate concentration at a given time. The speed is proportional to the substrate concentration. In other words, substrate hydrolysis is due to a first order process, with kobs as the first order rate constant. kobs = Vm / Km = (kcat.Et) / Km since Vm = kcat.Et.

A continuous record of substrate hydrolysis will allow the determination of kobs from the graph of lnS versus time. The kcat / Km ratio is simply inferred from kobs as long as the concentration of active enzyme is known:

kcat / Km = kobs / Et

Test method: use a starting substrate concentration well below the estimated Km, and a low enzymatic concentration to allow the hydrolysis of the substrate to be recorded. A first order curve will be obtained for product generation:

After total substrate hydrolysis, the absorbance (or fluorescence units) of the product will allow the exact determination of So, since Pt = So. kobs is determined from the slope of the graph of lnS versus time, or, as alternative, using an adjustment software (Enzfitter, SigmaPlot…).

Note: Do not forget to calculate the substrate concentration for any given time from the product concentration (S = So - P), since the graphical representation of P versus time would not provide the correct kobs (dP / dt = kobs.S is not integrated in the same way).

As an alternative, successive t1 / 2 (half time) can be measured from the product appearance curve, since in the first order process:

t1 / 2 = ln2 / kobs = 0.693 / kobs, then kobs = 0.693 / t1 / 2

Using this method allows verifying that there is a true first order decay (identical values for successive t1 / 2).

Example 3

Inactivation of protease genes in Aspergillus

The most convenient way to inactivate protease genes in the Aspergillus genome is the gene replacement technique (also called “one-stage gene breakdown”). The basic principles of this technique have been described by Rothstein RJ in Meth. Enzymol 101, p. 202, 1983. Essentially, the technique is based on homologous recombination of DNA fragments transformed with the genomic DNA of a fungal cell. Via double crossing, the gene to be inactivated is replaced (partially) by the DNA fragment with which the cell is transformed. Preferably, the transformed DNA fragment contains a selectable marker gene for Aspergillus niger. Basically, DNA manipulation and the generation of an inactivation construct are performed

using general molecular biology techniques. First, genomic DNA from the Aspergillus niger strain is isolated, which is later used for inactivation of the protease gene. The genomic DNA of A. niger can be isolated by any of the techniques described, for example by the method described by de Graaff et al. (1988) Curr. Genet 13, 315-321, and known to the person skilled in the art. This genomic DNA is used as a template for amplification of flanking regions of the protease gene, using the polymerase chain reaction (PCR; Sambrook et al. (1989) Molecular cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). By flanking regions it is meant here the non-coding regions in the 5 ’and 3 ′ direction of the protease gene that will be inactivated. Preferably, the flanking regions should each be more than 1.0 kb in length.

For priming the PCR amplification of each flanking region, two single stranded DNA oligonucleotides are used. For the 5 ’flank region, a primer is homologous to a 5 ′ DNA sequence from the beginning of the protease gene coding sequence. Preferably, the homologous region is located more than 1.0 kb in the 5 ’direction of the translation start site. The second primer is homologous to the complementary and reverse DNA sequence located immediately in the 5 ′ direction of the protease gene coding sequence.

For the 3 ’flanking region, a primer is homologous to the DNA sequence immediately in the 3 ′ direction of the protease gene coding sequence. The second primer is homologous to a complementary and inverse DNA sequence preferably located more than 1.0 kb in the 3 ′ direction of the protease gene coding sequence.

The DNA sequence included in all primers and homologous to the A. niger genome should be at least 15 nucleotides in length, preferably more than 18 nucleotides in length. More conveniently, all primers should contain a DNA sequence encoding the appropriate restriction enzyme recognition site in the 5 ′ direction of the sequence that is homologous to the A. niger genome. These additional recognition sites facilitate the cloning process.

Both primers and A. niger genomic DNA are used in a PCR reaction under conditions known to those skilled in the art. The hybridization temperature of the primers can be calculated from the part of the DNA sequence that is homologous to the A. niger genome. Both fragments containing the 5 ’flank region and the 3 ′ flank region were cloned into a vector that can be propagated in E. coli using general molecular biology techniques. A gene that can be used as a selection marker in Aspergillus niger is then cloned between the two flanking regions. More conveniently, the marker gene is under the control of a promoter that is expressed in A. niger, preferably an endogenous A. niger promoter. The orientation of the insertion of the marker gene is preferably in the same direction as the original protease gene. The final inactivation fragment contains the 5 ’flank region, a selection marker gene preferably under the control of an endogenous A. niger promoter, and the 3 ′ flank region, all in this direction and orientation. The DNA of the final construct is cloned into a vector, which can be propagated in E. coli.

