RU2423525C2 - New genes coding new proteolytic enzymes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: recovered polynucleotide coding aminopeptidase containing a nucleotide sequence presented in the description is presented. Also, polynucleotide hybridised with said polynucleotide in the high stiffness environment is presented. An expression vector containing said polynucleotide, and an applicable host cell are described. Polypeptide related to the sequence presented in the description and being aminopeptidase is presented. A method of producing said polypeptide involving the stages of applicable host cell transformation by said polynucleotide or vector, cell cultivations in the medium adequate for said polynucleotide expression, and optional polypeptide purification from said cell or culture medium is offered. Besides, there has been described diagnostic technique for Aspergillus-infection in an organism involving the stages: a) recovery of a biological sample from said organism which is supposed to be Aspergillus infected, b) recovery of nucleic acid from said sample, c) determination whether said recovered nucleic acid contains polynucleotides hybridised with polynucleotide recovered from Aspergillus niger.

EFFECT: invention allows diagnosing Aspergillus-infection in the organism.

13 cl, 3 tbl, 11 ex

Description

FIELD OF THE INVENTION

The present invention relates to new identified polynucleotide sequences, including genes encoding new proteases isolated from Aspergillus niger . The present invention relates to a full-sized nucleotide sequence of these new genes, to cDNA sequences including these full-length coding sequences of new proteases, as well as to amino acid sequences of full-sized functional proteins, as well as their fragments and variants. The present invention also relates to methods for using these enzymes in industry and to methods for diagnosing fungal infections. The present invention also relates to cells transformed with the polynucleotide of the present invention, and to cells in which the protease of the present invention is genetically modified in order to enhance or reduce its activity and / or expression level.

State of the art

Proteolytic enzymes

Proteins can be considered as heteropolymers, which consist of amino acid structural elements connected by a peptide bond. The repeating element in proteins is the central alpha-carbon atom with an amino group and a carboxyl group. The so-called amino acid side chain, with the exception of glycine, replaces one of the two remaining hydrogen atoms of alpha carbon. The amino acid side chain makes the central alpha carbon atom asymmetric. As a part of proteins, mainly L-enantiomers of amino acids are found. The various types of polymerized amino acids are defined in the following terms. “Peptides” are short chains of amino acid residues with a specific sequence. Although, in fact, it is impossible to specify exactly the maximum number of residues, this term usually refers to a chain that possesses properties determined mainly by its amino acid composition and which does not have a fixed three-dimensional conformation. The term “polypeptide” is usually used to mean longer chains having a generally defined sequence and length, which, in principle, is sufficient to form a three-dimensional structure. The term “protein” usually means polypeptides found in nature and having a certain three-dimensional structure. If the main function of proteins is the catalysis of a chemical reaction, then usually such proteins are called enzymes. Proteases are enzymes that catalyze the hydrolysis of a peptide bond in (poly) peptides and in proteins.

Under physiological conditions, proteases catalyze the hydrolysis of the peptide bond. The International Union for Biochemistry and Molecular Biology (1984) recommends the use of the term “peptidase” for a subgroup of hydrolazpeptide bonds (subclass E.C.3.4). The terms “protease” and “peptidohydrolase” are synonymous with the term “peptidase” and can also be used in the present description. Proteases include two classes of enzymes: endopeptidases and exopeptidases, which cleave peptide bonds at specific points in the protein and sequentially remove amino acids from either the N- or C-terminus, respectively. The term “proteinase” is used synonymously with the term “endopeptidase”. The peptide bond may be present in the context of di-, tri-, tetrapeptides, peptides, polypeptides or proteins. Basically, the amino acid compositions of natural peptides and polypeptides include 20 different amino acids that have an L configuration (with the exception of glycine, which lacks a chiral center). However, the proteolytic activity of proteases is not limited to peptides that contain only 20 natural amino acids. Peptide bonds between so-called unnatural amino acids and peptide bonds between modified amino acids or amino acid analogs can also be cleaved. Some proteases recognize the D-enantiomers of amino acids at certain positions. In general, the significant stereoselectivity of proteases is a very important factor that allows their use in chemical separation methods. Many proteases have other activities of interest, such as esterase activity, thiolesterase activity, and (de) amidase activity. These auxiliary activities are usually not limited to amino acids only and can be successfully used in biological transformation reactions in the field of fine chemical synthesis.

A special interest in the proteases of filamentous fungi, eukaryotic microorganisms, is caused by a number of reasons. Obviously, the main process of hydrolytic cleavage of peptide bonds in proteins is expensive, and if it is not properly regulated, it can have a negative effect on the microorganism. The desired limitation of proteolytic action can be achieved by the specificity of proteinases; compartmentalization of proteases and substrates in the cell; modification of substrates recognized by the corresponding proteases, regulation by activation of zymogens and due to the presence or absence of specific inhibitors, as well as by regulation of protease gene expression. In fungi, proteases also participate in other fundamental cellular processes, including intracellular protein metabolism, processing, translocation, sporulation, development and differentiation. Indeed, Aspergillus nidulans and Neurospora crassa were used as model microorganisms to analyze the molecular basis of a number of physiological and developmental processes. Their genetics provides direct access to biochemical and genetic research under certain nutritional and cultivation conditions. In addition, a large group of fungi pathogenic for humans, cattle, and crops was isolated, and it was suggested that their pathogenicity (penetration into the host organism, counteraction to the host defense mechanisms and / or infection during nutrition) plays a certain role proteolysis. Proteases are also often used in laboratory, clinical, and industrial processes; while both microbial and non-microbial proteases are widely used in the food industry (in baking, brewing, in the production of cheese and in the tendering of meat), in the leather industry and in the industrial production of biological detergents (Aunstrup, 1980). Recently, in connection with the industrial use of certain filamentous fungi, in particular Aspergillus , as hosts for the production of homologous and heterologous proteins, interest in fungal proteases has again increased (van Brunt, 1986ab). Proteases often impede heterologous expression and homologous overexpression of proteins in fungi. In particular, heterologous expression is adversely affected by proteolytic cleavage of expression products by homologous proteases. The indicated commercial interest was an incentive for a detailed study of the proteolytic spectrum of proteases and the construction of protease-deficient strains, which led to a deeper knowledge of the expression of proteases and their regulation in these microorganisms. Therefore, at present, there is a need to identify and eliminate new proteases in filamentous fungi.

Microorganisms, such as, for example, fungi, are especially suitable for large-scale production of proteins, and especially if such proteins are secreted into the medium. In these production processes, proteolytic enzymes play a role. On the one hand, specific proteolytic enzymes are mainly necessary for the “correct” processing of the target protein and the favorable implementation of the metabolic functions of the producer host. On the other hand, proteolytic cleavage can significantly reduce the yield of secreted proteins. Incorrect folding along the secretion pathway can lead to cleavage by intracellular proteases. In this regard, certain difficulties may arise in the production of heterologous proteins. The exact mechanisms of proteolytic processes that are responsible for the breakdown of proteins that deviate from the secretory pathway in fungi are not yet entirely clear. In eukaryotes, cellular protein degradation is achieved using the proteasome and is usually carried out by labeling the degradable proteins with ubiquitin. In fungi, proteosomal and vacuolar proteases are also likely candidates for proteolytic cleavage of poorly laid secretory proteins. Proteolytic degradation is probably cytoplasmic, but the existence of proteases in the endoplasmic reticulum cannot be ruled out. From the point of view of improving the host producer strain, the proteolytic system can serve as an interesting goal for improving the genetic construction and production of strains. Additional copies of protease genes, overexpression of certain proteases, modification of transcriptional regulation, as well as knock-out procedures for deletion of protease genes will help to better understand the functions of this protease. The deletion of genes encoding a protease can serve as a valuable strategy for the modification of the host strain, is carried out in order to increase the yield of production of homologous as well as heterologous proteins.

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

In addition to hydrolytic cleavage of bonds, proteases can also participate in the formation of bonds. In this aspect, the term “bonds” includes not only peptide and amide bonds, but also ester bonds. The ability of proteases to catalyze the cleavage or formation of a specific bond, primarily depends on the thermodynamics of the reaction. An enzyme such as protease does not affect the balance of the reaction. The equilibrium of the reaction depends on the specific conditions under which this reaction is carried out. Under physiological conditions, the thermodynamics of these reactions may be favorable for the hydrolysis of the peptide due to the highly thermodynamically stable structure of the zwitterionic product. Based on the physicochemical laws that affect the equilibrium of the reaction, either by changing the concentration or nature of the reagents and products, or by selecting the kinetic parameters of the enzymatic reaction, proteases can be used to synthesize peptide bonds. The addition of organic solvents miscible with water leads to a decrease in the degree of ionization of the carboxyl component and, thereby, to an increase in the concentration of the substrate available for the reaction. To increase the yield, biphasic systems, aqueous mimetics, inverted micelles, anhydrous media, or modified amino and carboxyl groups that stimulate the precipitation of products are often used. If proteases with suitable properties were available, the use of such proteases for synthesis would provide significant advantages. Since proteases are stereoselective as well as regioselective, sensitive groups on reagents usually do not need protection and these reagents do not have to be optically pure. If the conditions of enzymatic synthesis are mild, then racemization and decomposition of labile reagents or products can be avoided. Furthermore, in addition to the bonds between amino acids, suitably selected proteases can also bind other compounds having a primary amino group, a thiol group or a carboxyl group. In addition, esters, thiol esters and amides can be synthesized by some proteases. It was shown that proteases have regioselectivity in the acylation of mono-, di- and trisaccharides, nucleosides and riboflavin. Problems associated with stability, which sometimes arise under severe reaction conditions, can be solved by the proper selection of reagents. Encapsulation and immobilization not only stabilize the enzymes, but also make it easy to separate and isolate them from the reaction medium. Density of crosslinking, aldehyde treatment or coating of the surface with certain polymers such as dextrans, polyethylene glycol and polyimines can significantly increase the life of the biocatalyst.

The natural role of proteases

Traditionally, proteases were considered proteolytic enzymes that are able to break down proteins into small peptides and / or amino acids and whose role is to hydrolyze food proteins or to participate in the metabolism of cellular proteins. In addition, it has been shown that proteases also play key roles in a wide range of cellular processes in accordance with the mechanisms of selective modification by limited proteolysis, and therefore they have important regulatory functions (Holzer & Tschensche 1979; Holzer & Heinrich, 1980). It is assumed that the specificity of the proteinase is closely related to its physiological function and to the type of its expression. As for the function of a particular protease, its localization in most cases is very important; for example, a series of vacuolar and periplasmic proteases is involved in protein degradation, while many membrane-associated proteases play an important role in protein processing (Suarez Rendueles & Wolf, 1988). The different roles of proteases in many cellular processes can be divided into four main functions of proteases: 1) protein degradation, 2) post-translational processing and (in) activation of specific proteins, 3) morphogenesis and 4) pathogenesis.

The obvious role of proteases in organisms that use protein as a nutrient source is the hydrolysis of nutrients. In fungi, this should lead to their degradation outside the cell under the influence of extracellular proteases with broad specificity. Protein degradation is also important for the rapid metabolism of cellular proteins and allows you to remove unwanted proteins from the cell and adapt their protein complement to changing physiological conditions. Typically, proteases with sufficiently broad specificity should be very well regulated to protect cellular proteins that are not target proteins from accidental degradation.

In contrast to hydrolysis, the synthesis of polypeptides occurs in vivo through an ATP-driven process on the ribosome. Ultimately, the sequence in which amino acids are linked is determined by the information provided by the genome. This process is known as transcription. The primary translation products are usually longer than the final functional products, and after transcription, the processing of such precursor proteins by proteases is usually necessary. Proteases play a key role in the maturation of such precursor proteins and in the formation of the final functional protein. In contrast to the highly regulated “truncation” and “reconstruction” of proteins, proteases can have a highly destructive effect and can lead to the complete decomposition of polypeptides into peptides and amino acids. To prevent such free proteolytic activity, proteases must undergo intensive regulation before it is required. Many proteases are synthesized as larger precursors, known as zymogens, which, if necessary, become activated. It should be noted that such activation always occurs through proteolysis. In addition to direct participation in processing, the selective activation and inactivation of individual proteins are well-known processes catalyzed by specific proteases.

Obviously, the selectivity of limited proteolysis is almost always observed in the interaction of proteinase-substrate. This specificity can be provided by a proteolytic enzyme that recognizes only specific target amino acid sequences. On the other hand, it can also be due to the selective effect on the “processing site” under certain conditions, such as pH, ionic strength, or secondary modifications, which allows non-specific proteases, in one way or another, to catalyze a highly specific event. An example of this type of event is the activation of vacuolar zymogens with limited proteolysis.

Morphogenesis or differentiation can be defined as a regulated series of events leading to the transition from one state in the body to another state. Although, in many cases, a direct relationship between proteases and morphological effects has not been established, however, there is evidence to suggest a significant role for proteases in the morphogenesis of fungi, that is, in addition to the observed intense protein metabolism in the process of differentiation, sporulation and germination of spores, proteases , obviously, are directly involved in normal processes, such as branching of the tips of hyphae and the formation of septa (Deshpande, 1992).

Species of Aspergillus , in particular A. fumigatus and A. flavus , are believed to be the etiological factors of a number of diseases in humans and animals called aspergillosis (Bodey & Vartivarian, 1989). Again, it has been suggested that proteases play a role in the virulence of A. fumigatus and A. flavus , similar to what has been observed in many studies on the relationship of secreted proteases and virulence of bacteria. Indeed, most human infections caused by Aspergillus spp . Are characterized by intense degradation of the lung parenchyma, which consists mainly of collagen and elastin (Campbell et al., 1994). The studies were aimed at studying the alleged role of secreted proteases in the virulence of A. fumigatus and A. flavus , which are the main human pathogens and are known to have elastinolytic and collagenic activity (Kolattukudy et al., 1993). These elastinolytic activities have been shown to correlate in vitro with the infectivity of mice (Kothary et al., 1984). It is known that two secreted proteases, alkaline serine protease (ALP) and neutral metalloprotease (MEP), are produced by A. fumigatus and A. flavus . Both genes encoding these proteases from A. fumigatus were isolated, characterized, and disrupted (Reicherd et al., 1990; Tang et al., 1992, 1993; Jaton-Ogay et al., 1994). However, it was shown that double alp - mep mutants do not differ in their pathogenicity from wild-type strains. Therefore, it was concluded that secreted A. fumigatus proteases identified in vitro are not major factors for invasion of tissue (Jaton Ogay et al., 1994). Although A. fumigatus is responsible for the formation of only a small fraction of airborne mold spores, this fungus is most often secreted from the lungs and sputum (Schmitt et al., 1991). Another explanation for the virulence of fungi can be that the conditions in the bronchi (temperature and culture medium) are more favorable for the parasitic growth of A. fumigatus . Therefore, if the host’s defense against pathogens is weakened by the administration of immunosuppressants or diseases such as AIDS, then invasive aspergillosis can be caused by environmental factors.

Four main classes of proteases are known, which are determined by the main functional groups in their active site, namely, “serine”, “thiol” or “cysteine”, “asparagine” or “carboxyl” proteases and “metalloproteases”. A detailed description of these large and small classes of proteases and unclassified proteases can be found in Methods in Enzymology, part 244 and 248 (A.J. Barrett ed., 1994 and 1995).

Specificity of proteases

In addition to the catalytic mechanism of action of proteases, another important aspect of proteolytic enzymes is the specificity of proteases. The specificity of the protease indicates specific protease substrates, which, in all probability, this protease hydrolyzes. Twenty natural amino acids provide great opportunities for the construction of peptides. For example, using twenty amino acids, approximately 400 dipeptides and 800 different tripeptides and the like can be constructed. With the use of longer peptides, the number of possible constructs becomes almost unlimited. Some proteases hydrolyze only specific sequences in a highly specific position. The interaction of the protease with the peptide substrate may include from one to ten amino acid residues of the peptide substrate. In the case of large protein substrates, even a larger number of substrate residues can interact with proteases. However, this probably leads to less specific interactions with protease residues located outside the binding pocket of the active center. Basically, specific recognition is limited to a linear peptide that binds to the active site of the protease.

A nomenclature for describing the interaction of a substrate with a protease was developed in 1967 by Schechter & Berger (Biochem. Biophys. Res. Com. 1967, 27, 157-162) and is currently widely used in the literature. It is believed that in this system the amino acid residues of the polypeptide substrate bind to the so-called subsites in the active site. For convenience, these subsites on the protease were designated S (for subsites), and the corresponding amino acid residues were designated P (for peptide). Amino acid residues from the N-terminal side of the cleavage bond were designated P3, P2, P1, and residues from the C-terminal side were designated P1 ', P2', P3 '. Residues P1 or P1 ′ are amino acid residues located near the cleavable bond. Residues of the substrate located near the cleavage site can then be numbered to P8. The corresponding subsites on this protease, which complement the residues that bind to the substrate, were numbered S3, S2, S1, S1 ', S2', S3 ', etc. The preference of subsites at the peptide binding site is determined by the preference of the protease in the cleavage of specific specific amino acid sequences at a particular site. The amino acid sequence of the substrate should be consistent with the preference shown by the sub sites. The specificity with respect to a particular substrate clearly depends on the affinity of binding to the substrate and on the rate at which this cleavable bond hydrolyzes. Therefore, the specificity of the protease to a specific substrate is usually determined by the ratio kcat / Km, which is better known under the term “specificity constant”. In this specificity constant, kcat means metabolic rate, and Km means dissociation constant.

In addition to amino acid residues involved in catalysis and binding, proteases contain many other basic amino acid residues. Some residues are important for folding, some residues support the entire three-dimensional structure of the protease, some residues can be involved in the regulation of proteolytic activity, and some residues can target the protease to a specific site. Many proteases contain, outside the active site, one or more metal ion binding sites. These metal ions, in most cases, play a role in stabilizing the structure. In addition, secreted eukaryotic microbial proteases can be highly glycosylated. In this case, both N- and O-linked glycosylation can occur. Glycosylation can promote protein folding, increase solubility, and also prevent aggregation and thus stabilize mature protein. In addition, the degree of glycosylation can affect the secretion, as well as the binding to water under the action of protein.

Regulation of proteolytic activity

In order to avoid undesirable proteolytic cleavage, a large number of proteases undergo intensive regulation of proteolytic activity. To a certain extent, this regulation occurs at the transcriptional level. For example, in fungi, transcription of secreted protease genes is obviously sensitive to external carbon and nitrogen sources, while genes encoding intracellular proteases are not sensitive to such sources. Fungi are sensitive to extracellular pH, and some genes are regulated by pH. In this process, transcriptional regulatory proteins play a crucial role. The proteolytic processing of such regulatory proteins often serves as a “switch” that either “turns on” or “turns off” regulatory proteins.