The inactivation construct is digested with suitable restriction enzymes, to eliminate the sequences of the E. coli vector, and the inactivation fragment is isolated using standard techniques (Sambrook et al. (1989) Molecular cloning, a laboratory manual, 2nd edition , Cold Spring Harbor Laboratory Press, New York). Finally, Aspergillus niger is transformed with the inactivation fragment using a method described in the literature, for example by the method described by Kusters-van Someren et al. (1991) Curr. Genet 20, 293-299. Transformed cells are selected by plating the transformation mixture on agar plates that are selective for the growth of Aspergillus niger strains that express the marker gene. After purification of the Aspergillus strains transformed by plaque culture by replicas, a representative number of strains are analyzed by Southern blotting using standard methods (Sambrook et al. (1989) Molecular cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). Therefore, the mycelium genomic DNA of transformed strains is isolated and digested with suitable restriction enzymes. Restriction fragments are separated using agarose gel electrophoresis, transferred to microcellulose membranes and probed with a labeled fragment of the marker gene. Hybridization and washing are performed under restrictive conditions. Strains containing marked restriction fragments of the correct length are considered correct.

Using this method, strains of A. niger can be selected with an inactivated protease gene of choice.

Example 4

Protease isolation by ion exchange chromatography

Small amounts of the protease encoded by the nucleotide sequence as provided herein are obtained by constructing an expression plasmid containing the relevant DNA sequence, transforming a strain of A. niger with this plasmid, and growing the strain of A. niger into A suitable medium. After collecting the free broth from contaminating cells, the desired protease can be purified.

To isolate the protease as encoded by the nucleotide sequence provided in an essentially pure form, several strategies can be followed. All of these strategies have been adequately described in the relevant scientific literature (see, for example, the Protein Purification Handbook, 18-1132-29 Edition AA as published by Amersham Pharmacia Biotech, Uppsala, Sweden). Below is provided a procedure that is applicable to purify proteases from complex mixtures. It is essential that there is a suitable assay that is selective with respect to the enzymatic characteristics sought. For proteases, a synthetic chromogenic peptide substrate is typically used as described in Example 1. Such peptide substrates may be selective with respect to endoproteases, carboxypeptidases, aminopeptidases or omega peptidases. Example 11 describes the selectivity with respect to a specific tripeptidyl peptidase. By choosing the correct amino acid residues in the relevant synthetic peptide, proteases with the desired specificity can be selected.

First, it should be determined if the protease is excreted in the medium, depending on the expression system chosen to produce the protease, it can be excreted or contained in the cell. If the protease is excreted to the fermentation medium, the producing cells or fragments of these cells must be removed by centrifugation or filtration, and the resulting clear or clarified medium is the starting point for further purification. In those cases in which the sought protease is not excreted, the producing cells must be destroyed to allow purification of the protease. In such cases, the collected cell mass is best crushed with an abrasive, ground with beads, subjected to ultrasound, or subjected to a French press or a Manton-Gaulin homogenizer, and then filtered or centrifuged. In the case where the protease is hydrophobic or membrane bound, the addition of a non-ionic detergent may be necessary to solubilize the protease before the filtration step

or centrifugation.

After the clarification stage, a three-phase purification strategy can be applied to obtain the unknown proteases in an essentially pure state. In all or some of these three phases, the addition of a detergent may be necessary.

In the first or capture phase, the target protease is isolated, partially purified and concentrated. During the subsequent intermediate purification phase, most of the bulky impurities are removed, and, in the final polishing phase, trace amounts of impurities that are left of larger quantities of closely related substances are removed, and the enzyme is dissolved in The desired buffer. Depending on the nature and physical properties of the protease available, the person skilled in the art is able to optimize the three phases using slightly modified versions of the different protein binding materials, and apply these in somewhat changed conditions. However, in all cases a selective analytical test is indispensable, since it will allow continuous monitoring of the increasingly purified proteolytic activity. Analytical assays suitable for the purpose include the use of chromogenic peptide substrates as mentioned above.

In the first phase of capturing the purification, a powerful anionic ion exchange resin is preferably used to apply the medium containing the clarified and desalted enzyme. To ensure the union of the desired proteolytic activity to the resin, three or four different pH values of medium and resin are tested, under conditions of low conductivity. In these tests, the resin is always equilibrated with a buffer of the same pH and conductivity value as the medium containing the enzyme. The medium is then applied to the column under pH conditions which have been shown to allow adequate binding of the protease to the resin, that is, no desired enzymatic activity can be detected in the medium that has passed through. Subsequently, the desired enzymatic activity is eluted from the ion exchange resin using a continuous salt gradient that begins with the resin equilibration buffer and ends with this buffer to which 1 mol / liter of NaCl has been added. The eluted fractions containing the desired activity according to the assay are combined and then prepared for an additional purification step. This additional purification stage depends on the purity of the desired enzyme in the pooled fraction: if it is almost pure, an additional gel filtration stage will prove to be adequate. If it is not almost pure, chromatography is applied on a hydrophobic interaction resin, followed by a gel filtration step.

Chromatography on a hydrophobic interaction resin is carried out by first increasing the salt content of the pooled fraction obtained from the ion exchange resin to 4 moles / liter of NaCl, and eliminating any precipitate formed. If the resulting clear fraction does not contain the desired activity, this activity is obviously present in the precipitate, and can be recovered in an essentially pure state. If the resulting clear fraction still shows the desired activity in the test, then the liquid is applied as such to a phenyl resin of sepharose (Pharmacia), equilibrated in this buffer with high saline concentration with an identical pH and conductivity. If the desired enzyme activity binds to the sepharose phenyl resin, the activity is eluted with a continuous gradient of decreasing salt content, followed by a salt-free wash and, if necessary, with a chaotropic agent. As before, those fractions from the gradient that show activity in the assay are combined and finally subjected to a gel filtration step. If the desired enzymatic activity does not bind to the sepharose phenyl resin, many of the contaminants will do so, so that the desired proteolytic activity as present in the empty volume of the column requires only one additional ultrafiltration step to obtain the activity. in a more concentrated way before applying it to the column of

gel filtration The gel filtration column not only removes trace contamination, but also takes the enzyme to the buffer, which is necessary for subsequent use.

Although this method is generally applicable for the isolation and purification of proteases according to the invention, a more specific isolation technique is described in Example 4. In that example, the isolation of an Aspergillus protease using immobilized bacitracin, a peptide antibiotic known for its selective interaction with various types of proteases, is described.

Example 5

Protease isolation by affinity chromatography

An alternative method to purify small amounts of protease is by affinity chromatography. For

10 to obtain the protease in purified form, a 100 milliliter culture was grown in a well aerated shake flask. After centrifugation to remove any non-soluble matter, the supernatant was applied to a 40 milliliter bacitracin-sepharose column, equilibrated with 0.05 mol / liter of sodium acetate pH 5.0. Column bound proteases were eluted using acetate buffer supplemented with 1 mol / liter of NaCl and 10% (v / v) of isopropanol (J. Appl. Biochem., 1983 p. 420-428). The active fractions were collected, dialyzed against water

15 distilled and applied to a 20 milliliter bacitracin-sepharose column, again equilibrated with acetate buffer. As before, elution was carried out using the acetate buffer supplemented with NaCl and isopropanol. The active fractions, that is, the fractions exhibiting the activities sought, were collected, dialyzed against 5 mmol / L acetate buffer pH 5.0, and then concentrated by means of ultrafiltration with an Amicon PM-10 membrane. To obtain the protease in an essentially pure state, the

The concentrated liquid was chromatographed on a Superdex 75 column, equilibrated with the sodium acetate buffer 0.05 mol / liter pH 5.0 and supplemented with 0.5 mol / liter NaCl.

Additional experiments carried out with the enzyme purified on PAGE can confirm if the molecular weight is in line with what can be expected based on the available sequence data. Final confirmation can be obtained by carrying out a partial analysis of N-terminal amino acids.

25 Example 6

Properties of a new cysteine protease from A. niger

In this example, the Aspergillus nr 28 gene was cloned, and overexpressed in A. niger as described above. The enzyme obtained was purified according to procedures described in Example 4, and was used to destroy the trypsin inhibitory activity of soy beans under various conditions. As materials of

30 reference, papain and bromelain were used. Bromelain was obtained from Sigma, and papain was obtained from DSM Food Specialties Business Unit Beverage Ingredients, PO Box 1, 2600 MA Delft, The Netherlands.

Trypsin inhibition was measured according to the method of Kakade, M.L., Rackis, J.J., McGhee, J.E. and Puski, G. (1974):

J. Cereal Chemistry 51: 376-382. The degradation of the substrate N-benzoyl-L-arginine-p-nitroaniline to N-benzoyl-Larginine and p-nitroaniline was taken as a measure of trypsin activity. Trypsin was obtained from British Drug

35 Houses Ltd., and derived from cow pancreas containing more than 0.54 Anson units per gram of product. Kunitz inhibitor for soya beans was also obtained from Sigma.