Proteases undergo intramolecular as well as intermolecular regulation. This means that some amino acids in the molecule of the proteolytic enzyme play a major role in such regulation. Proteases are usually synthesized as larger precursors known as zymogens, which are catalytically inactive. Typically, an extension of the peptide chain that makes the precursor protease inactive is localized at the amino terminus of the protease. This precursor is better known as pro-protein. Since many proteases processed in this way are secreted from the cells, they also contain a signal sequence (pre-sequence) so that the full-sized precursor is synthesized as a pre-pro-protein. Besides the fact that such a protease is inactive, this pro-peptide often plays an important role in mediating product folding. Examples of proteases are serine proteases (alpha-lytic protease, subtilisin, aqualysine, prohormone convertase), thiol proteases (cathepsin L and cruzian), aspartic protease (proteinase A and cathepsin D), and metalloproteases. In addition, the pro-peptide can play a role in cell transport, either alone or in combination with signal peptides. It can facilitate interaction with cellular chaperones, or it can facilitate transport through the membrane. The size of the extension region in the pre-pro-protein precursor can vary significantly, from a short peptide fragment to a polypeptide, and this region can exist as an autonomous stacking unit. In particular, as is observed in most cases, these larger extension sites are strong protease inhibitors even after cleavage from the protease. It has been observed that even after cleavage, such pro-peptides can contribute to the corresponding folding of the protease. It can be assumed that such pro-peptides act as molecular chaperones and the separate or combined co-expression of such pro-peptides may be preferable for protease production.

There is a significant difference in the levels of regulation of proteases that are secreted into the environment and proteases that remain inside the cell. Usually, after activation, the proteases secreted into the medium no longer undergo regulation, and therefore they have a relatively simple molecular structure consisting of one globular module. Intracellular proteases must undergo continuous regulation to avoid cell damage. Unlike secreted protease zymogens, in more complex regulatory proteases, very large polypeptide segments can be introduced between the signal sequence and the zymogen activation domain of the proteolytic module. A study of the structure and function indicates that such non-protease parts can participate in interactions with macroscopic structures, membranes, cofactors, substrates, effectors, inhibitors and ions that regulate the activity and activation of the proteolytic module (s) or him (them) zymogen (s). Such non-proteolytic modules vary significantly in size and structure. Many of these modules can exist independently of the proteolytic module. Therefore, we can assume that such modules are independent of structural and functional units that are autonomous with respect to styling. The value of such a modular organization is that the acquisition of new modules can give the recipient protease a new binding specificity and can lead to a sharp change in its activity, regulation, and targeting. The principle of modular organization of proteolytic enzymes can also be used in molecular biology methods to provide new interactions, regulation, specificity and / or targeting by “shuffling” the modules. Although, basically, such additional modules are present as N- or C-terminal extensions, however, large inserts were also observed in the outer loops of the catalytic domain. It is obvious that in this case also the main protease folding represents the main topology for the formation of a functional proteolytic molecule and that this insert can be considered as a substructure laid on the surface of the proteolytic module.

Molecular structure

In principle, the main topic of discussion is the modular organization of larger natural proteins. In particular, in larger multimodular frameworks, typical proteolytic modules have an average size of 100 to 400 amino acids. This corresponds to the average size of most globular proteolytic enzymes that are secreted into the medium. As discussed above, polypeptide modules are polypeptide fragments that can be stacked and can function as independent molecules. Another name for such modules is “domains”. However, the term “domain” is used in a broader context than a module. As used herein, the term “domain” generally means a portion of a polypeptide chain that has a typical folding topology in a three-dimensional structure. In a protein, domains interact at various levels, but less intensively than structural elements within domains. Other terms are also used in the literature, such as a subdomain and a stacking unit. In fact, it has been observed that many proteins that have the same specific functional groups can have the same domains. Such domains can be recognized by their primary structure, which may have sequences of a certain type that are characteristic of a given domain. Typical examples are a mononucleotide binding fold; cellulose binding domains; DNA binding spiral-coil motif; zinc finger domains; EF shoulders; membrane anchors. The term “modules” means domains that are assumed to be capable of autonomous stacking and function. The specialist knows methods for identifying specific domains in the primary structure using generally available computer programs for the specified structure and homologous sequences originating from other organisms or species.

Although multimodular or multidomain proteins can be a string of beads, a much more complex architecture was observed. If different “beads” are present on the same polypeptide chain, then these “beads” are usually called modules or domains. If the “beads” are not present on the same polypeptide chain, but form an ordered structure through non-covalent interactions, then the term “subunit” is used. Subunits can be transcribed by the same gene or different genes. After transcription, a multimodular protein can undergo proteolytic processing, resulting in the formation of multiple subunits. Individual subunits may consist of multiple domains. Basically, smaller globular proteins containing 100-300 amino acids, usually consist of only one domain.

Molecular classification of proteolytic enzymes

Basically, proteases are classified by their molecular properties or by their functional properties. Molecular classification is based on the primary structure of the protease. The primary structure of a protein is its amino acid sequence, which may occur from the nucleotide sequence of the corresponding gene. Intensive studies regarding the similarity of primary structures can lead to the identification of similarities of the catalytic mechanism and other properties, which may even relate to functional properties. As used herein, the term “family” means a group of proteases that exhibit evolutionary kinship based on the similarity of their primary structures. Obviously, members of such a family descended from the same predecessor, but then underwent divergence in the process of evolution. In addition, within the family, these proteases are divided into subgroups according to their primary structures, which were established by a more detailed refinement of their sequence based on the results of comparing subfamilies. Three-dimensional folding of proteases may include a secondary structure, a tertiary structure, and a quaternary structure. In general terms, classification according to the secondary structure is limited by the content and approximate orientation of the elements of the secondary structure. The similarity of tertiary structures allows the identification of superfamilies or clans. A superfamily or clan is a group of families that obviously have a common “ancestor” because they exhibit a similar 3-dimensional styling. Basically, a three-dimensional structure is more conservative than the primary structure. Consequently, the similarity of the primary structure does not always reflect similar functional properties. Indeed, functional properties can undergo significant changes, which may lead to the emergence of new properties of interest. Currently, the quaternary structure is not used to classify various proteases. This may be due to some shift of structural databases towards simple globular proteases. Many proteolytic systems that undergo activation, regulation, or complex cascades of reactions are likely to consist of multiple domains or subunits. General trends in the structural organization of such protease systems may lead to new types of classification.

Specificity classification

In the absence of information on the sequence of proteases, they can be classified according to various types of their functions. The classification and names of enzymes according to the reactions that they catalyze are the main principle in the nomenclature of enzymes. This approach is also based on the principle of EC-enumeration of enzymes (Enzyme Nomenclature 1992 Academic Press, Orlando). Two types of proteases (EC 3.4) can be determined according to the Enzyme Nomenclature 1992 enzyme nomenclature, i.e. exopeptidases (EC 3.4.11-19) and endopeptidases (EC 3.4.21-24, 3.4.99). Endopeptidases cleave peptide bonds in the internal regions of the peptide chain, which are far from its end. Exopeptidases cleave residues only from the ends of the peptide chain. Exopeptidases acting at the free N-terminus can release one amino acid residue, dipeptide or tripeptide and are called aminopeptidases (EC 3.4.11), dipeptidyl peptidases (EC 3.4.14) and tripeptidyl peptidases (EC 3.3.14), respectively. Proteases that trigger peptide processing from the carboxyl end, leading to the release of one amino acid, are called carboxypeptidases (EC 3.4.16-18). Peptidyl dipeptidases (EC 3.4.15) cleave the dipeptide from the carboxyl end. Exo- and endopeptidases, in general, are dipeptidases (EC 3.4.13), which specifically cleave only dipeptides in their two amino acid halves. Omega-peptidases (EC 3.4.19) remove terminal residues that are substituted, cyclic or attached by isopeptide bonds.

In addition to the position in which this protease cleaves the peptide chain, an additional separation is possible for each type of protease in accordance with the nature of the preferred amino acid residues in the substrate. Basically, proteases can be classified by broad, medium and narrow specificity. Some proteases are simply named for specific proteins or polypeptides that they hydrolyze, for example, keratinase, collagenase, elastase. Narrow specificity may be limited to one specific amino acid or one specific sequence, which can be removed or cleaved, respectively. If the protease exhibits a particular preference for one amino acid at the P1 or P1'-position, then the name of this amino acid may be its identifier. So, for example, prolylamine peptidase removes proline from the amino terminus of the peptide (proline is residue P1). X-Pro or proline is used if a bond is cleaved on the side imino chain of this proline (the proline is the P1 ′ residue), for example, proline carboxypeptidase removes proline from the carboxy terminus. Prolyl endopeptidase (or Pro-X) cleaves in the position behind the proline, and proline endopeptidase (X-Pro) cleaves in the position in front of the proline. An amino acid residue in front of a cleavable peptide bond means an amino acid residue that introduces a carboxyl group into the peptide bond. An amino acid residue located behind a cleaved peptide bond means an amino acid residue that introduces an amino group into the peptide bond. According to the generally accepted agreement, the amino acid chain extends from the amino terminus (beginning) to the carboxy terminus (end), and amino acids are numbered in accordance with this. Endoproteases may also have a clear preference for a particular amino acid at the P1 or P1 ′ position, for example, glycyl endopeptidase, peptidyl lysine endopeptidase, glutamyl endopeptidase. In addition, proteases may favor a specific group of amino acids that share some similarities. Such a group of preferred amino acids may include hydrophobic amino acids, only bulk hydrophobic amino acids, small hydrophobic amino acids or only small amino acids, large positively charged amino acids, and the like. In addition to the preference for residues P1 and P1 ', there may also be a particular or exceptional preference for residues preferred by other subsites on this protease. Such multiple preferences may result in proteases being highly specific only to sequences that simultaneously satisfy multiple binding requirements. In general, it should be noted that proteases are more likely non-specific enzymes. Even a very specific protease can cleave peptides that are not the subject of a commonly observed protease preference. In addition, it should be noted that protease preferences may be influenced by environmental conditions such as pH, temperature, ionic strength, water activity, the presence of solvents, the presence of competing substrates or inhibitors. Environmental conditions can affect not only the protease, but also the way the protein substrate is represented by the protease.

Catalytic classification

Proteases can be classified based on their catalytic mechanisms. It should be noted that for each catalytic mechanism, the above classification of proteases, based on their specificity, leads to the subsequent division of proteases according to their mechanism of action. Four main classes of proteases are known, which were named according to their main functional groups in the active site, namely serine proteases (EC 3.4.21 endopeptidase, EC 3.4.16 carboxypeptidase), thiol or cysteine proteases (EC 3.4.22 endopeptidase, EC 3.4. 18 carboxypeptidase), carboxylic or aspartic protease (EC 3.4.23 endopeptidase) and metalloprotease (EC 3.4.24 endopeptidase, EC 3.4.18 carboxypeptidase). For the members of each catalytic type of protease, there are characteristic inhibitors. These small inhibitors reversibly modify the amino acid residue of the active site of the protease. For example, the serine protease is inactivated by phenylmethanesulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), which reacts with active serine, while the chloromethyl ketone derivatives react with the histidine of the catalytic triad. Phosphoramidone and 1,10-phenanthroline usually inhibit metalloproteases. Pepstatin inhibition usually indicates an aspartic protease. E64 specifically inhibits thiol protease. Amastatin and bestatin inhibit various aminopeptidases. In this case, there are significant differences in the susceptibility of proteases to inhibitors, even within the same catalytic class. To some extent, this may be due to the specificity of the protease. In the case where the architecture of the binding site interferes with the mechanism according to which the inhibitor acts on the catalytic site, such a protease “escapes” the inhibition, and identification by the type of inhibition mechanism cannot be carried out. For example, chymostatin is a potent serine protease inhibitor with a specificity similar to that of chymotrypsin. Elastatin inhibits elastase-like serine proteases and does not react with trypsin or chymotrypsin; 4-amido-PMSF (APMSF) inhibits only serine proteases with trypsin-like specificity. A wide discussion of the use of inhibitors in the classification of proteases is given in Barret and Salvesen, Proteinase Inhibitors, Elsevier Amstardam, 1986; Bond and Beynon (eds), Proteolytic Enzymes, A Practical Approach, IRL Press, Oxford, 1989; Methods in Enzymology, eds EJBarret, volume 244, 1994 and volume 248, 1995; E. Shaw, Cysteinyl proteinases and their selective inactivation, Adv Enzymol. 63: 271-347 (1990).

Classification by optimal operating conditions

The catalytic mechanism of action of proteases and the requirements for their conformational integrity mainly determine the conditions under which these proteases can be used. Establishing optimal conditions for the use of protease is the main task. Often the conditions under which the proteases must function are not optimal, and a compromise must be found between the ideal conditions for a given particular application and the conditions that should be most suitable for a given protease. In addition to the specific properties of the protease, it should also be noted that the presentation of protein substrates depends on the conditions, and, in essence, it also determines the conditions that are most effective for proteolysis. The parameters of an enzyme suitable for its use are, for example, pH dependence, temperature dependence, sensitivity to metal ions or dependence on metal ions, ionic strength, salt concentration and solvent compatibility. Another most important factor is the specific activity of the protease. The greater the specific activity of a given enzyme, the less it needs a specific conversion. The lower the requirements for the enzyme, the lower its cost and lower levels of protein impurities.

pH is the main parameter that determines the functioning of the protease used for certain purposes. Therefore, the dependence on pH is an important parameter for the protease group. The main groups that proteases are usually divided into are acid proteases, neutral proteases, alkaline proteases and highly alkaline proteases. The optimal pH corresponds to the proteolytic mechanism only to a certain extent, for example, in most cases, the aspartic protease optimally acts only at an acidic pH; metalloproteases and thiol proteases optimally act from about neutral to slightly alkaline pH; serine proteases are active mainly in alkaline and alkaline environments. Exceptions are known for each class of proteases. In addition, a certain role is played by the activity of water in this system. The optimal pH of the protease is defined as the pH range over which the prosthesis has the optimal hydrolysis rate for most of its substrates in a particular environment and in specific conditions. This range can be low, for example in one pH unit, and also quite wide, in 3-4 units. Basically, the optimal pH also depends on the nature of the protein substrate. The speed of the protease functional cycle, as well as its specificity, can vary with pH. For some purposes, it may be desirable to use a protease at a pH whose values are far from optimal, for example, when it is necessary to avoid the production of less desirable peptides. Such less desirable peptides can be, for example, very short peptides or peptides giving a bitter aftertaste. In addition, a narrower specificity can serve as the basis for the selection of conditions that do not correspond to optimal conditions with respect to the speed of the functional cycle. Depending on the pH, the specificity of the protease can be narrow, for example, it can split the peptide chain only in one specific position in front of or behind a specific amino acid, or it can be wider, for example, when the protease splits the chain in many positions, either in front or behind amino acids many types. Indeed, pH dependence can serve as an important tool for regulating proteolytic activity for specific purposes. In the case where the pH changes during proteolysis, such proteolysis may cease spontaneously and does not require additional processing to inactivate the protease. In some cases, proteolysis itself can be a regulator of pH changes.

When using proteases, their technological and functional stability is a very important factor. Since the stability of the prosthesis is largely dependent on the working temperature, this parameter is often also called thermostability. Generally speaking, protease stability indicates the shelf life of the proteolytic activity of the protease under these specific conditions. Such specific conditions may include fermentation conditions, conditions during isolation and subsequent processing of the enzyme, storage conditions, conditions for preparation and functioning of the enzyme, or conditions for its use. If specific conditions include stability at elevated temperatures, then such stability is mainly called thermal stability. In addition to the well-known causes of inactivation of the enzyme, such as chemical modification, the deployment of the enzyme molecule, its aggregation, etc., the main problem is that these enzymes easily undergo autodegradation. Therefore, when using proteases, the optimal temperature is an important parameter for the protease group. Although there are various definitions, however, from an economic point of view, the most commonly used determination of the optimum temperature is the temperature at which the protease is the most productive for a given application. Protease productivity depends on both stability and the speed of its functional cycle. While elevated temperatures generally increase cycle speed, rapid inactivation leads to a decrease in cycle speed and, ultimately, to low productivity. The conformational stability of the protease under these process conditions will determine its maximum operating temperature. The temperature at which a given protease loses its active conformation is often regarded as a deployment or melting point and can be determined by various methods, for example, by NMR, circular dichroism spectroscopy, differential scanning calorimetry, etc. Deployment of the protease molecule is usually accompanied by a sharp increase in the rate of its autodegradation.

In cases where low temperatures are required, then when choosing a protease, one should focus on its high internal activity at low or moderate temperatures. Since under such conditions protease inactivation is relatively slow, activity under these conditions can largely determine its productivity. In processes where protease activity is only required in a very short period of time, protease stability can be used to “enable” or “disable” the protease. In this case, instead of a highly thermostable protease, it may be preferable to use a more labile protease.

Other environmental parameters that may play a role in the selection of a suitable protease may be its sensitivity to salts. Compatibility with metal ions, which are often present in low concentrations in various natural substances, may be a decisive factor for some applications. As for metalloproteases, some ions can replace the catalytic metal ion and reduce or even completely inhibit protease activity. In some applications, to prevent leakage of metal ions coordinated with the protease, it is necessary to add these metal ions. It is well known that in order to maintain the stability of the enzyme and maintain its validity, it is necessary to add calcium ions in order to prevent the dissociation of calcium associated with the protein.

Most microorganisms exhibit a certain tolerance and adapt to changes in environmental conditions. Therefore, as was shown on the basis of the proteolytic spectrum of such a microorganism, it is capable of producing at least a similar tolerance. Such a proteolytic spectrum can be covered by many proteases, which together cover the “window” of the spectrum, or it can only be covered by several broad-spectrum proteases. If we take into account the entire proteolytic spectrum of the microorganism, then localization can be important.

Cellular localization and characterization of proteolytic processing and degradation

From the point of view of industrial applications, proteases that are excreted from the cell have particular advantages in terms of large-scale productivity and stress tolerance, since they are viable in the absence of cellular protection. A large group of cellular proteases can also be subdivided into soluble and membrane-associated proteases. The group of membrane-associated proteases may include proteases associated with the membrane from both the inside and the outside. Intracellular soluble proteases can also be subdivided according to the specific compartments of the cells in which they are present. Since the cell, to some extent, protects the proteases from the external environment and since the cell regulates the conditions in it, the intracellular protease may be more sensitive to significant changes in environmental conditions, and, in this case, the optimal parameters can better correlate with specific conditions inside the cell. Knowledge of the conditions of the cell compartment in which this protease is present may indicate its preference. If the extracellular protease, basically, does not require any regulation immediately after its excretion from the cell, then intracellular proteases are often subjected to finer control and regulation.

Regarding the function of a particular protease, its localization is often very important, for example, a large number of vacuolar and periplasmic proteases are involved in protein degradation, while membrane-associated proteases play an important role in protein processing (Suarez Rendueles & Wolf, 1988) .

A complete review of the biological properties and evolution of proteases was published in van den Homberg: Thesis Landbouwuniversitet Wageningen: An analysis of proteolytic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2, which is incorporated herein by reference.

Protease Use Concerns

An important reason for the interest in microbial proteases is the problems associated with the expression of proteases in some expression hosts used in the biological process industry. In recent years, due to the increasing use of heterologous hosts for the production of proteins by recombinant DNA methods, certain problems have arisen, since heterologous proteins have been shown to be more prone to proteolysis (Archer et al., 1992; van den Homberg et al., 1996b) .

Regarding the use of S. cerevisiae , back in the eighties there were problems and difficulties associated with the use of certain proteases, and therefore, to solve this problem, a method was developed that provided for the disruption of the target target gene. During secretion, the protein undergoes several proteolytic activities that act on this secretory pathway. In addition, in a prototrophic microorganism such as Aspergillus , a secreted protein may undergo several extracellular proteolytic activities.

The degradation of heterologously expressed proteins in Aspergillus is well documented (van den Hombergh: Thesis Landbouwuniversitet Wageningen: An analysis of the proteolitic system in Aspergillus in order to improve protein production ISBN 90-5485-545-2), and it has been reported that such degradation observed with expression of bovine prochymosin, human interferon α-2-tHA, GMCSF, IL6, lactoferrin, chicken egg protein lysozyme, porcine pIA2, A. niger pectin lyase B, E. coli enterotoxin B and β-glucuronidase and pectate lyase 3 Erwinia carobotovora .

The problem of proteolysis can be associated with several stages of protein production. Biological process specialists who face the problem of proteolysis can process at low temperatures, carry out early isolation of the product and protease (s), or use protease inhibitors. All this can lead to a successful solution to this problem. However, all these difficulties cannot be completely eliminated, since, in most cases, degradation occurs in vivo during protein production.

To regulate proteolysis in the cell, it is important to understand the mechanism of triggering proteolysis. To a certain extent, (heterologous) proteins are proteolytically sensitive and recognizable as aberrant proteins due to their incorrect folding or, if correctly inserted, as “foreign” proteins because they do not possess traits that are important for their stability, which are specific to the host. Various types of stresses can cause an overall significant increase in proteolysis in the cell. Known factors for increasing the rate of proteolysis are cellular “starvation” and various types of stresses (that is, fever, osmotic stress, toxic substances, and the expression of certain heterologous proteins). To successfully solve the problems associated with in vivo proteolysis, several methods have been proposed, which will be discussed below. However, the authors of the present invention draw attention to the fact that absolutely “non-proteolytic cells” cannot exist, since proteolysis by intracellular proteases is a link in many important metabolic and constitutive reactions (“housekeeping” reactions). Therefore, a decrease in the degree of proteolysis is always a process in which a change in the genetic background leads to a decrease in proteolytic activity, and this process must be analyzed for possible side effects that can lead to a decrease in protein production (for example, a decrease in growth rate or sporulation).

Dysfunction of proteases expressed in filamentous host fungi

Berka and colleagues (1990) described the cloning and dizruptsiya pep A gene A.awamori. More recently, three disruptive aspartyl proteases in A. niger have been described. Disruptants have been described for both major extracellular aspartyl proteases and major vacuolar aspartyl prostheses. Double and triple disruptants were generated by recombination and tested on the protease spectrum, as well as on the expression and secretion of pectin lyase of A. niger PELB protein, which is very sensitive to proteolytic degradation (van den Hombergh et al., 1995). The disruption of the Aβ and Bβ gene caused a decrease in the activity of these extracellular proteases by 80% and 6%, respectively. In the disruption Δ receptor E, the activities of other (vacuolar) proteases were also significantly reduced, due to the inactivation of the proteolytic cascade of reactions for other vacuolar proteases. Decreased extracellular activity correlated with a decrease in the level of in vitro degradation of PELB and an increase in the level of in vivo expression of PelB (van den Hombergh et al., 1996f).

Protease deficient ( prt ) filamentous mushroom mutants

Several protease-deficient Aspergillus mutants have been investigated to increase protein production. Archer and coworkers described a reduced level of proteolysis of chicken egg protein lysozyme in supernatants of the prt double mutant A. niger generated by Mattern and coworkers (1992), and it was concluded that although degradation did occur, it was significantly reduced. Van den Hombergh et al. (1995) showed that in vitro degradation of A. niger PELB was reduced in all the seven groups with prt complementation that they isolated. In fact, no degradation was observed in prt B-, prt F-, and prt G mutants. Recently, it was shown that the expression of the Rel b gene was enhanced in six tested complementary groups ( prt A-F), and the highest level of expression was observed in prt B, prt F and prt G mutants. In addition to single-gene deficient mutants that contained residual extracellular proteolytic activities ranging from 2% to 80% compared to wild-type activity, double mutants were generated through recombination and additional rounds of mutagenesis. Several double prt mutants were selected and characterized by such methods, and it was shown that they showed an even lower degree of degradation of PELB compared to the parent strains.

In addition to suppressing protease activity through disruption or mutagenesis, a decrease in proteolysis can be achieved by negatively regulating unnecessary proteolytic activity. This can be done by genetic modification of the promoter or other regulatory sequences of the gene. As shown by Fraissinet-Tachet et al. (1996), all extracellular proteases in A.niger were regulated by inhibition of carbon catabolite and inhibition of nitrogen metabolite. Cell starvation also led to an overall significant increase in the level of proteolysis in the cell, which was important for cells that did not contain nutrients, but contain proteins that are not necessary in fasting conditions or are necessary only in small quantities. It was reported that when using expression strategies that produce high levels of expression in media containing high concentrations of glucose and ammonium, proteolysis was reduced. Some constitutive glycolytic promoters ( gpd and pki A) provide a high degree of expression under these conditions and can also be used to regulate the expression of a (heterologous) gene during continuous fermentation. The type of forced cell starvation can affect various proteases to varying degrees, which indicates the importance of cell nutritional conditions in this process, depending on the type of proteolysis. Therefore, specific proteolysis can be induced by substrate restriction conditions, which are often used in large-scale industrial fermentation processes.

Currently, one or more of the above strategies are being addressed to solve a protease problem. However, the residual proteolytic activity of still unidentified proteolytic enzymes is still the main problem for specialists. To further reduce the level of undesired proteolysis, it is imperative to identify new proteases responsible for the degradation of homologously and heterologically expressed proteins. The present invention relates to such new protease gene sequences encoding new proteases. As soon as the primary sequence of the new protease gene becomes known, one or more of the above recombinant DNA strategies can be used to produce (knockout) mutants with reduced proteolytic activity.

Despite the widespread use of proteases in most industrial processes, known enzymes also have many disadvantages regarding at least one of the following properties.

When added to an animal’s food, the commonly used proteases do not have sufficient resistance to the degrading enzymes present in the gastrointestinal (GI) tract, such as pigs or poultry.

As for other aspects, the existing enzymes do not have sufficient resistance to high temperatures and high pressures, which are commonly used during displacement or precipitation operations.

In addition, the commonly used enzymes are not sufficiently active at a pH in the range of 3-7, that is, under the conditions of preparation of many foods and drinks, as well as in the LCD tract of most animals.

In accordance with another aspect, the specificity of commonly used proteases is very limited, which leads to the inability of the present enzymes to decompose or dissolve some “protease-resistant” proteins and, thus, to a low yield of peptides or amino acids. In addition, low specificity proteases may contribute to the synthesis of new peptides.

Another disadvantage of commonly used enzymes is their low specific activity.

Based on this, it is obvious that in most cases the use of proteases is required, which are more resistant to digestive enzymes, to high temperatures and / or high pressure and which possess new specificities with respect to hydrolysis sites. The present invention relates to such enzymes.

The purpose of the invention

An object of the present invention is to provide novel polynucleotides encoding novel proteases. Another objective of the present invention is to obtain natural and recombinantly produced proteases, as well as recombinant strains producing these proteases. Such strains can also be used to produce products in the classical fermentation process at a higher rate or in higher yield. Another objective of the present invention is to obtain a strain of filamentous fungi deficient in the production of proteases of the present invention. Such strains can be used to more efficiently produce heterologous or homologous proteins. The present invention also includes antibodies and fusion proteins, as well as methods for the preparation and use of polynucleotides and polypeptides of the present invention.

SUMMARY OF THE INVENTION

The present invention relates to new polynucleotides encoding new proteases.

More specifically, the present invention relates to polynucleotides having a nucleotide sequence that hybridizes (preferably under high stringency conditions) with a sequence corresponding to 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 - SEQ ID NO: 114. In addition, the present invention relates to nucleic acids, which are approximately 60%, preferably 65%, more preferably 70% and even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% are homologous to sequences corresponding to a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO : 114.

In a more preferred embodiment, the present invention relates to a polynucleotide isolated from filamentous fungi, preferably from Aspergillus , and in particular from A. niger .

In one embodiment, the present invention relates to an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, or a functional equivalent thereof.

In another preferred embodiment, the present invention relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide corresponding to a sequence selected from the group consisting of sequences SEQ ID NO: 115 - SEQ ID NO: 171 or their functional equivalents.

In a preferred embodiment, the present invention relates to a protease gene having a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57. In another aspect, the present invention relates to a polynucleotide, preferably to a cDNA encoding an A. niger protease selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, or to variants or fragments of this polypeptide. In a preferred embodiment, said cDNA has a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 or their functional equivalents.

A genomic clone encoding the polypeptide of the present invention can also be obtained by selecting suitable probes for specific amplification of the genomic region corresponding to any of the sequences SEQ ID NO: 1 to SEQ ID NO: 57 or fragments thereof; hybridization of this probe under suitable conditions with genomic DNA obtained from a suitable organism such as Aspergillus , e.g. A.niger , amplification of the desired fragment, for example, by PCR (polymerase chain reaction), followed by purification and cloning of the amplified fragment.

In an even more preferred embodiment, the present invention relates to a polynucleotide comprising the coding sequence of the genomic polynucleotides of the present invention, with a polynucleotide sequence selected from the group consisting of the sequences SEQ ID NO: 58 - SEQ ID NO: 114 being preferred.

In another preferred embodiment, the present invention relates to cDNA obtained by cloning and expression of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 in a suitable host organism such as A.niger .

The polypeptide of the present invention can also be obtained by cloning and expression of a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57 in a suitable host organism such as A.niger .

The present invention also relates to vectors containing the polynucleotide sequence of the present invention, and to primers, probes and fragments that can be used for amplification or for DNA detection of the present invention.

In another preferred embodiment, the present invention relates to a vector in which the polynucleotide sequence of the present invention is operably linked to regulatory sequences suitable for expression of the encoded amino acid sequence in a suitable host cell, such as A.niger or A.oryzea . The present invention also relates to methods for producing polynucleotides and vectors of the present invention.

In addition, the present invention relates to recombinantly produced host cells that contain the heterologous or homologous polynucleotides of the present invention.

In one embodiment, the present invention relates to recombinant host cells where the expression of the protease of the present invention is significantly reduced or its activity is reduced, or where said protease is even inactivated. Such recombinants are particularly preferred for the expression of homologous or heterologous proteins.

In another embodiment, the present invention relates to recombinant host cells in which the level of expression of the protease of the present invention is significantly increased or its activity is increased. Such recombinants are particularly preferred for the expression of homologous or heterologous proteins, the maturation of which is significantly hindered when the required proteolytic cleavage becomes a speed-limiting step.

In another embodiment, the present invention relates to a recombinantly produced host cell that contains the heterologous or homologous DNA of the present invention, and preferably DNA encoding proteins that carry signal sequences, where the cell is capable of producing a functional protease of the present invention, and preferably overexpressing the protease of the present invention wherein said cell is, for example, a strain of Aspergillus, containing a large number of gene copies yl cDNA of the present invention.

In another embodiment, the present invention relates to a recombinantly produced host cell that contains the heterologous or homologous DNA of the present invention, wherein said cell is capable of secreting the functional protease of the present invention, and preferably overexpressing and secreting the protease of the present invention, and where said cell is, for example, Aspergillus strain containing an increased copy number of a gene or cDNA of the present invention.

In another aspect, the present invention relates to a purified polypeptide. The polypeptides of the present invention are polypeptides encoded by the polynucleotides of the present invention. Particularly preferred is a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, or functional equivalents thereof.

The present invention also relates to antibodies that interact with the polypeptide of the present invention. These antibodies may be polyclonal, and preferably monoclonal. Such antibodies are particularly suitable for purification of the polypeptides of the present invention.

Hybrid proteins comprising the polypeptide of the present invention are also within the scope of the present invention. The present invention also relates to methods for producing the polypeptides of the present invention.

In addition, the present invention relates to a method for diagnosing aspergillosis by detecting the presence of a polypeptide of the present invention or its functional equivalents, or by detecting the cDNA of the present invention, or fragments thereof, or functional equivalents.

The present invention also relates to the use of the protease of the present invention in the industrial process described herein.

DETAILED DESCRIPTION OF THE INVENTION

Polynucleotides

The present invention relates to polynucleotides encoding proteases having an amino acid sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, or functional equivalents thereof. The sequence of these genes is determined by sequencing a genomic clone derived from Aspergillus niger . The present invention relates to polynucleotide sequences containing a gene encoding these proteases, as well as their complete cDNA sequence and its coding sequence. Accordingly, the present invention relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 or their functional equivalents.

More specifically, the present invention relates to an isolated polynucleotide hybridizing under stringent conditions with a polynucleotide selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or with a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114, preferably under high stringency conditions. Preferably, such polynucleotides can be obtained from filamentous fungi, and in particular from Aspergillus niger . More specifically, the present invention relates to an isolated polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO : 114.

The present invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171 or their functional equivalents.

As used herein, the terms “gene” and “recombinant gene” mean nucleic acid molecules that can be isolated from chromosomal DNA and which include an open reading frame encoding a protein, for example, A. niger protease. A gene may include coding sequences, non-coding sequences, introns, or regulatory sequences. In addition, the term “gene” may mean an isolated nucleic acid molecule as defined in the present description of the invention.

A nucleic acid molecule of the present invention, such as a nucleic acid molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 or its functional equivalents, can be isolated by standard methods used in molecular biology, and using the information provided here related to this sequence. So, for example, using all or part of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57 or a nucleotide sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114, as a hybridization probe, the nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, EF, & 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 comprising all or part of a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on sequence information contained in a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114.

The nucleic acid of the present invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and using appropriate oligonucleotide primers in accordance with standard PCR amplification methods. The nucleic acid thus amplified can be cloned into the corresponding vector and characterized by DNA sequence analysis.

In addition, oligonucleotides corresponding to the nucleotide sequences of the present invention or hybridizable with them can be obtained by standard methods of synthesis, for example, using an automatic DNA synthesizer.

In a preferred embodiment, the isolated nucleic acid molecule of the present invention comprises a nucleotide sequence represented in the sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114. A sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 corresponds to the coding region of the A. niger protease cDNA . This cDNA comprises sequences encoding A.niger protease polypeptide having a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171.

In another preferred embodiment, the isolated nucleic acid molecule of the present invention includes a nucleic acid molecule that is complementary to the nucleotide sequence represented in the sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, or in a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114, or the functional equivalent of these nucleotide sequences.

A nucleic acid molecule complementary to another nucleotide sequence is a molecule that is substantially complementary to another nucleotide sequence so that it can hybridize with this other nucleotide sequence to form a stable duplex.

In one aspect, the present invention relates to isolated nucleic acid molecules encoding a polypeptide of the present invention or its functional equivalent, such as a biologically active fragment or domain, as well as to nucleic acid molecules suitable for use as hybridization probes to identify nucleic acid molecules encoding the polypeptide of the present invention, and to fragments of such nucleic acid molecules suitable for use as PCR primers in amplification or mutagenesis of nucleic acid molecules.

The terms “isolated polynucleotide” or “isolated nucleic acid” means DNA or RNA that is not directly adjacent to both coding sequences with which it is directly adjacent (from one at the 5'-end and the other at the 3'-end) in the natural the genome of the body from which it comes. Thus, in one embodiment of the invention, the isolated nucleic acid comprises some or all of the 5'-non-coding (eg, promoter) sequences that are directly adjacent to the coding sequence. Therefore, the term includes, for example, recombinant DNA that is embedded in a vector, in an autonomously replicating plasmid or virus, or in the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, as a cDNA or a fragment of genomic DNA produced by PCR or by treatment with restriction enzymes) independent of other sequences. This term also includes recombinant DNA, which is part of a hybrid gene encoding an additional polypeptide, essentially free of cellular material, viral material or culture medium (when produced by recombinant DNA methods), or chemical precursors or other chemical compounds (in chemical synthesis) . In addition, the term “isolated nucleic acid fragment” means a nucleic acid fragment that usually does not occur as a fragment and is not found in nature.

As used herein, the terms “polynucleotide” or “nucleic acid molecules” mean DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), and DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably, it is a double-stranded DNA. A nucleic acid can be synthesized using oligonucleotide analogues or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to produce nucleic acids that have an altered ability to form base pairs or increased resistance to nucleases.

In another embodiment, the present invention relates to an isolated nucleic acid molecule that is antisense to a protease nucleic acid molecule, for example, to the coding chain of a protease nucleic acid molecule. The complementary strands of nucleic acid molecules described herein are also within the scope of the present invention.

Sequencing Errors

The sequence information presented here should not be construed in the narrow sense as a requirement to include erroneously identified bases. The specific sequences described here can be successfully used to isolate a complete gene from filamentous fungi, and in particular from A. niger , which, in turn, can be easily subjected to subsequent sequence analysis, thereby identifying sequencing errors.

Unless otherwise indicated, all nucleotide sequences determined here by sequencing a DNA molecule were determined using an automatic DNA sequencer, and all amino acid sequences of the polypeptides encoded by the DNA molecules defined here were predicted by translation of the DNA sequence as defined above. Therefore, as is known to those skilled in the art, any nucleotide sequence defined herein for any DNA sequence determined in such an automated manner may contain a certain number of errors. Typically, nucleotide sequences determined by an automated method are at least about 90%, more preferably at least about 95% -99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other methods, including manual DNA sequencing methods well known to those skilled in the art. As is also known to those skilled in the art, a single insertion or deletion in a specific nucleotide sequence compared to the actual sequence will lead to a shift in the reading frame during translation of the nucleotide sequence, as a result of which the predicted amino acid sequence encoded by a specific nucleotide sequence will be completely different from the actual amino acid sequence, encoded by a sequenced DNA molecule, starting at the point of such insertion or deletion.

Each specialist can identify such incorrectly identified grounds and correct such errors.

Nucleic acid fragments, probes, and primers

The nucleic acid molecules of the present invention may include only a part or fragment of a nucleic acid sequence represented in the sequence selected from the group consisting of SEQ ID NO: 1-57, or a sequence selected from the group consisting of SEQ ID NO: 58 - 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 protein. The nucleotide sequence determined by cloning the protease gene and cDNA allows the generation of probes and primers designed to identify and / or clone other members of the protease family, as well as protease homologs of other species. The probe / primer typically includes a substantially purified oligonucleotide, typically containing a region of a nucleotide sequence that hybridizes, preferably under high stringency conditions, to at least about 12 or 15, preferably about 18 or 20, more preferably about 22 or 25, and most preferably with about 30, 35, 40, 45, 50, 55, 60, 65 or 75 nucleotides, or with more closely spaced nucleotides of the nucleotide sequence represented in the sequence selected from the group consisting of SEQ ID N O: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58-114, or their functional equivalents.

Probes based on protease nucleotide sequences can be used to detect transcripts or genomic protease sequences encoding the same or homologous proteins, originating, for example, from other organisms. In preferred embodiments of the invention, said probe further includes a tagging group attached thereto, for example, the tagging group may be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can also be used as part of a diagnostic test kit to identify cells expressing a protease protein.