The trypsin inhibitor was pre-incubated at a concentration of 2 mg / ml with the cysteine protease enzymes mentioned above, at pH 3, in 50 mM sodium acetate buffer, before measuring trypsin inhibition. Enzymes were added at an enzyme protein to trypsin inhibitor ratio of 1: 100 (w / w). The

40 albumin served as a negative control for enzymes. The remaining trypsin activity was measured after incubation for 3 hours at 37 ° C. Results are shown in table 2.

Table 2. Effects of various cysteine proteases on the enzymatic inactivation of the Kunitz-type trypsin inhibitor from soybeans.

one

2 3 4 5

Enzyme tested

IT activity remaining (%) IT activity that remains after pepsin treatment IT activity left after heat treatment at 75 ° C IT activity left after heat treatment at 90 ° C

Papain

 25 55 78 95

Bromelain

 30 62 86 99

A. niger

26  26 28 35

Albumin (control)

100 100 100 100

TI: Trypsin inhibitor activity

Experiments were repeated in the presence of pepsin during preincubation of cysteine proteases with the trypsin inhibitor. Pepsin was added at a final concentration of 1.3 mg / ml. The results are shown in column 3.

5 Another series of experiments was carried out to check thermal stability. The cysteine proteases were incubated at 75 and 90 ° C for 5 minutes before adding these enzymes to preincubation with trypsin inhibitors. The results are shown in columns 4 and 5.

These results clearly demonstrate the superior activity of these new cysteine proteases from Aspergillus niger with respect to the currently available cysteine proteases for the inactivation of inhibitors of

10 trypsin in animal feed.

Example 7

Exopeptidases that promote cheese maturation and cheese flavor

The aminopeptidases encoded by genes nr 20 and 54 (see Table 1) were overexpressed in A. niger according to methods described above. The purification of these enzymes was carried out according to procedures as described in Example 4. The activity of the purified enzyme samples was determined at pH 7.2 in an aqueous phosphate buffer (50 mM) containing the para derivative. -nitroanilide of a number of hydrophobic amino acids (3 mM) as a substrate. Conversion of the substrate by aminopeptidase was determined by monitoring the change in optical density at 400 nm as a result of substrate conversion, using a solution that does not contain the enzyme as a reference. Activity (A) was calculated as the change in OD per minute, and expressed as, for example, units of Phe-AP, Leu-AP or Val-AP, depending on the substrate used. Normal cheese milk was inoculated with ferment culture of the Delvo-tecTM DX 31 interval (DSM Food Specialties Delft, The Netherlands), to obtain a Gouda cheese, and the coagulation was run with a medium dose of coagulant (50 IMCU per liter of cheese milk). In addition, 25 Phe units of each exoprotease were added to two experimental cheeses, while the control did not contain any of the exoproteases. Cheese production parameters were used according to the procedure applied for semi-hard cheese for both cheeses. A difference in terms of flavor and aroma development was observed between the experimental cheeses and the control cheese to such an extent that the experimental cheeses obtained the majority of their organoleptic properties after three (3) weeks, while the cheese of control obtained a similar classification after six (6) weeks. It was shown that the level of free amino acids after three weeks was twice as high in experimental cheeses; after

30 of six weeks of maturation, the levels were again comparable. An amino acid analysis was carried out according to the Waters Picotag method (Milford MA, USA).

These data suggest that the product is ready for sale three weeks before, without decreasing the maintenance of cheese quality. The organoleptic nature of the experimental cheeses differed from that of the control to such an extent that the taste of soft cheese with a slight tendency to bitterness of the control cheese was exceeded in the cheese

Experimental in the presence of aminopeptidase. It was found that the texture of the cheeses was also somewhat softer.

Example 8

New specificity of a protease encoded by gene 55

As explained above, certain proteins can resist enzymatic hydrolysis as a result of

40 specific amino acid compositions or specific tertiary structures. In such cases, the amount of peptides that can be solubilized from protease resistant proteins can be drastically improved using proteases that have new specificities.

Beta-casein is a protein with a very limited tertiary structure, but with an extraordinary high level of proline residues. Many proteases have difficulties in cleaving proline-containing sequences, so that hydrolysis of beta-casein with available proteases normally produces a hydrolyzate that is relatively rich in large protease-resistant peptides. These latter resistant peptides can contribute to a number of undesirable properties of the hydrolyzate. For example, it is well known that these larger peptides have a relatively strong effect on allergenicity and bitterness. In addition, these peptides support subsequent degradation to free amino acids, so that, in certain processes, the appearance of these peptides

50 large, protease resistant, is synonymous with performance losses. Therefore, the availability and use of proteases that are capable of cleaving the protease-resistant parts of the proteins translate into important technical and economic benefits.