Identification and homology

As used herein, the terms “homology” or “percent identity” are used interchangeably. For the purposes of the present invention, to determine the percent identity of two amino acid sequences or two nucleic acid sequences, an optimal comparison of the sequences was carried out (for example, gaps may be introduced in the sequence of the first amino acid sequence or nucleic acid sequence in order to optimally compare it with the second amino acid sequence or sequence nucleic acid). Then, a comparison was made of amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as in the corresponding position of the second sequence, then these molecules are identical in this position. The percent identity between two sequences is a function of the number of identical positions for the two sequences to be compared (i.e.,% identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100).

Preferably, these two sequences have the same length.

Each specialist knows that to determine the homology between two sequences, there are several different computer programs. For example, comparison of sequences and determination of percent identity between two sequences can be carried out using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman & Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)), which was introduced into the GAP program GAP software (available at http : //www.gcg.com) using either the Blossom 62 matrix or the RAM250 matrix and using a “gap” weight of 16, 14, 12, 10, 8, 6 or 4 and a weight of length 1, 2, 3, 4, 5 or 6. It is obvious to the person skilled in the art that all these different parameters give slightly different results, but the overall percentage of identity is two x sequences is not significantly changed when using different algorithms.

In another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program, the GCG software (available at http://www.gcg.com) using the NWSgapdna.CMP matrix, and using a “gap” weight of 40, 50, 60, 70, or 80 and weights of length 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 of E. Meyer & W. Miller (CABIOS, 4: 11- 17 (1989), which was included in the ALIGN program ( version 2.0) (available at http: //vega/igh.cnrs.fr/bin/align-guess.cgi), using the table of the weight of residuals RAM120, a penalty for a “gap” -extraction 12 and a penalty for a “gap-pass” four.

The nucleic acid and protein sequences of the present invention can also be used as the “requested sequence” to search available databases, for example, to identify other family members or related sequences. Such a search can be carried out using the NBLAST and XBLAST programs (version 2.0), Altschul et al. (1990) J. Mol. Biol. 215: 403-10. Nucleotides can be searched in BLAST using the NBLAST program, reference label = 100, word length = 12 to obtain nucleotide sequences homologous to the protease nucleic acid molecules of the present invention. Search for proteins in BLAST can be performed using the XBLAST program, reference label = 50, word length = 3 to obtain amino acid sequences homologous to the protease protein molecules of the present invention. To make comparisons with the introduction of “gaps”, the BLAST program with the introduced “gaps” can be used, as described by Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and BLAST programs with introduced gaps, the parameters of the corresponding default programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nim.nih.gov.

Hybridization

As used herein, the term “hybridization” means hybridization and washing conditions in which nucleotide sequences that are at least about 50%, at least about 60%, at least about 70%, more preferably at least 80%, even more preferably at least about 85-90%, and most preferably at least 95% homologous to each other, hybridize to each other.

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

High stringency conditions are, for example, hybridization at 68 ° C in 5 × SSC / 5 × Denhardt's solution / 1.0% SDS and washing at 0.2 × SSC / 0.1% SDS at room temperature. Alternatively, washing may be carried out at 42 ° C.

Each specialist knows in which cases it is necessary to apply stringent conditions and conditions of high stringency of hybridization. Further information on such conditions can be found in the literature, 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.).

It goes without saying that a polynucleotide that hybridizes only with a poly (A) sequence (such as a 3'-terminal poly (A) tail of mRNA) or with a complementary fragment of T (or U) residues is not included in the polynucleotide of the present invention used for specific hybridization with a portion of the nucleic acid of the present invention, since such a polynucleotide must hybridize with any nucleic acid molecule containing a poly (A) tail, or with a complementary sequence (for example, with any double-stranded DNA clone )

Obtaining full-sized DNA from other microorganisms

Using a conventional technique, cDNA libraries constructed from other microorganisms, such as filamentous fungi, and in particular, Aspergillus species, can be screened.

For example, Aspergillus strains can be screened for homologous protease polynucleotides using Northern blot analysis. After detection of transcripts homologous to the polynucleotides of the present invention, cDNA libraries can be constructed from RNA isolated from the corresponding strain using standard methods well known in the art. Alternatively, the entire genomic DNA library can be screened using a probe hybridizable to the protease polynucleotide of the present invention.

Homologous gene sequences can be isolated, for example, by PCR using two oligonucleotide primers or two pools of degenerate oligonucleotide primers constructed based on the nucleotide sequences described herein.

The matrix for the reaction may be cDNA obtained by reverse transcription of mRNA isolated from strains that are known or supposed to express the polynucleotide of the present invention. The PCR product can be subcloned and sequenced to ensure that these amplified sequences are nucleic acid sequences of a new protease or its functional equivalent.

The PCR fragment can then be used to isolate a full-sized cDNA clone by various known methods. For example, an amplified fragment can be isolated and used for screening a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment can be used to screen the genomic library.

PCR technology can be used to isolate full-length cDNA sequences from other microorganisms. So, for example, RNA can be isolated in accordance with standard procedures from a suitable cellular or tissue source. To start the synthesis of the first chain, the reverse transcription reaction can be carried out on RNA using an oligonucleotide primer, specific, mainly to the 5'-end of the amplified fragment.

The resulting RNA / DNA hybrid can then be “extended” (for example, with guanines) by a standard terminal transferase reaction, and this hybrid can be cleaved with RNase H, after which the second strand synthesis can be started (for example, using a poly-C primer) . Thus, cDNA sequences located above the amplified fragment can be easily isolated. A review of suitable cloning strategies can be found, for example, in Sambrook et al., See above; and Ausubel et al., see above.

Vectors

In another aspect, the present invention relates to vectors, and preferably, expression vectors containing a nucleic acid encoding a protease protein or its functional equivalent. As used herein, the term “vector” means a nucleic acid molecule capable of transferring another nucleic acid to which it has been attached. One type of vector is a “plasmid,” which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (for example, bacterial vectors having a bacterial replication source and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the cell, and thus are replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes with which they are functionally linked. Such vectors are referred to herein as “expression vectors”. Basically, expression vectors are often used in the technique of recombinant DNA in the form of plasmids. The terms “plasmid” and “vector” can be used interchangeably since plasmid is the most common form of vector. However, the present invention also relates to other forms of expression vectors, such as viral vectors (for example, replication defective retroviruses, adenoviruses and adeno-associated viruses) that have equivalent functions.

Recombinant expression vectors of the present invention include a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that this recombinant expression vector includes one or more regulatory sequences selected in accordance with the host cells used for expression and functionally linked to an expressed nucleic acid sequence. As used in relation to a recombinant expression vector, the term “operably linked” means that the nucleotide sequence present in it and of interest is attached to the regulatory sequence (s) in a manner that expresses the nucleotide sequence (for example, in an in vitro transcription / translation system or in a cell -host, if the specified vector is introduced into this host cell). The term “regulatory sequence” means promoters, enhancers, and other expression regulatory elements (eg, polyadenylation signal). Such regulatory elements are described, for example, by Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences are sequences that direct the constitutive expression of a nucleotide sequence to many types of host cells, and sequences that direct the expression of a nucleotide sequence only to certain host cells (e.g., tissue-specific regulatory sequences). It should be noted that the construction of the expression vector may depend on factors such as the choice of the transformed host cell, the level of expression of the desired protein, etc. The expression vectors of the present invention can be introduced into host cells, as a result of which they will produce proteins or peptides encoded by the nucleic acids described herein (e.g., protease proteins, mutant forms of protease proteins, fragments thereof, variants or functional equivalents, hybrid proteins, etc.). .P.).

Recombinant expression vectors of the present invention can be designed to express protease proteins in prokaryotic or eukaryotic cells. For example, protease proteins can be expressed in bacterial cells such as E. coli , in insect cells (using baculovirus expression vectors), in yeast cells or in mammalian cells. Suitable host cells are discussed in detail 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 using the T7 promoter regulatory sequences and T7 polymerase.

Expression vectors used in the present invention are chromosomal, episomal and viral vectors, for example, vectors derived from bacterial plasmids, bacteriophage, yeast episoma, yeast chromosome elements; viruses such as baculoviruses, papovaviruses, vaccine viruses, adenoviruses, poultry smallpox viruses, pseudo-rabiviruses and retroviruses, as well as vectors derived from their combinations, such as vectors derived from plasmids and bacteriophage genetic elements such as cosmids and phagemids.

The DNA insert should be operably linked to a suitable promoter, such as PL promoters of the lambda phage, lac , trp, and E. coli tac promoters, early and late SV40 promoters, and retroviral LTR promoters and the like. Other suitable promoters are well known to those skilled in the art. In a particular embodiment, promoters capable of providing a high level of expression of proteases in filamentous fungi are preferred. Such promoters are known to those skilled in the art. Expression constructs may contain transcription initiation sites, transcription termination sites, and a ribosome binding site for translation in the transcribed region. The coding part of the mature transcripts expressed by these constructs includes the AUG translation initiation site at the beginning of the sequence and the translation termination codon located at the corresponding position at the end of the translated polypeptide.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by standard methods of transformation or transfection. As used herein, the terms “transformation” and “transfection” mean a number of methods known to those skilled in the art for introducing foreign nucleic acid (eg, DNA) into a host cell, including co-precipitation with calcium phosphate or calcium chloride, 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 the manual by 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 laboratory manuals .

Regarding stable transfection of mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector used and the transfection method. To identify and select these integrants, a gene encoding a selective marker (for example, a marker of antibiotic resistance) is usually introduced into the host cells along with the desired gene. Preferred selective markers are markers that confer resistance to drugs such as G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker can be introduced into the host cell on the same vector that encodes the protease protein, or it can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug resistance selection (for example, cells into which the selective marker gene has been introduced survive and other cells die).

The expression of proteins in prokaryotes, in most cases, is carried out in E. coli cells, including vectors containing constitutive or inducible promoters that regulate the expression of hybrid or non-hybrid proteins. Hybrid vectors attach a number of amino acids to the protein encoded in it, for example, to the amino terminus of a recombinant protein. Such hybrid vectors usually serve three purposes, namely: 1) enhance expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) facilitate the purification of the recombinant protein, acting as a ligand in affinity purification. In most cases, in hybrid expression vectors, the proteolytic cleavage site is introduced at the junction of the hybrid molecule and the recombinant protein, which allows the recombinant protein to be separated from the hybrid molecule, and then the fusion protein is purified. Such enzymes and their cognitive recognition sequences are factor Xa, thrombin and enterokinase.

As indicated above, expression vectors preferably contain selective markers. Such markers are the dihydrofolate reductase gene or the neomycin resistance gene for eukaryotic cell cultures and the tetracycline or ampicillin resistance gene for cultivation in E. coli and other bacteria. Representative examples of a suitable host are 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. Suitable culture media and culturing conditions of the above host cells are known to those skilled in the art.

Vectors preferred for use in bacteria are the vectors pQE70, pQE60 and PQE-9, 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. Preferred eukaryotic vectors are the vectors PWLNEO, pSV2CAT, pOG44, pZT1 and pSG, available from Stratagene; and vectors pSVK3, pBPV, pMSG and pSVL, available from Pharmacia. Other suitable vectors are well known in the art.

Known bacterial promoters used in the present invention are the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the PR lambda promoters, the PL promoter and the trp promoter, the HSV thymidine kinase promoter, SV40 early and late promoters, LTR promoters of retroviruses such like Routh sarcoma virus (“RSV”), and metallothionein promoters, such as the mouse metallothionein-1 promoter.

Transcription of DNA encoding the polypeptides of the present invention in higher eukaryotes can be enhanced by introducing an enhancer sequence into the vector. Enhancers are cis-acting DNA elements that typically have a length of approximately 10-300 bp. and which enhance the transcriptional activity of the promoter in a host cell of this type. Examples of enhancers are the SV40 enhancer located in the late portion of the replication initiation site and having a length of 100-270 bp, the cytomegalovirus early promoter enhancer, the polyoma enhancer located in the late portion of the replication initiation site, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum into the periplasmic space or into the extracellular space, the corresponding secretion signal can be introduced into the expressed polypeptide. These signals may be endogenous to the polypeptide, or they may be heterologous.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For example, a region consisting of other amino acids, and in particular charged amino acids, can be added to the N-terminus of a given polypeptide to increase its stability and stability in the host cell during purification or during subsequent processing and storage. In addition, peptide molecules can be added to the polypeptide to facilitate purification.

The polypeptides of the present invention

The present invention relates to an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171; the amino acid sequence obtained by expression of the polynucleotide of the present invention, or, in a preferred embodiment of the invention, a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57 in the corresponding host, as well as the amino acid sequence obtained by expression of polynucleotide sequences selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114 in the corresponding host. In addition, a peptide or polypeptide comprising a functional equivalent of the above polypeptides is included within the scope of the present invention. The above polypeptides are generally referred to as “polypeptides of the present invention”.

The terms “peptide” and “oligopeptide” are regarded as synonyms (in the common sense), and these terms are used interchangeably depending on the context where it is required to indicate that the chain consists of at least two amino acids linked by peptidyl bonds. As used herein, the term “polypeptide” means chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or their sequences are written from left to right, in the direction from the amino terminus to the carboxy terminus. The single-letter code of the amino acids used is well known to those skilled in the art and can be found in the manual of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

The term “isolated” polypeptide or protein means a polypeptide or protein extracted from its natural environment. Thus, for example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as was determined for native or recombinant polypeptides, which were essentially purified by any suitable methods, such as, for example, one-step purification described by Smith & Johnson, Gene 67: 31-40 (1988).

The proteases of the present invention can be isolated and purified from recombinant cultures by well known methods, including precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, and hydroxyapatite chromatography. For analytical purification, it is most preferable to use high performance liquid chromatography (“HPLC”).

The polypeptides of the present invention are natural purified products, chemical synthesis products, and products produced by recombinant methods from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells. Depending on the host used in recombinant production methods, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the present invention may also include a modified initiating methionine residue, which, in some cases, is formed as a result of cell-mediated processes in the host.

In addition, the protein of the present invention may be a precursor protein, such as a zymogen; fusion protein; and a protein obtained as a pro-sequence or pre-pro-sequence or as another immature form of any type.

Protein fragments

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

Biologically active fragments of the polypeptides of the present invention are polypeptides comprising amino acid sequences that are substantially identical to or derived from the amino acid sequence of a protease protein (e.g., the amino acid sequence contained in a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171), and which include fewer amino acids than a full-sized protein, and have at least one biological activity, respectively constant activity of a full-sized protein. Typically, biologically active fragments include a domain or motif having at least one protease protein activity. The biologically active fragment of the protein of the present invention may be a polypeptide that has a length of, for example, 10, 25, 50, 100 or more amino acids. In addition, other biologically active parts in which other regions of the protein are deleted can be obtained by recombinant methods and evaluated for one or more biological activities inherent in the native form of the polypeptide of the present invention.

The present invention also relates to nucleic acid fragments that encode the biologically active protease protein fragments described above.

Hybrid proteins

The proteins of the present invention or their functional equivalents, for example, their biologically active parts, can be functionally linked to a non-protease polypeptide (for example, heterologous amino acid sequences) to produce fusion proteins. As used herein, the term “chimeric protein” or “fusion protein” of a protease means a protease polypeptide operably linked to a non-protease polypeptide. The term “protease polypeptide” means a polypeptide having an amino acid sequence corresponding to the polypeptide sequence of the present invention, and the term “non-protease polypeptide” means a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to a protein of the present invention, for example, a protein that differs from a protease protein and comes from the same or another organism. In a protease fusion protein, the protease polypeptide may correspond to all or part of the protein of the present invention. In a preferred embodiment, the protease fusion protein comprises at least one biologically active protein fragment of the present invention. In another preferred embodiment, the protease fusion protein comprises at least two biologically active portions of the protein of the present invention. As used with respect to a fusion protein, the term “operably linked” indicates that the protease polypeptide and non-protease polypeptide are fused to each other while maintaining a reading frame. The non-protease polypeptide may 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, wherein said protease sequences are fused to the C-terminus of the GST sequences.

Such fusion proteins may facilitate purification of the recombinant protease. In another embodiment, the fusion protein is a protease protein containing a heterologous signal sequence at its N-terminus. In some host cells (e.g., mammalian and yeast host cells), protease expression and / or secretion can be enhanced using a heterologous signal sequence.

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

The signal sequence can be used to facilitate the secretion and isolation of the protein or polypeptide of the present invention. Signal sequences are typically characterized by core hydrophobic amino acids, which are usually cleaved from a 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 along the secretory pathway. The signal sequence directs the secretion of the protein, for example, from a eukaryotic host into which the expression vector has been introduced, and then, or at the same time, the signal sequence is cleaved. Then the protein can be easily purified from the extracellular medium by known methods. Alternatively, the signal sequence can be attached to the desired protein using a sequence that facilitates purification, for example, using the GST domain. Thus, for example, a sequence encoding a polypeptide can be fused to a marker sequence, for example, a sequence encoding a peptide that facilitates the purification of a hybrid polypeptide. In some preferred embodiments of this aspect of the present invention, the marker sequence is a hexahistidine peptide, such as a label present in the pQE vector (Qiagen, Inc.), and other labels, many of which are commercially available. As, for example, described by Gentz et al., Proc.Natl. Acad. Sci., USA, 86: 821-824 (1989), hexahistidine provides convenient purification of the fusion protein. The HA tag is another peptide used for purification that corresponds to an epitope derived from the influenza virus hemaglutinin protein and which has been described, for example, by Wilson et al., Cell 37: 767 (1984).

Preferably, the chimeric or fusion protease protein of the present invention is prepared using standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together in the same reading frame by standard methods, for example, using blunt or sticky ends for ligation, then digested with restrictase enzymes to obtain the corresponding ends and, if necessary, sticky ends are completed , treated with alkaline phosphatase to avoid unwanted addition and subjected to enzymatic ligation. In another embodiment, the fusion gene can be synthesized by standard methods, including methods using automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using “anchor” primers designed so that they are complementary to the protruding ends between two adjacent gene fragments, which can then be hybridized and re-amplified to obtain a chimeric gene sequence (see, e.g. Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons, 1992). In addition, many expression vectors are commercially available and encode a hybrid portion (e.g., GST polypeptide). A nucleic acid encoding a protease can be cloned into said expression vector so that this hybrid moiety is attached to the protease protein while maintaining a reading frame.

Functional Equivalents

The terms “functional equivalents” and “functional variants” are used interchangeably. The functional DNA equivalents of the present invention are isolated DNA fragments encoding a polypeptide that has the specific function of the A. niger protease defined herein. The functional equivalent of the polypeptide of the present invention is a polypeptide that has at least one function of the A. niger protease defined herein.

Functional equivalents of a protein or polypeptide may contain only conservative substitutions of one or more amino acids in the sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, or substitution, insertion, or deletion of replaceable amino acids. Accordingly, a substitutable amino acid residue is a residue that can be substituted in the sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, without significant change in biological function. For example, it is believed that the conserved amino acid residues of the protease proteins of the present invention are particularly unsuitable for modification. In addition, conservative amino acids in the protease proteins of the present invention and in other proteases are also not suitable for modification.

The term “conservative substitution” means a substitution in which the amino acid residue is replaced by an amino acid residue having a similar side chain. These families of amino acids are known to those skilled in the art, and include amino acids with major side chains (e.g., lysine, arginine and histidine), with acidic side chains (e.g., aspartic acid, glutamic acid), with uncharged polar side chains (e.g., glycine, asparagines , glutamine, serine, threonine, tyrosine, cysteine), with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), with beta-branched side chains (e.g. threonine, valine, isoleucine ) and with ar aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

Functional equivalents of nucleic acids can mainly contain silent mutations or mutations that do not alter the biological function of the encoded polypeptide. Accordingly, the present invention relates to nucleic acid molecules encoding protease proteins that contain amino acid residues that substantially do not affect a particular biological activity. Such protease proteins in their amino acid sequence differ from the sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, however, they retain at least one biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein said protein contains an amino acid sequence substantially homologous to at least about 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence represented in the sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171.

For example, guidance on phenotypically silent amino acid substitutions can be found in Bowie J.U. et al., Science, 247: 1306-1310 (1990), where the authors indicate that there are two main methods for studying the substitution tolerance of an amino acid sequence. The first method is based on the evolutionary process, in which mutations are either acquired or suppressed as a result of natural selection. The second method involves genetic engineering for introducing amino acid substitutions at specific positions of the cloned gene, as well as selection or screening to identify sequences that retain their functional properties. As the authors indicate, these studies unexpectedly revealed that proteins are tolerant to amino acid substitutions. In addition, the authors indicate that these substitutions are likely to be valid at a particular position in the protein. So, for example, the most hidden amino acid residues require non-polar side chains, while some elements of the surface side chains are mostly conservative. Other such phenotypically silent substitutions are described, for example, in Bowie et al., Supra, and in the references cited therein.

An isolated nucleic acid molecule encoding a protease protein homologous to a protein selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171 can be constructed by introducing one or more nucleotide substitutions, additions or deletions to the coding nucleotide sequences selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58 - SEQ ID NO: 114, so that one or more amino acid substitutions, deletions or insertions were introduced into the encoded protein. These mutations can be introduced by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis.

The term “functional equivalents” also encompasses orthologs of the protease protein A.niger . Orthologs of the protease protein A.niger are proteins that can be isolated from other strains or species and which have similar or identical biological activity. Such orthologs can be easily identified as containing an amino acid sequence essentially homologous to a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171.

As used herein, the term “substantially homologous” refers to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of amino acids or nucleotides identical or equivalent (eg, with a similar side chain) to amino acids or nucleotides of a second amino acid or nucleotide sequence, such that the first and second amino acid or nucleotide sequences had a common domain. For example, amino acid or nucleotide sequences that contain a common domain that is approximately 60% identical, preferably 65%, more preferably 70%, and even more preferably 75%, 80%, 85%, 90%, 95%, 96 %, 97%, 98% or 99%, and are, as defined above, fairly identical.

In addition, nucleic acids encoding other members of the protease family that have a nucleotide sequence different from a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 57, or a sequence selected from the group are within the scope of the present invention. consisting of SEQ ID NO: 58 - SEQ ID NO: 114. In addition, nucleic acids encoding protease proteins from other species that have a nucleotide sequence different from the 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 - SEQ ID NO: 114.

Nucleic acid molecules corresponding to variants (e.g., natural allelic variants) and DNA protease homologs of the present invention can be isolated based on their homology to the protease nucleic acids described herein, using the cDNA described herein or their suitable fragments as a hybridization probe in accordance with standard hybridization methods, preferably under hybridization conditions of high stringency.

Specialists know that in addition to using natural allelic variants of the protease sequence, the nucleotide sequences contained in the sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO : 58 - SEQ ID NO: 114, modifications may be made that result in changes in the amino acid sequence of the protease protein, but do not significantly affect the function of the protease protein.

In another aspect, the present invention relates to improved protease proteins. “Improved” protease proteins are proteins that have at least one improved biological activity. Such proteins can be obtained by introducing non-specific mutations along the entire coding sequence of the protease or its part, for example, by saturation mutagenesis, and the obtained mutants can be recombinantly expressed and screened for their biological activity. Thus, for example, standard analyzes for measuring the enzymatic activity of proteases are described in the literature, and thus improved proteins can be easily selected.

In a preferred embodiment, the protease protein has an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171. In another embodiment, the protease polypeptide is substantially homologous to an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 115 to SEQ ID NO: 171, and retains at least one biological activity of the polypeptide corresponding to the sequence selected from the group consisting of SEQ ID NO: 115-171, but it differs in its amino acid sequence due to natural changes or mutagenesis, as described above.

In another preferred embodiment, the protease protein has an amino acid sequence encoded by an isolated nucleic acid fragment capable of hybridizing with a nucleic acid of the corresponding 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 - SEQ ID NO: 114, preferably under conditions of hybridization of high stringency.

Accordingly, a protease protein is a protein comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or more homologous to the amino acid sequence represented in the sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, and retains at least one functional activity of the polypeptide corresponding to the sequence, selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171.

The functional equivalents of the protein of the present invention can also be identified, for example, by screening combinatorial libraries of mutants, for example, truncated protein mutants of the present invention, for protease activity. In one embodiment, a discrete library of variants is generated by combinatorial mutagenesis at the nucleic acid level. Such a discrete library of variants can be produced, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into a gene sequence so that a degenerate series of potential protein sequences is expressed as individual polypeptides or, alternatively, as a set of larger fusion proteins (e.g., for phage presentation ) A number of methods can be used to produce libraries of potential variants of the polypeptides of the present invention from a degenerate oligonucleotide sequence. Methods for the synthesis of degenerate oligonucleotides are known to those skilled in the art (see e.g. 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, libraries of fragments of the coding sequence of the polypeptide of the present invention can be used to generate a discrete population of polypeptides for screening for subsequent selection of options. So, for example, a library of fragments of a coding sequence can be generated by processing a double-stranded PCR fragment of the desired coding sequence with nuclease under conditions in which only one single-strand break (“nick”) falls on one molecule; by denaturing double stranded DNA; DNA renaturation with the formation of double-stranded DNA, which may include sense / antisense pairs from various nick products; removing single-stranded fragments from newly formed duplexes by treatment with S1 nuclease; and ligating the resulting library of fragments into an expression vector. Using this method, an expression library can be obtained that encodes the N-terminal and internal fragments of the desired protein of various sizes.

Several methods for screening gene products of combinatorial libraries obtained by truncated point mutations and screening cDNA libraries of gene products having a selected property are known to those skilled in the art. Methods suitable for high-performance assays and most commonly used for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming the corresponding cells with the resulting vector library, and expressing the combined genes under conditions in which the detection of the desired activity facilitates the isolation of a vector including the gene whose products have been detected. Recursive multiple mutagenesis (REM), which is a method of increasing the frequency of functional mutants in libraries, can be used in combination with screening assays to identify protein variants of the present invention (Arkin & 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 represented in the sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 57, it is obvious to a person skilled in the art that DNA polymorphism may be present in this population, which can lead to changes in the amino acid sequence protease protein. Such genetic polymorphism may be present in cells of different populations or within the same population, due to natural allelic variation. Allelic variants may also include functional equivalents.

Fragments of a polynucleotide of the present invention may also include polynucleotides that do not encode functional polypeptides. Such polynucleotides may function as probes or primers for the PCR reaction. Such polynucleotides can also be used if it is desirable to prevent the functional activity of the protease in a particular organism (“knockout” mutants).

The nucleic acids of the present invention, whether they encode functional or non-functional polypeptides, can be used as hybridization probes or primers for the polymerase chain reaction (PCR). The use of nucleic acid molecules of the present invention that do not encode a polypeptide having protease activity includes, but is not limited to, (1) isolating a gene encoding a protease protein or its allelic variants from a cDNA library, for example, from other organisms other than A. niger ; (2) in situ hybridization (e.g., FISH) with metaphase chromosome preparations to ensure accurate chromosome localization of the protease gene, as described by Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis to detect protease mRNA expression in specific tissues and / or cells; and (4) the possible use of probes and primers as a diagnostic tool for analyzing the presence of a nucleic acid hybridizing with a protease probe in a given biological sample (e.g. in tissue).

A method for preparing a functional equivalent of a protease gene or cDNA is also within the scope of the present invention. Such a method involves the preparation of a labeled probe that comprises an isolated nucleic acid encoding all or part of a sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, or a variant thereof; screening a library of nucleic acid fragments using a labeled probe under conditions that allow hybridization of the probe with nucleic acid fragments in the library to form nucleic acid duplexes; and producing a full-sized gene sequence from nucleic acid fragments in any labeled duplex to produce a gene related to the protease gene.

In one embodiment of the invention, the protease nucleic acid of the present invention is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the nucleic acid sequence represented in the sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 57, or a sequence selected from the group consisting of SEQ ID NO: 58-SEQ ID NO: 114, or its complementary sequences.

In another preferred embodiment, the protease polypeptide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more is homologous to the amino acid sequence represented in the sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171.

Host cells

In another embodiment, the present invention relates to cells, for example, transformed host cells or to recombinant host cells that contain a nucleic acid within the scope of the present invention. The term “transformed cell” or “recombinant cell” means a cell (or its precursor) into which, using recombinant DNA methods, the nucleic acid of the present invention has been introduced. Prokaryotic and eukaryotic cells are, for example, bacteria, fungi, yeast and the like, and cells of filamentous fungi, in particular Aspergillus niger , are particularly preferred.

The host cell can be selected so that it modulates the expression of the inserted sequences or modifies and processes the gene product in accordance with the specific desired mechanism. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate the optimal functioning of the protein.

Various host cells have characteristic and specific mechanisms of post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems known to those skilled in the field of molecular biology and / or microbiology can be selected so as to provide the desired and correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells with a cellular mechanism providing the appropriate processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. 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, MDKK, 293, 3T3, WI38, and choroid plexus cell lines.

If necessary, the polypeptides of the present invention can be produced using stably transfected cell lines. There are a number of vectors suitable for stable transfection of mammalian cells and available to those skilled in the art, and methods for constructing such cell lines are described, for example, described by Ausubel et al. (see above).

Antibodies

In addition, the present invention relates to antibodies, such as monoclonal or polyclonal antibodies, which specifically bind to the protease proteins of the present invention.

As used herein, the terms “antibody” (Ab) or “monoclonal antibody” (mAb) mean intact molecules as well as antibody fragments (such as, for example, Fab and F (ab ') ₂ fragments) that are capable of specifically binding to a protease protein. Fab and F (ab ') ₂ fragments do not contain an Fc fragment of an intact antibody; more rapidly excreted from the bloodstream and may have a lower degree of nonspecific binding than an intact antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). Therefore, these fragments are preferred.

Antibodies of the present invention can be obtained by any methods. Thus, for example, cells expressing a protease protein or antigenic fragment thereof can be administered to an animal to stimulate the production of serum containing polyclonal antibodies. In a preferred method, a protease protein preparation is prepared and purified that is substantially free of natural impurities. Then, such a preparation is administered to an animal to produce a polyclonal antiserum with higher specific activity.

In a most preferred method, the antibodies of the present invention are monoclonal antibodies (or fragments thereof that bind to a protease protein). Such monoclonal antibodies can be obtained using the hybridoma technique (Kohler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol. 6: 511 (1976); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, pp. 563-681 (1981)). In general, such procedures include immunizing an animal (preferably a mouse) with a protease protein antigen or a cell expressing a protease protein. Splenocytes of such mice are isolated and fused to a suitable myeloma cell line. Any suitable myeloma cell line can be used in accordance with the present invention, however, it is preferable to use the parent myeloma cell line (SP ₂ O) available from the American Type Culture Collection, Rockville, pc. Maryland. After fusion, the resulting hybridoma cells were incubated in a selective NAT medium and then cloned by limited dilution, as described by Wands et al. (Gastro-enterology, 80: 225-232 (1981)). Hybridoma cells obtained by this selection are then analyzed to identify clones that secrete antibodies capable of binding to the protease protein antigen. Generally speaking, these polypeptides can be attached to a carrier protein, such as KLH, as described by Ausubel et al. (see above), mixed with adjuvant and injected into the host mammal.

In particular, various host animals can be immunized by injection of the desired polypeptide. Examples of suitable host animals are rabbits, mice, guinea pigs and rats. Various adjuvants may be used to enhance the immunological response depending on the type of host, including, but not limited to, Freund's adjuvant (complete and incomplete), mineral gel adjuvants such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, snail lymph hemocyanin, dinitrophenol, BCG (Calmet-Guerin Bacillus) and Corynebacterium parvum . Polyclonal antibodies are heterogeneous populations of antibody molecules present in the serum of immunized animals.

Such antibodies can be any immunoglobulin of classes IgG, IgM, IgE, IgA, IgD and any of their subclasses. Hybridomas producing the mAbs of the present invention can be cultured in vitro or in vivo .

After production, polyclonal or monoclonal antibodies are tested for specific recognition of the protease polypeptide or its functional equivalent in an immunoassay, such as Western blot analysis or immunoprecipitation analysis using standard techniques, such as those described by Ausubel et al. (see above). Antibodies that specifically bind to protease proteins or their functional equivalents can be used in the present 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 Aspergillus extracts).

Antibodies of the present invention are preferably produced using polypeptide fragments of the protease, which are probably also antigenic, as indicated by a criterion such as a high frequency of charged residues. For example, such fragments can be generated by standard PCR methods and then cloned into the pGEX expression vector (Ausubel et al., Supra). Fusion proteins can then be expressed in E. coli and purified using a glutathione agarose affinity matrix as described by Ausubel et al. (see above). If necessary, for each protein several (for example, two or three) hybrids can be generated, and each hybrid can be injected into at least two rabbits. An antiserum can be produced by successive injections, usually at least three booster injections. Basically, an antiserum is evaluated for its ability to immunoprecipitate a recombinant protease polypeptide or its functional equivalents, while unbound proteins can serve as a control to assess the specificity of the immune response.

Alternatively, the methods described for the production of single chain antibodies (US Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce single chain antibodies against the protease polypeptide or its functional equivalents. There are commercially available phage presentation library generation and screening kits available, for example, from Pharmacia.

In addition, examples of methods and reagents particularly suitable for use in generating and screening an antibody presentation library can be found, for example, in US Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 20791; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio / Technology, 9: 1370-1372; Hay et al. (1992) Hum. Antibod Hybridomas 3: 81-85; 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 expression of a protease gene or its functional equivalents, for example, in another Aspergillus strain.

For example, a protease polypeptide can be easily detected in standard immunoassays of cells or Aspergillus extracts. Examples of suitable assays include, but are not limited to, Western blotting, ELISA, radioimmunoassays, and the like.

The term “specific binding” means that the antibody recognizes a specific antigen and binds to it, for example, a protease polypeptide, and, essentially, does not recognize other unrelated molecules in the sample and does not bind to them.

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 present invention (e.g., a monoclonal antibody) can be used to isolate a polypeptide by standard methods, such as affinity chromatography or immunoprecipitation. In addition, such an antibody can be used to detect a protein (for example, in a cell lysate or in a cell supernatant) to evaluate the level and nature of expression of this polypeptide. These antibodies can also be used for diagnostic purposes to monitor the level of proteins in cells or tissues, as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen or to establish a diagnosis of aspergillosis.

Detection can be facilitated by binding the antibody to a detectable substance. Examples of detectable substances are various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes are horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetyltransferase; examples of suitable fluorescent materials are umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, danzyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials are luciferase, luciferin and equorin, and suitable examples of radioactive materials are ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ N.

Preferred epitopes encompassed by the antigenic peptide are regions located on the surface of the protein, for example, hydrophilic regions. The hydrophobicity plots of the proteins of the present invention can be used to identify hydrophobic regions. The antigenic protein peptide of the present invention includes at least 7 (preferably 10, 15, 20 or 30) contiguous amino acid residues of an amino acid sequence selected from the group consisting of SEQ ID NO: 115 - SEQ ID NO: 171, and contains an epitope of the protein , which stimulates the production of antibodies against the peptide, forming a specific immune complex with a protein.

Preferred epitopes encompassed by the antigenic peptide are protease regions that are localized on the surface of the protein, such as hydrophilic regions, hydrophobic regions, alpha regions, beta regions, helical regions, turn regions, and flexible regions.

Immunoassays

Qualitative or quantitative determination of the polypeptide of the present invention in a biological sample can be carried out by any known method. An antibody technique is particularly preferred for evaluating specific levels of polypeptides in a biological sample.

In this case, specific recognition is carried out by the “first” antibody (polyclonal or monoclonal antibody), and antibodies conjugated with fluorescent substances, enzymes, or other conjugated “second” antibodies can be used in the secondary detection system. The result is an immunocomplex.

In accordance with this, the present invention relates to a method for the diagnosis of Aspergillus infection in a particular organism, comprising the steps of:

- isolation of a biological sample from the specified organism, which is supposed to be infected with Aspergillus ,

- the interaction of the specified biological sample with the antibody of the present invention,

- assessment of the formation of immunocomplexes.

To release protein for Western blot analysis or dot / blot analysis, tissues can also be extracted, for example, with urea and a neutral detergent. This technique can also be applied to physiological fluids.

Other antibody-based methods used to detect protease gene expression are immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA). For example, protease-specific monoclonal antibodies can be used as an immunoabsorbent and as an enzyme-labeled probe for the detection and quantification of a protease protein. The amount of protease protein present in a given sample can be calculated from the amount of protein present in a standard preparation using a linear regression computer algorithm. In another ELISA assay, two different specific monoclonal antibodies can be used to detect a protease protein in a biological fluid. In this assay, one of the antibodies is used as an immunoabsorbent, and the other as an enzyme-labeled probe.

The above methods can be implemented mainly in the form of a “one-stage” or “two-stage” analysis. “One-step analysis” involves contacting the protease protein with an immobilized antibody without washing and contacting this mixture with a labeled antibody. A “two step analysis” involves washing and then contacting this mixture with a labeled antibody. Other suitable methods may also be used. It is usually preferable to immobilize one component of the analytical system on a carrier, which allows other components of this system to come into contact with the specified component and easily stand out from the sample.

Suitable enzyme labels are, for example, labels derived from an oxidase group that catalyzes the production of hydrogen peroxide by reaction with a substrate. The activity of an oxidase tag can be analyzed by measuring the concentration of hydrogen peroxide formed by the interaction of an enzyme-labeled antibody with a substrate.

In addition to enzymes, other suitable labels are radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ N), indium ( ¹¹² In) and technetium ( ^99m Tc), as well as fluorescent labels such as fluorescein and rhodamine, and biotin.

Specific binding of the test compound to the protease polypeptide can be detected, for example, in vitro , by reversibly or irreversibly immobilizing the protease polypeptide on a substrate, for example, on the surface of a well of a 96-well polystyrene microtiter plate. Methods for immobilizing polypeptides and other small molecules are well known in the art. So, for example, microtiter plates can be sensitized with a protease polypeptide by adding to each well a polypeptide in solution (usually at a concentration of 0.05 to 1 mg / ml in a volume of 1-100 μl) and incubating these plates at a temperature ranging from room temperature up to 37 ° C for 0.1-36 hours. Polypeptides not associated with the plate can be removed by “shaking out” the excess solution from the plate, followed by washing the plate (single or multiple) with water or buffer. Typically, the polypeptide is contained in water or in buffer. Then the tablet is washed with a buffer that does not contain a bound polypeptide. To block sites that bind to free protein on the tablets, these plates are blocked with a protein that is not related to the associated polypeptide. For example, 300 μl of bovine serum albumin (BSA) at a concentration of 2 mg / ml in Tris-HCl is suitable for this purpose. Suitable substrates are substrates that contain a specific crosslinking chemical agent (for example, plastic substrates such as polystyrene, styrene or, for example, polypropylene substrates from Corning Costar Corp. (Cambridge, MA). If necessary, granular can be used as a substrate a particle, for example, granular agarose or granular sepharose.