Beta-casein represents one of the main casein fractions of bovine milk. The protein has been well characterized in terms of its amino acid sequence, and is commercially available in almost pure form. As such, beta-casein offers an excellent assay substrate to study the relationship between enzymatic cleavage sites and the length of various peptides formed during enzymatic hydrolysis.

This Example demonstrates that, despite the broad spectrum cleavage nature of the subtilisin endoprotease, the addition of a very specific enzyme such as a prolyl endopeptidase, as encoded by gene 55 (see Table 1), has an important impact on the size of the beta-casein fragments formed.

Bovine milk beta-casein (lyophilized powder, essentially free of salts) was obtained with a minimum of 90% beta-casein from Sigma. B. licheniformis subtilisin (Delvolase®, 560000 DU per gram) was obtained from DSM Food Specialties (Seclin, France). The proline specific endoprotease as encoded by gene 55 was overexpressed in A. niger and purified using procedures described in Example 4.

The beta-casein powder was dissolved at a concentration of 10% (w / w) together with 0.1% (w / w) of DelvolaseTM powder in a 0.1 mol / liter phosphate buffer pH 7.0 . After a 24 hour incubation at 45 ° C in a stirred water bath, the reaction was stopped by heating the solution for 15 minutes at 90 ° C. Half of the solution (1 ml containing 100 milligrams of beta-casein) was added 100 microliters of proline specific protease, and the reaction was continued for another 24 hours at 45 ° C. After another thermal shock at 90 ° C, samples of beta-casein material treated with both DelvolaseTM and DelvolaseTM + proline-specific endoprotease were analyzed by LC / MS equipment to study the precise distributions of peptide sizes in the two samples.

LC / MS analysis

HPLC, which uses an ion trap mass spectrometer (ThermoquestTM, Breda, The Netherlands) coupled to a P4000 pump (ThermoquestTM, Breda, The Netherlands), was used to characterize the enzymatic protein hydrolysates produced by the enzymatic mixture of the invention. The formed peptides were separated using a PEPMAP C18 300A column (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands), in combination with a gradient of 0.1% formic acid in Milli Q water (Millipore, Bedford, MA, USA; Solution A) and 0.1% formic acid in acetonitrile (Solution B) for elution. The gradient started at 100% of Solution A and increased to 70% of solution B in 45 minutes, and maintained at the latter ratio for another 5 minutes. The injection volume used was 50 microliters, the flow rate was 50 microliters per minute, and the column temperature was maintained at 30 ° C. The protein concentration of the injected sample was approx. 50 micrograms / milliliter.

Detailed information on the individual peptides was obtained using the "scan-dependent" MS / MS algorithm, which is a characteristic algorithm for an ion trap mass spectrometer. The full scan analysis was followed by a zoom scan analysis, to determine the state of the most intense ion charge in the full scan mass range. Subsequent MS / MS analysis of this latter ion resulted in partial peptide sequence information, which could be used for searching databases using the SEQUEST application of Xcalibur Bioworks (ThermoquestTM, Breda, The Netherlands). The data banks used were extracted from the OWL.fasta data bank, available at the NCBI (National Center for Biotechnology informatics), which contains the proteins of interest for the application used.

Using this technique as an identification method, only peptides with a mass ranging from approx. 400 to 2000 Daltons were considered suitable for subsequent analysis by MS sequencing.

To adjust the optimum sensitivity in the MS mode, angiotensin (M = 1295.6) was used, and for optimal fragmentation in the MS / MS mode, carrying out a constant infusion of 60 Pg / ml, mainly resulting Double and triple charged species in the MS mode, and an optimal collision energy of about 35% in the MS / MS mode.

In the sample digested with Delvolase alone, the LC / MS / MS analysis identified 40 peptides covering various parts of the beta-casein molecule. Together, these peptides accounted for 79% of the total beta-casein sequence. Retention times different from the peptides in column C18 could be traced back to peptide lengths ranging from 2 to 23 amino acid residues. Together, <15% of the peptides found were smaller than 6 amino acids. The sample digested with DelvolaseTM and the proline specific protease also generated a large number of identifiable peptides from beta-casein. Together, these peptides covered> 50% of the total beta-casein protein sequence. In this sample, the size distribution of the peptides was remarkably homogeneous, since the peptides ranged in length only between 2 and 6 residues. The results show that, in the hydrolyzate obtained with the proline specific protease, it contains a large fraction of dipeptides, tripeptides, and peptides with up to 6 AA, which show the beneficial effect other than co-incubation with an endoprotease that has an unusual specificity. . It is also clear from these experiments that the endoprotease according to gene 55 encodes an endoprotease that cleaves the peptide chain in the carboxy terminus of the proline moiety.

Example 9

The selective release of specific amino acids to promote flavor formation.

Free amino acids such as leucine and phenylalanine have not only been implicated in Maillard reactions, but also as precursors for desirable aromas in various food fermentations. To promote the formation of such aromas in food fermentations or during the heating, roasting or cooking phase of the food, it would be advantageous to incorporate in these products a protein hydrolyzate containing relatively high levels of these specific amino acids in a free form. This Example describes the production of yeast extracts selectively enriched in leucine and phenylalanine. This enrichment is obtained by combining an endoprotease with a cleavage preference for a selected set of amino acid residues with an exoprotease that favors the release of a similar set of amino acid residues. The preference of the endoprotease should match the preference of the exoprotease used. For example, it has been established that aminopeptidases encoded by genes 20 and 54 (see Table 1) have a definite preference for releasing leucine and phenylalanine residues that match the thermolysin cleavage preferences. Carboxypeptidases encoded by genes 23 and 24 have a preference for releasing arginine and lysine residues, which

15 matches the trypsin cleavage preferences. The carboxypeptidase encoded by gene 5 has a very unusual preference for releasing glycine, which could be combined with certain endoproteases present in papain. The carboxypeptidase encoded by gene 51 is capable of eliminating glutamate residues, which coincides with the specific glutamate protease encoded by gene 43.

It is known that the thermolysin endoprotease (commercially available as Thermoase) C180 from Daiwa Kasei KK (Osaka, Japan) cleaves peptide bonds on the aminoterminal side of bulky hydrophobic amino acids such as Leu and Phe. To release the amino acids thus exposed from the newly formed peptides, the aminopeptidases encoded by genes nr 20 and 54 were used (see Table 1). These genes were overexpressed in A. niger according to methods described above, and the purification of these enzymes was carried out according to procedures as described in Example 4.

25 To release as much leucine and phenylalanine as possible without the concomitant release of unwanted amino acids with this combination of enzymes, it is clear that the conditions used during enzymatic hydrolysis should be carefully selected. In addition, the endogenous (and probably specific) proteases of the yeasts themselves must be inactivated. After a number of test incubations, a protocol was obtained that leads to a surprisingly selective and effective release of leucine and phenylalanine from yeast proteins using these two new enzymes.

To inactivate the endogenous yeast proteases, the yeast suspension was maintained for 5 minutes at 95 degrees C. Then, the suspension was rapidly cooled to the required temperature, and the pH was adjusted to 7.0 using 4N NaOH. Yeast, thermolysin and one of the aminopeptidases were all incubated simultaneously under the following conditions. After thermal shock, the pH of the 2000 milliliters of yeast suspension is

35 adjusted to 7.0, after which 680 milligrams of Thermoase were added and, after stirring, the purified aminopeptidase. The mixture was incubated with stirring at 50 degrees C for 3 hours, and centrifuged. To stop all enzymatic activities, the pH of the supernatant was adjusted to 4 and underwent another 45-minute heat treatment at 95 degrees C. After another centrifugation, a sample for amino acid analysis was obtained from the supernatant. The precipitated or undissolved matter was removed by centrifugation for 15 minutes at 3500 rpm in a Hereaus Megafuge 2.0 R centrifuge. The supernatant was removed and kept frozen at -20 ° C. Samples of the supernatant were analyzed for amino acid content according to the Waters Picotag method (Milford, MA, USA), immediately after thawing.

In the amino acid analysis, Trp and Cys values were omitted, and Asp and Asn values were added as one value. According to the data obtained, in the resulting hydrolyzate the ratio between alanine and leucine (21.3: 11.7) was

45 1: 0.5. Commercially available yeast hydrolysates typically have alanine to leucine ratios of 1: 0.3.

In a second experiment, a yeast extract was prepared that was enriched in free glutamate. To achieve this, an endoprotease was used that exhibits a preference for cleavage at the C-terminal end of glutamate residues (encoded by the nr 43 gene in Table 1), and a carboxypeptidase (encoded by the nr 51 gene in Table 1) capable of eliminating these glutamate residues thus exposed. The endoprotease encoded by the nr 43 gene and the carboxypeptidase encoded by the gene 51 (see Table 1) were overexpressed in A. niger according to methods described above. The purification of these enzymes was carried out according to procedures as described in Example 4.