The binding of test compounds to the polypeptides of the present invention can be detected by any of the known methods. So, for example, a specific antibody can be used in an immunoassay. If necessary, this antibody can be labeled (e.g., with a fluorescent label or radioisotope) and detected by the direct method (see, for example, West & 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 tagged (e.g., tagged with a radioactive isotope, fluorophore, chromophore or the like) and the label is detected. In yet another method, the protease polypeptide is produced as a fusion protein that can be optically detected, for example, with a protein that fluoresces in the green spectrum (which can be detected by UV radiation). In an alternative method, the protease polypeptide may be covalently attached to an enzyme or fused to an enzyme having detectable enzymatic activity, such as horseradish peroxidase, alkaline phosphatase, alpha-galactosidase or glucose oxidase. Genes encoding all of these enzymes have been cloned and readily available to every person skilled in the art. If necessary, the fusion protein may include an antigen, and such an antigen can be detected and measured using a polyclonal or monoclonal antibody by standard methods. Suitable antigens are enzymes (e.g., horseradish peroxidase, alkaline phosphatase and α-galactosidase) and non-enzymatic polypeptides (e.g., whey proteins such as BSA and globulins, as well as milk proteins such as caseins).

Epitopes, antigens and immunogens

In another aspect, the present invention relates to a peptide or polypeptide comprising an epitope-carrying portion of a polypeptide of the present invention. The epitope of this portion of the polypeptide is the immunogenic or antigenic epitope of the polypeptide of the present invention. The term “immunogenic epitope” is defined as the part of a protein that produces a humoral response if all of the protein is immunogenic. Obviously, these immunogenic epitopes on a molecule are limited to several loci. On the other hand, the region of a protein molecule that an antibody can bind to is defined as an “antigenic epitope”. The number of immunogenic protein epitopes is usually less than the number of antigenic epitopes. See, for example, Geysen H.M. et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984).

Regarding the choice of peptides or polypeptides that carry an antigenic epitope (i.e., containing a region of a protein molecule that an antibody can bind to), it is well known to those skilled in the art that relatively short synthetic peptides that mimic part of a protein sequence are usually capable of producing an antiserum that reacts partially simulated protein. See, for example, Sutcliffe J.G. et al., Science 219: 660-666 (1984). Peptides capable of producing a protein-reactive serum and often presented in the primary protein sequence can be characterized according to simple chemical rules and are not limited to either immunodominant regions of an intact protein (i.e. immunogenic epitopes) or amino or carboxy terminus. Peptides that are highly hydrophobic, and peptides consisting of six or less residues, are usually ineffective in inducing antibodies that bind to the simulated protein, while longer soluble peptides, especially peptides containing proline residues, are usually effective. Sutcliffe et al., Supra. So, for example, 18-20 peptides designed in accordance with the above guidelines and containing 8-39 residues, covering 75% of the sequence of the hemagglutinin HAI polypeptide chain of the influenza virus, induced antibodies that reacted with the HAI protein or with the intact virus; and peptides 12/12 derived from MuLV polymerase and peptide 18/18 derived from rabivirus glycoprotein induced antibodies that precipitated the corresponding proteins.

Peptides and polypeptides of the present invention carrying an antigenic epitope can be used to produce antibodies, including monoclonal antibodies that specifically bind to the polypeptide of the present invention. Thus, most hybridomas obtained by fusion of spleen cells from donors immunized with a peptide carrying an antigenic epitope usually secrete an antibody that reacts with the native protein. Sutcliffe et al., Supra, 663. Antibodies produced by an antigenic epitope carrying peptides or polypeptides can be used to detect an imitated protein, and antibodies against different peptides can be used to track the “fate” of different regions of the precursor proteins, which undergo post-translational processing. Peptides and antibodies against peptides can be used in various qualitative or quantitative assays to determine the mimic protein, for example, competitive binding assays, since it has been shown that even short peptides (for example, consisting of approximately 9 amino acids) can bind and replace larger peptides in immunoprecipitation assays. See, for example, Wilson I.A. et al., Cell 37: 767-778, at 777 (1984). Antibodies against the peptides of the present invention can also be used to purify a simulated protein, for example, by adsorption chromatography by methods well known in the art.

Antigenic epitope-carrying peptides and polypeptides of the present invention, constructed in accordance with the above instructions, contain a sequence consisting of at least seven, more preferably at least nine, and most preferably about 15-30 amino acids present in the amino acid polypeptide sequences of the present invention. However, peptides or polypeptides comprising a larger portion of the amino acid sequence of the polypeptide of the present invention, containing from about 30 to 50 amino acids or having any length, including the full amino acid sequence of the polypeptide of the present invention are also considered epitope-carrying peptides or polypeptides of the present invention and can be used to induce antibodies reacting with simulated protein. Preferably, the amino acid sequence of the epitope-carrying peptide is chosen so that it provides good solubility in aqueous solvents (that is, so that this sequence includes relatively hydrophilic residues and, preferably, mainly does not contain a high degree of hydrophobic residues); and particularly preferred are sequences containing proline residues.

The epitope-carrying peptides and polypeptides of the present invention can be produced by any standard method for producing peptides or polypeptides, including recombinant methods using the nucleic acid molecules of the present invention. For example, a short epitope-carrying amino acid sequence can be fused to a larger carrier-acting polypeptide in the process of recombinant production and recombinant purification, as well as in the immunization process to produce antibodies against peptides.

Epitope-carrying peptides can also be synthesized by known chemical synthesis methods. For example, Houghten described a simple method for the synthesis of a large number of peptides, such as 10-20 mg from 248 different peptides, consisting of 13 residues and representing variants of the HAI polypeptide segment, which differ by one amino acid and which were obtained and characterized (in the analyzes ELISA binding) in less than four weeks. Houghten R.A. Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985). This process of “simultaneous synthesis of multiple peptides (SMPS)” was described in detail in US patent No. 4631211, Houghten et al. (1986). In this procedure, individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable bags, which allows many identical repeating steps to be carried out optimally in solid-state methods.

A manual procedure allows 500-1000 or more syntheses to be performed simultaneously. Houghten et al., Supra, 5134.

Epitope-carrying peptides and polypeptides of the present invention are used to induce antibodies in accordance with methods well known in the art. See, for example, Sutcliffe et al., See above; Wilson et al., Supra; 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).

In general terms, animals can be immunized with a free peptide; however, the anti-peptide antibody titer can be increased by binding the peptide to a macromolecular carrier such as cochlear lymphocyte hemocyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine can be coupled to the carrier via a linker, such as maleimidobenzoyl-N-hydroxysuccinimidoester (MBS), and other peptides can be coupled to the carrier via a more general binding agent, such as glutaraldehyde.

Animals, such as rabbits, rats, and mice, are immunized with either free or carrier-bound peptides, for example, by intraperitoneal and / or intradermal injection of emulsions containing 10 μg of peptide or carrier protein and Freund's adjuvant. It may be necessary to administer several booster injections, for example, at intervals of about two weeks, to obtain the desired antibody titer against the peptide, which can be detected, for example, by ELISA analysis using a free peptide adsorbed on a solid surface. The anti-peptide antibody titer in the serum of the immunized animal can be increased by selecting anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and eluting the selected antibodies in accordance with methods well known in the art.

The immunogenic epitope-carrying peptides of the present invention, that is, those parts of a protein that produce a humoral response if the entire protein is an immunogen, are identified by methods known to those skilled in the art. For example, Geysen et al., 1984, supra, described a procedure for the rapid competitive synthesis on solid supports of hundreds of peptides with a purity sufficient for the reaction in enzyme-linked immunosorbent assay. The interaction of the synthesized peptides with antibodies can then be easily detected without separating them from the carrier. In this method, a peptide carrying an immunogenic epitope of the desired protein can be identified by standard methods known to those skilled in the art. For example, the localization of the immunologically important epitope in the envelope protein of foot and mouth disease virus with a resolution of seven amino acids was determined by Geysen et al. By synthesizing an overlapping series of all 208 possible hexapeptides covering the entire protein sequence of 213 amino acids. Then, a complete series of substitution peptides was synthesized, in which all 20 amino acids, in turn, were replaced at each position within the epitope, and specific amino acids were identified that reported specificity for the reaction with the antibody. Thus, peptide analogs of the epitope carrying peptides of the present invention can be easily obtained by this method. In US patent No. 4708781, Geysen (1987), in addition, describes a method for identifying a peptide that carries an immunogenic epitope of the desired protein.

In addition, US Pat. No. 5,194,392 to Geysen (1990) describes a general method for detecting or determining the sequence of monomers (amino acids or other compounds), which is the topological equivalent of an epitope (that is, a mimotope) that is complementary to a particular paratope (angigen binding site) the desired antibody. More specifically, US Pat. No. 4,433,092 to Geysen (1989) describes a method for detecting or determining the sequence of monomers, which is the topographic equivalent of a ligand complementary to a particular ligand binding site of a particular receptor. Similarly, in US patent No. 5480971, Houghten, R.A. et al. (1996) relating to mixtures of peralkylated oligopeptides, linear peralkylated C1-C7 alkyl oligopeptides are described; kits and libraries of such peptides, as well as methods for using such kits and libraries of oligopeptides to determine the sequence of a peralkylated oligopeptide that binds predominantly to the desired acceptor molecule. Thus, non-peptide analogs of the epitope-carrying peptides of the present invention can also be routinely made by these methods.

Suppression or reduction of protease activity

The present invention also relates to methods for producing a mutant cell derived from a parent cell, which include disruption or deletion of a nucleic acid sequence encoding a protease or its regulatory sequence, which leads to the formation of a mutant cell producing less protease than the parent cell.

The construction of strains that have reduced protease activity can be mainly carried out by modifying or inactivating the nucleic acid sequence necessary for expression of protease activity in the cell. A modifiable or inactivable nucleic acid sequence may be, for example, a nucleic acid sequence encoding a protease or part thereof that plays an important role in providing protease activity, or this nucleic acid sequence may have the regulatory function necessary for expression of a protease from a nucleic acid encoding sequence. An example of such a regulatory or control sequence may be a promoter sequence or its functional part, that is, a part that is sufficient to influence protease expression. Other regulatory sequences for possible modification include, but are not limited to, 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 accomplished by mutagenesis of a given cell and selection of cells in which the ability to produce a protease has been reduced or suppressed. Mutagenesis, which may be specific or non-specific, can be carried out, for example, using a suitable physical or chemical mutagenizing agent, using a suitable oligonucleotide or by PCR-generated mutagenesis of a DNA sequence. In addition, mutagenesis can be carried out using any combination of such mutagenizing agents. Examples of physical or chemical mutagenizing agents suitable for use in the present invention are ultraviolet (UV) radiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS) , sodium bisulfite, formic acid and nucleotide analogues. When using such agents, mutagenesis is usually carried out by incubating the mutagenized cell in the presence of the selected mutagenizing agent under suitable conditions and selecting cells that have reduced protease activity or do not express such protease activity.

Modification or inactivation of the protease production of the present invention can be carried out by introducing, replacing or removing one or more nucleotides in the nucleic acid sequence encoding the protease, or in the regulatory element necessary for its transcription or translation. So, for example, nucleotides can be inserted or deleted so that this leads to the introduction of a stop codon, removal of the start codon or to a change in the open reading frame. Such modification or inactivation can be carried out using site-directed mutagenesis or PCR-generated mutagenesis by methods known in the art.

Although, in principle, the modification can be carried out in vivo , that is, directly in the cell expressing the modified nucleic acid sequence, it is preferable that the modification was carried out in vitro , as described below.

An example of a suitable way to inactivate or reduce protease production by a selected host cell is a method based on gene replacement or gene disruption. So, for example, in a gene interruption method, a nucleic acid sequence corresponding to an endogenous gene or a fragment of a desired gene is subjected to in vitro mutagenesis to produce a defective nucleic acid sequence, which is then transformed into a host cell to produce a defective gene. Through homologous recombination, a defective nucleic acid sequence replaces an endogenous gene or gene fragment. It may be desirable for the defective gene or gene fragment to also encode a marker that can be used to select transformants in which the gene encoding the protease has been modified or destroyed.

Alternatively, the modification or inactivation of the nucleic acid sequence encoding the protease of the present invention can be carried out by known methods using the antisense nucleotide sequence complementary to the protease encoding sequence. More specifically, the production of a protease by a cell can be reduced or suppressed by introducing a nucleotide sequence complementary to a protease encoding nucleic acid sequence that can be transcribed in a cell and capable of hybridizing with the mRNA of the protease produced in that cell. Under conditions that allow a complementary antisense nucleotide sequence to hybridize with protease mRNA, the amount of protease translated in this way is reduced or suppressed.

Moreover, it is preferable that the cell modified by the methods of the present invention have a microbial origin, for example, comes from a fungal strain suitable for producing the desired protein products that are homologous or heterologous to the cell.

In addition, the present invention relates to a mutant cell derived from a parent cell and comprising disruption or deletion in a nucleic acid sequence encoding a protease or in a regulatory sequence, as a result of which this mutant cell produces less protease than the parent cell.

Thus obtained protease-deficient mutant cells are particularly suitable as host cells used to express homologous and / or heterologous polypeptides. Therefore, the present invention relates to methods for producing a homologous or heterologous polypeptide, comprising (a) culturing a mutant cell under conditions suitable for producing a polypeptide, and (b) isolating the polypeptide. As used herein, the term “heterologous polypeptides” means polypeptides that are not native to the host cell; a native protein that has been modified to modify the native sequence, or a native protein whose expression has been quantified by modifying the host cell by recombinant DNA methods.

The methods of the present invention for the production of a substantially protease-free product are of particular interest for 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 or pharmaceutical industries.

The use of proteases in industrial production

The present invention also relates to the use of the protease of the present invention in certain methods used in industry and in pharmaceuticals. Despite many years of experience using these methods, the protease of the present invention has a number of significant advantages compared to the currently used enzymes. Depending on the particular application, such advantages may include factors such as lower product costs, higher substrate specificity, lower antigenicity, lower levels of undesirable side effects, higher yields when produced in a suitable microorganism, more suitable pH and temperature ranges, better taste of the final product, as well as higher quality food and its kosher.

In large-scale industrial production of feed or food products, proteolytic enzymes are usually used to improve parameters such as protein solubility, yield of extraction products, viscosity or taste, texture, nutritional value, minimizing antigenicity or level of harmful factors, color or functionality, as well as to improve technological parameters, such as filterability of protein raw materials. For these applications, protein feed can be obtained from an animal or plant, and plant proteins such as soy protein, wheat gluten, rapeseed protein, pea protein, alfalfa protein, sunflower protein, legume protein, cotton or sesame protein can serve as examples thereof. , corn protein, barley protein, sorghum protein, potato protein, rice protein, coffee proteins and animal proteins such as milk protein (e.g. casein, whey protein), egg white, fish protein, meat protein, including gelatin, call gene, blood protein (e.g. hemoglobin), hair proteins, feathers and fish meal.

An important property of the proteases of the present invention is that they operate at an optimal pH range and optimum temperature, which are ideal for various purposes. So, for example, to minimize the risk of microbial infections, many large-scale processes are preferably carried out at relatively high processing temperatures, i.e. at 50 ° C or higher. Some proteases of the present invention satisfy these requirements, but they do not have excessive thermal stability, such as would resist all attempts to inactivate this enzyme by additional heat treatment. The last of these distinguishing features allows for production, which gives the final products that do not contain residual proteolytic activity. Similarly, many feeds and food products have slightly acidic pH values, and therefore, it is preferable to use proteases with an optimal acidic pH or an almost neutral pH to process them. The protease of the present invention also satisfies these requirements.

The specificity of endoproteases is usually determined by the preferred cleavage of the bonds between the carboxyl of the amino acid residue at position P1 and the amino group of this residue at position P1 ', respectively. Preference can be determined predominantly either by P1 (e.g., positively charged residues in trypsin substrates), or P1 '(e.g., hydrophobic residues when cleaved by thermolysin), or P1 and P2 (e.g., specific cleavage of bonds between two positively charged residues of a serine endoprotease of the brain substance adrenal gland). In some cases, more distant residues may determine cleavage preference, for example, P2 is preferred for streptococcal peptidase A. Some residues are known to adversely affect cleavage, and it is also well known that proline bonds at position P1 'are resistant to many proteases. Most endoproteases are cleaved predominantly either in a hydrophobic environment or in the vicinity of negatively charged residues. For example, endoproteases used in industry, such as chymotrypsin (derived from bovine pancreas) or subtilisin, neutral metalloendoprotease or thermolysin (obtained from Bacillus species), tend to cleave hydrophobic amino acids, such as -Phe, in the area behind. -Leu and -Tyr. Other endoproteases used in industry are trypsin (derived from bovine pancreas), which cleaves predominantly behind -Arg and -Lys, and papain (a complex mixture of various enzymes, including proteases derived from papaya fruit), which predominantly cleaves behind -Arg. In contrast, peptide bonds formed by small residues such as Ala, Gly, Ser, Thr, as well as Ile and Pro are poor substrates (Keil B. et al., Protein Seq Data Anal. (1993), 5; 401-407). This situation creates certain difficulties for the pharmaceutical industry, namely in the production of food and beverages, in agriculture and in the chemical industry. The protease of the present invention has an unusual preference for cleavage.

Exopeptidases act only near the ends of the polypeptide chains. Such an action of peptidases at the free N-terminus results in the release of one amino acid residue (the so-called aminopeptidases), or a dipeptide, or a tripeptide (the so-called dipeptidyl peptidases and tripeptidyl peptidases). Such an action of peptidases at the free C-terminus leads to the release of one amino acid residue (the so-called carboxypeptidases) or a dipeptide (the so-called peptidyl dipeptidases). According to their catalytic mechanism, carboxypeptidases can be divided into three groups, namely, serine-type carboxypeptidases, metallocarboxypeptidases, and cysteine-type carboxypeptidases. Other exopeptidases are specific for dipeptides (the so-called dipeptidases) or capable of cleaving peptide bonds that are not bonds of alpha-carboxyl or alpha-amino groups (so-called omega-peptidases). Examples of such new omega-peptidases are pyroglutamyl peptidase and acytinoacyl peptidase identified in the present invention (see table 1, genes 18 and 45, respectively).

Typical examples of industrial applications that depend on the use of pure endoproteases and in which the protease of the present invention is expected to have superior properties are the processing of materials of plant or animal origin. These processing steps can be designed to modify a large array of raw material characteristics or (partially) purified protein fractions. For example, these processing steps can be carried out to maximize product solubility, filterability, release ability, protein yields after extraction and digestibility, or to minimize toxicity, extraneous taste and viscosity. In addition, such processing can be aimed at changing the physicochemical properties of the raw material or purified (or partially purified) protein. These advantages can be used not only in cases where the endoprotease of the present invention is used to facilitate processing in industrial production, but also in cases where it is used as an active enzyme component in animal feed. In particular, the endoprotease of the present invention can be used as a bread improver in the baking industry, for example, to slow down stale bread or to reduce the viscosity of the dough. Or endoprotease can be used in the brewing and wine industries to prevent or minimize the formation of unwanted protein clouding. Alternatively, it can be used in the brewing industry to optimize the yields of cereal protein extraction products used to produce wort. In addition, it can also be advantageously used in the dairy industry as a milk coagulation agent with improved properties or to optimize the texturing, foaming or coagulation properties of various milk components. Another application of the new protease of the present invention in the dairy industry is its use for the preparation of modified fermented cheeses (MFS).

In addition, various protein substrates can be exposed to the endoprotease of the present invention, usually in combination with other proteolytic enzymes to produce hydrolysates intended for use in medical and non-medical purposes. It was unexpectedly discovered that the endoprotease of the present invention is effective for the complete hydrolysis of the protein substrate, such that even protease-resistant parts are completely hydrolyzed, and in addition, it was unexpectedly found that this endoprotease also has activity aimed at minimizing allergenicity of the final hydrolyzate or to suppress the formation of a bitter taste.

More specifically, the endoprotease of the present invention is distinguished by its preferences in cleaving unusual protein peptide bonds, especially in small amino acid residues such as Ala, Gly, Ser and Thr, or in Ile and Pro residues, at position P1 or at P1 '(Keil B . et al., Protein Seq Data Anal. (1993) 5; 401-407). As a result of this, the obtained fractions of protein starting materials that are resistant to hydrolysis after using known endoproteases can be dissolved and hydrolyzed using the endoprotease of the present invention. Non-limiting examples of such protease-resistant fractions are so-called extensins in plant materials, collagen, gelatin, as well as specific dairy components in animal materials.

Various foods, such as soybeans, contain trypsin inhibitors. These proteins inhibit trypsin activity in the gastrointestinal tract, for example, in pigs and poultry. This trypsin-inhibiting activity leads to insufficient protein digestibility by these animals, and therefore to an increase in waste and an increase in material costs. This problem can be partially solved by frying soybeans at high temperatures. Two different types of trypsin inhibitors have been identified in soybeans, namely Bowman-Birk trypsin inhibitors and Kunitz trypsin inhibitors.

The present invention also relates to an alternative way of destroying trypsin-inhibitory activity during frying, which is provided by cysteine proteases (EC 3.4.22, table 1), capable of cleaving peptide bonds leucine 176 - aspartate 177 near the carboxyl end of the Kunitz trypsin inhibitor (as described by Wilson (1988 ), CRC Critical Reviews in Biotechnology 8 (3): 197-216). This pathway leads to the inactivation of the indicated trypsin inhibitor in soy. It was unexpectedly discovered that cysteine proteases secreted by Aspergillus niger fungi satisfy these criteria much better than similar enzymes originating from other organisms.

Proteases are also widely used in the production of cheese. In the production of cheese, milk for cheese making must be coagulated in order to separate cheese from milk whey, such as casein. Some milk coagulation enzymes, also called coagulants, have been described, and these enzymes are (bovine) chymosin, bovine pepsin, pork pepsin, as well as microbial enzymes such as Rhizomucor miehei protease, Rhizomucor pusillus protease and Cryptonectria parasitica protease . Chymosin can be obtained from the calf’s stomach, but it can also be produced by microbes, such as Kluyveromyces lactis . All these enzymes are characterized by the fact that they have specificity for the peptide bond between residue 105 (phenylalanine) and residue 106 (methionine) or to a bond adjacent to the bond in ĸ-casein. This means that when using such enzymes in the production of cheese, ĸ-casein is cleaved at the junction between the para-ĸ-casein and the macropeptide part, called glycomacropeptide (GMP), which carries negative charges. If the macropeptide diffuses into whey, then its stabilizing effect on the solubility of casein micelles is lost, and these casein micelles can begin to aggregate after sufficient hydrolysis of kappa-casein. A detailed description of the enzymatic coagulation of milk can be found, for example, in DG Dalgleish, Advanced Dairy Chemistry, vol. 1 ed. by PF Fox, Elsevier, London, 1992.

The coagulants currently used make it possible to obtain a sufficiently high yield of cheese, however, it should be taken into account that this is due to the huge volumes of cheese produced, and an increase in output by about one tenth of a percent can give tangible economic benefits. Therefore, there is currently an urgent need for coagulants giving an increase (even a small) yield.

Coagulants are characterized by their high specificity for the substrate, which usually depends on pH and temperature. In conventional cheese production processes, the pH can vary from an initial value of 6.3 to lower values in the range of 4.5-5.5, the final pH value depending on the conditions used in the cheese preparation process. Some coagulants are more susceptible to pH changes than others. For example, the protease Rhizomucor pusillus is more sensitive to pH changes than chitosin. In addition to pH, other parameters, such as temperature and water content, can influence protease specificity. It is well known that most coagulants exhibit a change in substrate specificity with a change in pH, which leads to a change in proteolytic activity in the final stages of cheese preparation. It is also very well known that coagulants differ in the degree of proteolysis of casein; and, in addition, they can detect differences in the types of peptides produced during proteolysis. They are important factors in the ripening process of the cheese and can affect the properties of the cheese, such as taste, smell and texture. In some cases, coagulants produce undesirable effects, such as the appearance of a bitter taste caused by peptides or an extraneous taste. In addition, a change in proteolytic specificity can lead to a decrease in yield. Pepsin, a well-known component found in many preparations of bovine chymosin, is an example of a protease that produces lower yields and poorer flavoring effects than pure chymosin. Therefore, it is necessary to obtain coagulants that would give the cheese a new improved texture and taste. Such new coagulants will lead to an accelerated formation of taste characteristics and consistency profiles associated with the ripening of cheese, which can give tangible economic benefits.

It is well known that free amino acids are very important for the formation of taste and smell. In particular, the amino acids leucine, phenylalanine, methionine and valine play an important role in the formation of the components of a typical cheese flavor and odor. The conversion of free amino acids occurs through fermentation under the influence of microorganisms, which are added during the preparation of cheese to compounds that form the desired smell and taste, such as methanediol, dimethyldisulfide, methylpropanoic acid and methylpropanal. Exopeptidases play an important role in the formation of free amino acids. However, they can only be effective if they act together with an endoprotease of appropriate specificity. In the preparation of cheeses, appropriate combinations of exo- and endopeptidases can be used, as a result of which cheeses with new and improved palatability can be obtained.

The enzymes of the present invention can be used to hydrolyze protein materials of animal origin, such as whole milk, separated milk, casein, whey protein or a mixture of casein and whey protein. Such mixtures of casein and whey protein can be used, for example, in relationships corresponding to those found in human milk. In addition, the enzyme mixture of the present invention can be used to hydrolyze proteinaceous materials of plant origin, such as, for example, wheat gluten, malt or non-malt barley or other cereals used for the production of beer, soy milk, its concentrates or isolates, corn protein, its concentrates or isolates and rice proteins.

For large-scale industrial production, some applications are based on the use of endoproteases alone, while other applications are mainly based on combinations of endoproteases and exoproteases. Typical examples of processes that rely on the use of pure endoproteases in which the proteases of the present invention can have excellent productivity are processes such as treating soy, pea or cereal proteins to minimize viscosity or optimize foaming or other physicochemical properties; the use of bread improvers in the baking industry, which also helps to reduce the viscosity of the dough; the use of technological additives in the brewing and wine industries, which helps prevent protein clouding or optimize yield after grain extraction; the use of feed additives in the production of bio-feed, which helps to increase the intestinal absorption or modulation of microbial activity in the intestine; the use of technological additives in the dairy industry, which helps to optimize the coagulation, foaming or coagulation properties of various components of milk. In addition, for specific market segments, milk or soy proteins or collagen are exposed to proteases in order to produce so-called protein hydrolysates. Although the hydrolysates of these proteins are mainly used for the manufacture of baby food and food products for hospitalized patients, they can also be intended for individuals who do not need medical attention, such as athletes or people on a diet for weight loss, and are An object of rapidly growing market demand. In all these applications, protein hydrolysates have such very attractive properties as reduced allergenicity, improved digestibility in the gastrointestinal tract, low level of chemical decomposition of the desired amino acids, such as glutamine and cysteine, and, finally, the absence of protein precipitate in acidic drinks during long-term storage. All these advantages can be combined if the hydrolyzate is a mixture of di- and tripeptides. However, all modern commercially available hydrolysates are prepared using a combination of several endoproteases. This latter method gives heterogeneous and incomplete decomposition of the protein. To obtain the desired mixture of di- and tripeptides, it would be ideal to use a combination of different di- and tripeptidyl peptidases in the hydrolysis process. Unfortunately, there are only a few food-grade enzymes and microorganisms suitable for industrial use, not to mention those actually available for industrial use. In accordance with the present invention, several widely used di- and tripeptidyl peptidases can be obtained in a cost-effective way and in relatively pure form. Preferred are those di- and tripeptidyl peptidases that have a low selectivity for cleavable substrate, that is, they prefer only to cleave a minimum number of amino acid residues. Combinations of those di- and tripeptidyl peptidases that hydrolyze a high percentage of naturally occurring peptide bonds are preferred. Despite such high activity with respect to natural peptide bonds, the complete hydrolysis of all amino acids depends on the nature of di- and tripeptidyl peptidases. Di- and tripeptidyl peptidases, which have optimal activity at pH 4-8 and have adequate thermal stability, are also preferred. Adequate thermal stability means that at least 40%, preferably at least 60%, and more preferably 70-100% of the initial hydrolytic activity is retained after heating the enzyme together with the substrate for 1 hour at 50 ° C.

Although a process aimed at efficiently producing mixtures of di- or tripeptides depends on the availability of the enzymes of the present invention, the endoprotease is usually the initial enzyme for incubation with a protein substrate. A preferred endoprotease with a wide range of endopeptidase activity suitable for this case is, for example, subtilisin (Delvolase from DSM), neutral metalloprotease (neutral from NOVO) or thermolysin (thermase from Daiwa Kasei) used under conditions close to neutral, and pepsin or aspegillopepsin (e.g., Sumizyme AP from Shin Nihon, Japan) used under acidic conditions. The purpose of this initial cleavage is to increase solubility, lower viscosity, and decrease the heat-shrinkability of a mixture of water and protein. In addition, this pretreatment with an endonuclease is important to create sufficient starting points for di- and tripeptidyl peptidases and accelerates the formation of a di- or tripeptide. The protease used to eliminate the bitterness of the hydrolyzate may be, but is not necessarily, included in this step of the process under consideration or later together with di- and tripeptidyl peptidases.

The main purpose of using the latest hydrolysates is to minimize the allergenicity of the product or to stimulate digestibility in the gastrointestinal tract. In the production of such hydrolysates, the use of dipeptidyl and tripeptidyl peptidases is particularly important and is an effective way to produce hydrolysates.

Another application in the production of food and feed is mainly based on combinations of one or more endoproteases with one or more exoproteases. Such combinations of endoprotease with exoprotease are commonly used in industry to improve parameters such as taste and color of the final product. The reason for this is that the formation of taste and color depends mainly on the presence of free amino acids. Free amino acids can be produced not only by proteases, such as carboxypeptidases and aminopeptidases, but also peptidyldipeptidases. When combined with endoproteases or even with dipeptidyl or tripeptidyl peptidases, carboxypeptidases, aminopeptidases and peptidyl dipeptidases can produce more free amino acids in a shorter period of time. However, in all these processes, the uncontrolled release of amino acids or even non-protein components must be prevented to minimize unwanted side reactions.

At the same time, free amino acids by themselves can produce a number of taste sensations, and these taste sensations are the most important (bitter, sweet, sour and “umami”), but a very high concentration of amino acids is necessary to distinguish between these tastes. Despite the high threshold values, free amino acids are able to create significant sensory effects at lower concentrations due to a number of taste-enhancing mechanisms. One of these mechanisms is the combination of free amino acids with sugars in the so-called Maillard reactions. Compared to free amino acids, these Maillard products with a very complex bouquet of taste and smell can be produced with threshold values that are several orders of magnitude lower than those produced by free amino acids. Maillard products can form at elevated temperatures, usually during cooking, baking, or frying when cooking food or feed. During this treatment, color and a large bouquet of aromas develop. In these reactions, in the first stage, amino groups react with reducing compounds, which ultimately leads to the implementation of a cascade of reactions. Amino compounds predominantly present in foods or feeds are free amino acids that are released from proteinaceous materials by various proteases, and the necessary reducing compounds are mainly reducing sugars. It follows that to minimize the extraneous taste that is generated during the subsequent heating stages, such as during spray drying or sterilization, it is necessary to prevent the undesired release of amino acids and sugars during processing of raw materials. This once again indicates that it is desirable to use the enzymes of the present invention, which have a high degree of purity and low cost.

In addition to Maillard reactions, amino acids can also undergo important chemical transformations at room temperature. Later types of conversion are enzyme-dependent processes and processes common to all fermented foods, such as the maturation of beer, yogurt and cheese, as well as the maturation of meat and wine. In these fermentation processes, free amino acids are released from the raw material used by the proteases or proteolytic enzymes present in the used raw material or microbial starter. Then, in the ripening phase, free amino acids under the influence of microbial metabolic activity turn into their derivatives having enhanced sensory properties. So, for example, L-leucine, L-isoleucine and L-valine during beer fermentation contribute to the formation of valuable fusel alcohols, such as amyl alcohols and isobutanol. Similarly, it was found that cheese volatile substances such as methanethiol and dimethyl disulfide indicate the presence of methionine in the cheese, and methylpropanoic acid and methylpropanal indicate the presence of valine. And finally, the free amino acid glutamate can give the effect of enhancing a pleasant spicy taste as a result of its synergistic action together with RNA cleavage products, the so-called 5'-ribonucleotides. It is known that when combined with appropriate concentrations of 5'-ribonucleotides, such as 5'-IMP and 5'-GMF, the threshold for detection of taste formation by umami generated by glutamate is reduced by almost two orders of magnitude.

To obtain distinct and well-defined taste effects in all of these procedures, protein substrates must be hydrolyzed using a combination of endo- and exoproteases, where at least one endo- or exoprotease, and preferably both endoprotease and exoprotease, are purified and preferably selective with respect to a particular series of amino acids or, preferably, preferred amino acids are cleaved. Such preferred proteases are highly selective for amino acid sequences that can be cleaved by Aspergillus enzymes, known as “maturases” and of particular value.

Besides being used in the food and feed industries, proteases are also commonly used in the chemical and pharmaceutical industries, as well as in the production of diagnostic reagents and personal care products. In the manufacture of personal care products, proteases are used to produce peptides that are added to various products to improve properties such as skin sensitivity and gloss, or to protect the skin. In addition, there is a new trend in the direct local use of proteases. Similarly, this enzyme can be used in the leather industry, and the main purpose of this application is to clean, remove wool from the skin and soften the skin.

In the chemical and pharmaceutical industries, proteases are seen as an important tool for the production of valuable ingredients or intermediates. In these industries, proteases are used not only because of their hydrolytic activity, but also because of their ability to synthesize peptides from natural or unnatural amino acids. The last possible use has been clearly demonstrated by the ability to synthesize aspartame from structural elements consisting of amino acids using an endoprotease such as thermolysin.

In contrast to the conditions used in the production of food and feed, the stereoselectivity and regioselectivity of proteases are very important for the desired chemical transformation, although in this case non-standard reaction conditions may be required. Typical examples of the use of proteases in such an industry are the use of endoproteases, aminopeptidases, as well as carboxypeptidases in the production of various intermediates for the manufacture of drugs such as insulin, antibiotics, renin and ACE inhibitors. A review of this application is presented in Industrial Biotransformations, A. Liese, K. Seelbach, C. Wandrey, Wiley-VCH; ISBH 3-527-30094-5.

With regard to the desired specificity, stereo- and regioselectivity, the important advantages of the protease of the present invention is the absence of side effects and resistance to unusual reaction conditions, such as high solvent concentrations, and increased productivity.

From a pharmaceutical point of view, the role of proteases is presented in a wide range of works by Martindale, “The Extra Pharmacopoeia” (Pharmaceutical Press, London, UK). In addition, the important role of highly specific proteases in the regulation of all types of biological processes is confirmed by the fact that many hormones become active only after processing, mainly, an inactive precursor molecule by this protease with very high specificity. Inhibitors that are active against certain categories of such specific proteases have been used in the development of all kinds of new drugs. Therefore, new and effective protease inhibitors can be identified using the sequences described here.

A complete description of each document cited herein is incorporated into this application by reference.

Table 1 SEQ ID Encoded protein function EU number Gene cDNA Protein one 58 115 Pepsin A ₃ EC3.4.23.1 2 59 116 Metalloprotease EC3.4.24.56 3 60 117 Acylaminoacyl peptidase EC3.4.19.1 four 61 118 Tripeptidylamine Peptidase EC3.4.14.- 5 62 119 Serine Carboxypeptidase EC3.4.16.6 6 63 120 Serine Endoprotease EC3.4.21.- 7 64 121 Carboxypeptidase Y EC3.4.16.5 8 65 122 Aspergillopepsin II. EC3.4.23.19 9 66 123 Tripeptidyl peptidase EC3.4.14.9 10 67 124 Tripeptidyl peptidase EC3.4.14.9 eleven 68 125 Aspergillopepsin II. EC3.4.23.19 12 69 126 Tripeptidyl peptidase EC3.4.14.9 13 70 127 Metalloprotease EC3.4.24.- fourteen 71 128 Aspergillopepsin I EC3.4.23.18 fifteen 72 129 Pepsinogen E EC3.4.23.25 16 73 130 Aspergillopepsin I-hom. EC3.4.23.18 17 74 131 Aspergillopepsin II EC3.4.23.19 eighteen 75 132 Pyro-glu-peptidase EC3.4.19.3 19 76 133 Dipeptidyl peptidase EC3.4.14.2 twenty 77 134 Secreted aminopeptidase EC3.4.11.10 21 78 135 Alkaline D-peptidase EC3.4.16.4 22 79 136 Carboxypeptidase EC3.4.16.1 23 80 137 Carboxypeptidase EC3.4.16.1 24 81 138 Carboxypeptidase-II EC3.4.16.1 25 82 139 Aspartic protease 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.- 29th 86 143 Metallocarboxypeptidase EC3.4.17.- thirty 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 34 91 148 Metalloprotease EC3.4.24.- 35 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 A ₃ 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 Keh EC3.4.21.61 42 99 156 Serine protease EC3.4.21.- 43 one hundred 157 Glutamyl Endoprotease EC3.4.21.82 44 101 158 Aspergillopepsin II. EC3.4.23.19 45 102 159 Acylaminoacyl peptidase EC3.4.19.1 46 103 160 Tripeptidylamine Peptidase 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 Secreted aminopeptidase EC3.4.11.10 55 112 169 Prolyl endopeptidase EC3.4.21.26 56 113 170 Aspergillopepsin I - hom. EC3.4.23.18 57 114 171 Aminopeptidase EC3.4.11.-

EXAMPLES

Example 1

Analysis for proteolytic activity and specificity

The specificity of prostheses can be investigated using various peptide substrates. Synthetic substrates are widely used for the detection of proteolytic enzymes in screening, in fermentation, during the isolation process, for the analysis of enzymatic activity, for determination of enzyme concentrations, for specificity studies, or for studies of interactions with inhibitors. Peptide p-nitroanilides are preferably used for the analysis of protease activity, and since such activity can be continuously monitored, this allows kinetic measurements. The cleavage of peptide p-nitroanilides can be monitored by measuring the increase in adsorption at 410 nm after the release of 4-nitroanilide. Paranitroanilide substrates are commonly used for serine and cysteine proteases. In addition, peptide thioethers and derivatives of 7-amino-p-methylcoumarin peptides are used. Peptide thioesters are very sensitive substrates for serine protease and metalloproteases, which have a relatively high speed of the functional cycle, since the thioether bond cleaves more easily than the amide bond. Thioether cleavage can be traced using a thiol reagent such as 4,4-dithiopyridine (324 nm) or 5,5-dithiobis-2-nitrobenzoic acid (405 nm). The same increased speed of the functional cycle is usually observed with the cleavage of ester bonds compared with the speed with cleavage of amide bonds. The most well-known substrates for the analysis of esterase activity of proteases are derivatives of p-nitrophenol. The release of p-nitrophenol can be observed at different wavelengths depending on the pH used; for example, at an almost neutral pH, a wavelength of 340 nm is used, and at a pH above 9, monitoring is performed at about 405 nm. In addition, monitoring the hydrolysis of esters can also be carried out by titration using pH-stat equipment. In the case of a qualitative measurement of esterase activity, pH-sensitive dyes can be used.