The essential role of free glutamate in a number of aroma-forming processes is well documented, and

55 MSG, the sodium salt of glutamic acid, is recognized as the single most important flavor enhancer component.

In this Example, the pH of 200 ml of yeast suspension subjected to thermal shock was adjusted to 8.0, then the purified enzyme product encoded by gene 43 was added, and the mixture was incubated for 4 hours at 50 degrees

C. Then, the pH was reduced to 5.0, and the suspension was centrifuged. The purified gene product of gene 51 was added to 100 milliliters of supernatant. Incubation with this carboxypeptidase took place for 30 minutes at 50 degrees C, with continuous pH adjustments. After stopping the enzymatic incubation by a 5-minute heat treatment at 95 degrees C, the material was centrifuged again (see above), and a

5 sample for amino acid analysis.

According to the amino acid data obtained (see above), in the resulting hydrolyzate the ratio between alanine and glutamate (30.0: 48.7) was 1: 1.6. Commercially available yeast hydrolysates typically have alanine to glutamate ratios of 1: 1.

Example 10

10 Evaluation of the flavor of yeast hydrolysates enriched in specific amino acids.

To demonstrate that a protein hydrolyzate enriched in specific amino acids according to the invention can generate specific aromas, a number of experiments with the yeast hydrolysates described in the previous Example were carried out. To that end, larger portions of these hydrolysates were prepared and lyophilized. The behavior of the resulting powders was compared with the behavior of a yeast extract

15 commercially available (Gistex LS, available from DSM Food Specialties, Delft, The Netherlands) in a standardized mixture under various reaction conditions. The standardized mixture consisted of one of the hydrolysates, base mix and water.

The base mixture contained 22 grams of Maxarome Plus Powder (a specialized yeast extract, with a high content of natural nucleotides, also available from DSM Food Specialties), 29.2 grams of glucose, 9

20 grams of REFEL-F fat (hydrogenated soybean oil, obtainable from Barentz, Hoofddorp, The Netherlands) and 0.2 grams of calcium stearoyl lactylate (emulsifier, obtainable from Abitec, Northampton, UK), mixed thoroughly in a mortar .

All standardized mixtures contained 5 grams of yeast hydrolyzate powder (i.e., leucine or glutamate enriched material, or commercial yeast extract), 3 grams of the base mixture, and 3 grams

25 of water After thorough mixing, these three suspensions were subjected to different heating regimens, that is, 65 minutes at 90-95 degrees C in a reaction vial (liquid reaction), or dried at 20 millibars at 120 degrees C in a vacuum oven (roasting reaction under vacuum), or they were heated in an open reaction vial at 120 degrees C for 10 minutes after dissipating all the water (roasting reaction).

After the heat treatment, the three products assumed colors ranging from dark brown to

30 almost black. In the case of the roasting reaction under vacuum, only the upper layers of light color were used. The evaluation of the taste of the heated products was carried out by grinding the darkened cakes to fine powders, and dissolving these powders to a concentration of 2% (w / w) in water containing 0.6% (w / w) NaCl . The flavor panel observations are specified in Table 3.

Table 3

 Reference

Leucine Glutamate

Liquid

Broth, lightly toasted Cold tea, slightly flowery, similar to yeast More broth, meaty, similar to yeast

Vacuum roast

Burned french fries Astringent, beans, similar to yeast Burned, broth, similar to yeast

Roast

Dark toast, broth, umami Less roast, flowery, umami Toast, more broth, more umami

35 Example 11

Non-allergenic whey protein hydrolysates formed with tripeptidyl peptidases.

Dipeptidyl peptidases encoded by genes 19 and 55, as well as tripeptidyl peptidases encoded by genes 4, 9, 10, 12, 26, 35, 46, and 50 (see Table 1), can be overproduced as described, and you can

Purify according to the methods provided in Example 4. After purification, the optimum pH and temperature stability of each individual enzyme can be established by any of the methods available and known to the skilled person. In addition, the specificity of each individual enzyme can be determined using the methods explained in Example 1. The following experiment illustrates the selectivity shown by tripeptidyl peptidases.

The enzyme encoded by gene 12 was overproduced in an Aspergillus niger host cell, and purified by procedures described in Example 4. The enzyme thus obtained was incubated at pH 5 and at 50 degrees C with

different synthetic chromogenic substrates, that is, Ala-Ala-Phe-pNA and Ala-Phe-pNA (both from Bachem, Switzerland). Incubation with the Ala-Ala-Phe-pNA substrate led to a significant increase in absorbance at 410 nm, while incubation with Ala-Phe-pNA did not. This observation clearly demonstrates that tripeptidyl peptidases cleave tripeptides and have no aminopeptidase activity that can lead to an undesirable increase in free amino acids.

In addition, the enzyme encoded by gene 12 shows favorable enzyme stability characteristics, as shown in the following experiment. Four enzyme samples were incubated at pH 5 for one hour at 0, 40, 50 and 60 degrees C, respectively. Then, each enzyme sample was incubated with the above-mentioned Ala-Ala-Phe-pNA substrate in a citrate buffer at pH 5, and the residual activity in each individual sample was determined by measuring the absorbance increase at 410 nm. The sample at 0 degrees C showed a 100% activity, the sample at 40 degrees showed a residual activity of 96%, the sample at 50 degrees showed a residual activity of 92%, and the sample at 60 degrees showed a residual activity of 88%

In a typical process aimed at producing a hydrolyzate with a high proportion of tripeptides, whey protein (WPC 75) can be dissolved / suspended in a concentration of 100 grams of protein / liter, in an aqueous medium having a pH of 8, 5. The first enzymatic incubation is performed with the broad spectrum subtilisin endoprotease (Delvolase®, 560000 DU per gram, of DSM). After a pre-digestion of the serum with this enzyme in a concentration of 0.5% of enzyme concentrate per gram of protein for 2 hours at 60 ° C, the mixture was heat treated to inactivate the endoprotease used. Then, the temperature was adjusted to 50 degrees C and tripeptidyl peptidase was added, and the entire mixture was incubated until the desired level of tripeptides was reached. The additional processing steps of the hydrolyzate thus obtained depend on the specific application, but may incorporate microfiltration or centrifugation followed by evaporation and spray drying.

SEQUENCE LIST

<110> DSM NV

<120> New genes encoding new proteolytic enzymes

<130> 20095WO

<160> 171

<170> PatentIn version 3.1

<210> 1

<211> 2520

<212>. DNA

<213> Aspergillus niger

<400> 1

Claims (18)

one.

 An isolated polynucleotide hybridizable under conditions very restrictive to polynucleotide SEQ ID NO: 10 or to the sequence SEQ ID NO: 67, wherein said polynucleotide encodes a protein that has protease activity.

2.

 An isolated polynucleotide according to claim 1, obtainable from a filamentous fungus.

3.

 An isolated polynucleotide according to claim 2, obtainable from A. niger.

Four.

 An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence according to SEQ ID NO: 124 or an amino acid sequence that is at least 80% homologous to the amino acid sequence according to SEQ ID NO: 124, and having protease activity.

5.

 An isolated polynucleotide comprising a nucleotide sequence according to SEQ ID NO: 10 or the sequence SEQ ID NO: 67.

6.

 An isolated polynucleotide according to the sequence SEQ ID NO: 10 or the sequence SEQ ID NO: 67.

7.

 A vector comprising a polynucleotide sequence according to claims 1 to 6.

8.

 A vector according to claim 7, wherein said polynucleotide sequence according to claims 1 to 6 is operably linked with regulatory sequences suitable for the expression of said polynucleotide sequence in a suitable host cell.

9.

 A vector according to claim 8, wherein said suitable host cell is a filamentous fungus.

10.

 A method of manufacturing a polynucleotide according to claims 1 to 6 or a vector according to claims 7 to 9, comprising the steps of culturing a host cell transformed with said polynucleotide or said vector, and isolating said polynucleotide or said vector of said host cell .

eleven.

 An isolated polypeptide according to the sequence SEQ ID NO: 124 or an amino acid sequence that is at least 80% homologous to the amino acid sequence according to SEQ ID NO: 124, and that has protease activity.

12.

 An isolated polypeptide according to claim 11, obtainable from Aspergillus niger.

13.

An isolated polypeptide obtainable by expressing a polynucleotide according to claims 1 to 6 or a vector according to claims 7 to 9 in an appropriate host cell, preferably Aspergillus niger.

14.

 A method for manufacturing a polypeptide according to claims 11 to 13, comprising the steps of transforming a suitable host cell with an isolated polynucleotide according to claims 1 to 6 or a vector according to claims 7 to 9, culturing said cell under conditions that allow the expression of said polynucleotide, and optionally purify the encoded polypeptide of said cell or culture medium.

fifteen.

 A recombinant host cell comprising a polynucleotide according to claims 1 to 6 or a vector according to claims 7 to 9.

16.

 A recombinant host cell expressing a polypeptide according to claims 11 to 13.

17.

 A recombinant host cell according to claims 15 or 16, wherein said host cell is derived from an Aspergillus species, preferably A. niger.

18.

 Fusion protein comprising a polypeptide sequence according to claims 11 to 13.