Alternatively, the peptides may be attached to a fluorescent leaving group. Proteolysis is accompanied by an increase in fluorescence when monitored at appropriate wavelengths. The most commonly used are peptidyl-2-naphthylamides and peptidyl-4-methyl-7-coumarilamides. The release of, for example, 7-amino-4-methylcoumarin was measured at 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 chromogenic (absorbance at 380 nm), as well as fluorogenic (excitation at 400 nm, radiation at 505 nm). If it is necessary that an amino acid is present on both sides of the cleavable bond, it may be useful to introduce a group that will quench fluorescence. The general properties of such substrates are that in them the fluorescent donor group is separated from the acceptor group by a peptide sequence that acts as a fluorescence quencher. Cleavage of the peptide bond between the quenching group and the fluorophore can lead to a significant increase in fluorescence. Several donor-acceptor pairs have been described, including o-aminobenzoic acid (Abz) used as a donor and 2,4-dinitrophenyl (Dnp) used as an acceptor, or 5 - [(2'-aminoethyl) amino ] naphthalenesulfonic acid (EDANS) used as a donor, and 4 - [[4 '- (dimethylamino] phenyl] azo] benzoic acid (DABCYL) used as an acceptor. Abz / EDDnp is the most suitable donor-acceptor pair ”, Because after complete hydrolysis, the fluorescence intensity increases by 7-100 times, and the absorption spectrum of EDDnp does not change The peptide sequence may contain up to 10 residues, but at the same time lose its quenching effect. As the size of the connecting peptides increases, the position of this cleavable bond may become less specific. Therefore, in addition to establishing a proteolysis event, it may be necessary to carry out additional analysis of these products.This can be done by analyzing and isolating the produced peptides using HPLC and determining the amino acid sequence of these fragment ov. In addition, the peptide composition of the hydrolyzate can be directly analyzed by a combined method using HPLC / mass spectroscopy.

In addition to using peptides of a specific sequence, synthetic peptide libraries can also be used to study protease specificity. Peptides were synthesized using solid-phase synthesis in a randomized or semi-randomized manner. For example, Meldal et al. (PNAS USA 91, 3314, 1994) report the preparation of a family of substrates for proteases by attaching an H-Lys (Abz) resin, lengthening the resin with peptides up to 6 amino acids in length, and finally attaching Tyr (NO ₂ ) to these peptides. Each resin granule has a unique sequence, and after treatment with proteases, the most sensitive granules become fluorescent as the Tyr (NO ₂ ) -containing peptide is released. An analysis of the sequence of these peptides for their sensitivity will provide information on the specificity of the protease.

Protease activity is usually expressed in units. Generally speaking, an “international standard unit” (ME) is defined as the amount of an enzyme that, under certain conditions, transfers one micromole of substrate per minute. In particular, with regard to proteases, ME should correspond to the hydrolysis of one micromole of peptide bond per minute. However, in the case of a protease, deviations in the definition of its units from the international standard are the rule rather than the exception. In the case of model peptides that specifically cleave on one bond, the calculation of ME is a narrowly targeted procedure, and for protein substrates where cleavage by this protease can be carried out in different positions and to different degrees, another definition can be adopted that differs significantly from the standard one. Besides the definition of activity units used, any hydrolysis experiment requires an adequate description of the conditions under which these units are measured. Such conditions include, for example, substrate concentration, enzyme-substrate ratio, pH and temperature. Typical assays to determine the specific activity of proteases include the use of a protein substrate, such as, for example, denatured hemoglobin, insulin or casein. The polypeptide substrate is cleaved by a protease under fixed conditions for a strictly defined period of time. Unsplit and large polypeptides are precipitated using TCA, and the TCA-soluble product is evaluated by measuring the optical density at 220 or 280 nm or by titration of soluble peptides with folin reagent, ninhydrin, fluoro-2,4-dinitrobenzene / danzil chloride, using the method of TNBS or fluorescein. Instead of labeling the product after hydrolysis, polypeptide substrates that have already been labeled with specific dyes or fluorophores, such as, for example, fluorescein, can also be used. In addition to standard methods, amino acid analysis can be carried out using standard laboratory analyzers. To assess the size distribution of the peptides generated by the protease, experiments using gel chromatography can be performed. In addition, HPLC using reverse phase techniques can be used to better isolate the types of peptides generated by the protease. The course of hydrolysis of protein substrates is usually evaluated by the degree of hydrolysis or DH. In the case of using a pH-stat to monitor the progress of hydrolysis, DH can be determined based on the absorption of the base during hydrolysis (Enzymatic Hydrolysis of Food Protein, J. Adler-Nissen, 1986, Elsevier Applied Science Publishers LTD). DN means various useful functional properties of the hydrolyzate, such as solubility, emulsifying ability, foaming and foam stability, increase in volume when whisking, organoleptic qualities. In addition, taste is an important parameter of food hydrolysates. Bitterness can be a major problem when using protein hydrolysates. The completion of the hydrolysis reaction can be carried out by changing the pH, thermal inactivation and the use of denaturing agents such as SDS, acetonitrile, etc.

The polypeptides shown in table 1 were expressed and at least partially purified by standard methods known in the art. These polypeptides were analyzed by at least one of the methods described above, and it was found that they have the activities listed in table 1.

Example 2

Direct determination of the kcat / Km ratio for protease substrates

Synthetic substrates were used to monitor the enzymatic activity during the purification process, to determine the concentration of the substrate, to determine the inhibition constants, or to study the substrate specificity. Determination of the kcat / Km ratio allows the measurement of substrate specificity. It also allows comparisons of the specificities of different substrates for the same enzyme or a comparison of the hydrolysis rates of different enzymes that cleave the same substrate. This ratio is a rate constant expressed in second order units and is defined as 1 / (concentration · time). Substrates having a kcat / Km ratio in the range of 10.5-10.6 M ^-1 · sec ^-1 are considered very good substrates, that is, having good affinity and a fast functional cycle. However, some substrates can be very specific, that is, have kcat / Km values of about 10.4 M ^-1 · sec ^-1 .

The ratio kcat / Km can be calculated after determining the individual parameters. In this case, Km and Vm can be determined from various linear curves (constructed, for example, by the Hans or Cornish-Bowden method) or by the nonlinear regression method. It is known that Vm = kcat · Et (where Et means the final concentration of the active enzyme), and therefore, kcat = Vm / Et. Determination of the kcat / Km ratio by the method described above can be difficult if the product or substrate is inhibited or if the substrate precipitates at a high concentration. However, it is possible to obtain the exact value of the kcat / Km ratio under first-order reaction conditions, that is, when the substrate concentration is significantly lower than the calculated Km. Under these conditions, the Michaelis-Menten equation: v = (Vm · S) / (Кm + S) has the form:

v = (Vm · S) / Km, if S << Кm

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

and after integration get: lnS = -kobs · t + lnSо, where So means the initial concentration of the substrate, and S means the concentration of the substrate at a given time. The speed is proportional to the concentration of the substrate. In other words, the hydrolysis of the substrate proceeds in accordance with a first-order reaction, where kobs is the first-order rate constant: kobs = Vm / Кm = (kcat · Et) / Кm, if Vm = kcat · Et. Continuous registration of substrate hydrolysis makes it possible to graphically determine kobs from the curve of lnS versus time. The ratio kcat / Km is simply determined based on kobs, provided that the concentration of the active enzyme is known:

kcat / Km = kobs / Et.

Analysis method: the use of an initial concentration of the initial substrate, which is much lower than the calculated Km, and a low concentration of the enzyme allows us to record the course of substrate hydrolysis. Based on this, a first-order curve is obtained to generate the product. After complete hydrolysis of the substrate, the optical density (or fluorescence units) of the product can be accurately determined on the basis of So, because Pt = So · kobs is determined from the time dependence of lnS, or alternatively, it can be determined using the point curve program (Enzfitter , SigmaPlot ...).

NB: It should be remembered that it is necessary to calculate the concentration of the substrate for a given point in time based on the concentration of the product (S = So-P), since the dependence of P on time does not give the correct value kobs (dP / dt = kobs · S is not integrated in a similar way).

Alternatively, successive t _1/2 values (half period) can be measured from the product production curve during the first order reaction:

t _1/2 = ln2 / kobs = 0.693 / kobs, and kobs = 0.693 / t _1/2

Using this method allows you to control the true attenuation of the first order (identical values for successive values of t _1/2 ).

Example 3

Inactivation of protease genes in Aspergillus

The most suitable method for inactivating protease genes in the Aspergillus genome is the technique of gene replacement (the so-called “one-step gene disruption”). The principles of this technique are described by Rothstein RJ, Meth. Enzymol. 101, p.202, 1983. Basically, this technique is based on homologous recombination of transformed DNA fragments with the genomic DNA of fungal cells. Through double crossing over, the inactivated gene is replaced (partially) by the DNA fragment that transforms the cell. Preferably, the transforming DNA fragment contains the Aspergillus niger selective marker gene. DNA modification and the generation of an inactivating construct are carried out mainly using general methods of molecular biology. First, genomic DNA is isolated from a strain of Aspergillus niger , which is then used to inactivate the protease gene. A.niger genomic DNA can be isolated by any of the methods described, for example, by the method described by Graaff et al. (1988) Curr. Genet. 13, 315-321, and methods well known in the art. This genomic DNA is used as a template for amplification of the flanking regions of the protease gene by polymerase chain reaction (PCR; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). The term “flanking regions” means non-coding regions located above and below the inactivated protease gene. Preferably, each flanking region has a length of more than 1.0 TPN

For the PCR amplification of each flanking region, two single-stranded DNA oligonucleotides were used as primers. For the 5'-flanking region, one primer of the homologous DNA sequence located above the initiation codon of the coding sequence of the protease gene was used. Preferably, the homologous region is localized in a region located more than 1.0 kb. above the translation initiation site. The second primer was homologous to the complementary and inverted DNA sequence located immediately above the coding sequence of the protease gene.

For the 3'-flanking region, one primer was used that was homologous to the DNA sequence located immediately below the coding sequence of the protease gene. The second primer was homologous to the complementary and inverted DNA sequence, localized, preferably, in the area located more than 1.0 TPN below the coding sequence of the protease gene.

The DNA sequence, included in all primers and homologous to the A.niger genome, should have a length of at least 15 nucleotides, and preferably more than 18 nucleotides. Basically, all primers should contain a DNA sequence encoding a recognition site for suitable restriction enzymes, located above the sequence homologous to the A.niger genome. These additional recognition sites facilitate the cloning process.

A.niger primers and genomic DNA were used in the PCR reaction under conditions known to those skilled in the art. The primer annealing temperature can be calculated for the part of the DNA sequence that is homologous to the A.niger genome. Both fragments containing the 5'-flanking region and the 3'-flanking region were cloned into a vector that can be replicated in E. coli using general molecular biology techniques. Then the gene, which can be used as a selective marker in Aspergillus niger , was cloned between two flanking regions. Typically, the marker gene is under the control of a promoter that regulates expression in A.niger , and preferably the endogenous A.niger promoter. The orientation of the insertion of the marker gene preferably coincides with the orientation of the original protease gene. The final inactivating fragment contains a 5'-flanking region, a selective marker gene, preferably under the control of the endogenous A.niger promoter, and a 3'-flanking region, all of which have the same direction and the same orientation. The final construct DNA is cloned into a vector that can replicate in E. coli .

The inactivating construct is cleaved with suitable restriction enzymes to remove E. coli vector sequences and the inactivating fragment is isolated by standard methods (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). Finally, Aspergillus niger is transformed with an inactivating fragment using the method described in the literature, for example, 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 expressing the marker gene. After purification of the transformed Aspergillus strains by plating replicas, a number of representative strains were analyzed by Southern blotting using a standard technique (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York). For this, the genomic DNA of the mycelium of the transformed strains is isolated and digested with suitable restriction enzymes. Restriction fragments are separated using agarose gel electrophoresis, blotted on nitrocellulose membranes and probed with a labeled fragment of the marker gene. Hybridization and washing are carried out in harsh conditions. Suitable strains are those that contain labeled restriction fragments of the desired length. Using this method, A.niger strains containing the desired inactivated protease gene can be selected.

Example 4

Isolation of proteases by ion exchange chromatography

Small amounts of the protease encoded by the nucleotide sequence described herein were obtained by constructing an expression plasmid containing the relevant DNA sequence; transforming A.niger strain with a plasmid; and culturing A.niger strain in a suitable medium. After collecting the broth that does not contain contaminating cells, the desired protease can be purified.

Various strategies can be used to isolate the protease encoded by the nucleotide sequence in question in essentially pure form. All of these strategies are well described in the relevant scientific literature (see, for example, the Protein Purification Handbook, 18-1132-29, Edition AA, Amersham Pharmacia Biotech, Uppsala, Sweden). This manual also describes a procedure suitable for purifying proteases from complex mixtures. It is important that a suitable assay is available and selective with respect to the properties of the enzymes in question. For proteases, the chromogenic, synthetic peptide substrate described in Example 1 is usually used. Such peptide substrates can be selective for endoproteases, carboxypeptidases, aminopeptidases or omega-peptidases. Example 11 describes selectivity for a specific tripeptidyl peptidase. Proteases with the desired specificity can be selected by selecting the appropriate amino acid residues in the relevant synthetic peptide.

First, it is necessary to determine whether a given protease is secreted into the medium, since depending on the expression system selected for the production of the protease, it can be secreted from the cell or contained in the cell. If the protease is secreted into the fermentation medium, the producing cells or fragments of these cells must be removed by centrifugation or filtration, and the obtained clear or clarified medium can be used as a starting material for subsequent purification. In cases where the protease in question is not secreted, then the producing cells must be subjected to disruption to isolate the protease. In such cases, the collected cell mass is carefully ground using an abrasive, milled in a ball mill, subjected to ultrasonic treatment and processing on a French press or in a Manton-Golin homogenizer, and then filtered or centrifuged. If this protease is hydrophobic or membrane-associated, before the stage of filtration or centrifugation, it may be necessary to add a nonionic detergent to solubilize the protease.

To obtain unknown proteases, essentially in pure form, a three-phase purification strategy can be implemented after the clarification step. In all or some of these three phases, it may be necessary to add detergent.

In the first phase, or in the phase of immobilization, the desired protease is isolated, partially purified and concentrated. In the subsequent intermediate purification phase, most of the total volume of impurities is removed, and in the final purification phase, trace amounts of the remaining impurities of larger closely related substances are removed and the enzyme is dissolved in the desired buffer. The specialist can, depending on the nature and physical properties of the studied protease, optimize these three phases using slightly modified versions of various materials that bind to the protein and change the conditions to a certain extent. However, in all cases, it is necessary to conduct a selective analytical test, since this makes it possible to continuously monitor the increasing activity of the purified proteolytic enzyme. Analytical tests suitable for these purposes involve the use of the chromogenic peptide substrates mentioned above.

In the first immobilizing purification phase, it is preferable to use a strong anionic ion exchange resin to clarify and desalinate the enzyme-containing medium. To ensure the binding of the desired proteolytic activity to the resin, a test is carried out at three or four different pH values of the medium and the resin under conditions of low conductivity. In these tests, the resin is always equilibrated with a buffer with the same pH and conductivity as in the enzyme-containing medium. Then the medium is applied to the column under pH conditions, which, as has been shown, allow adequate protease binding to the resin, that is, so that none of the desired enzymatic activities are detected in the passing medium. The desired enzymatic activity is then eluted from the ion exchange resin using a continuous salt gradient, where gradient elution is started with a resin balancing buffer and terminated with the same buffer to which 1 mol / liter NaCl is added. According to this analysis, the eluted fractions containing the desired activity are collected and then prepared for an additional purification step. Carrying out this additional stage of purification depends on the purity of the desired enzyme in the collected fraction: if it is almost pure, then it is necessary to carry out an additional adequate stage of gel filtration; and if it is not pure enough, then it is necessary to carry out chromatography on a hydrophobic resin, and then the gel filtration step.

Chromatography on a hydrophobic resin was first carried out by increasing the salt content in the collected fraction obtained from the ion exchange resin to 4 mol / liter NaCl, followed by the removal of any precipitate formed. If the obtained clarified fraction does not contain the desired activity, then this activity is obviously present in the precipitate and cannot be isolated in essentially pure form. If in this analysis the obtained transparent fraction continues to possess the desired activity, then this liquid is applied to a phenylsepharose resin (Pharmacia), balanced in a buffer with a high concentration of salt, having the same pH and conductivity. If a fraction with the desired enzymatic activity binds to a phenylsepharose resin, this fraction is eluted with a continuous gradient with a decrease in salt content, and then with a salt-free wash and, if necessary, a chaotropic agent. As described previously, the fractions obtained by gradient elution of the fraction and showing activity in the analysis were collected and finally subjected to gel filtration. If the fraction with the desired enzymatic activity does not bind to the phenylsepharose resin, then in this case there are many impurities, and therefore, to obtain the desired proteolytic activity present in the empty column volume, only additional ultrafiltration steps are required to obtain activity in a more concentrated form, which then applied to a gel filtration column. Column gel chromatography allows not only to remove trace impurities, but also to enter the enzyme into the buffer, which is necessary for subsequent use.

Although this method is mainly suitable for the isolation and purification of the protease of the present invention, however, Example 4 describes a more specific isolation method. This example describes the isolation of Aspergillus protease using immobilized bacitracin, an antibiotic peptide that is known to selectively interact with various types of proteases.

Example 5

Affinity Protease Isolation

An alternative method for purifying small amounts of protease is affinity chromatography. To obtain protease in purified form, 100 milliliters of culture were grown in a well-ventilated shaker flask. After centrifugation, to remove any insoluble matter, the supernatant was applied to a 40 ml bacitracin-sepharose column, equilibrated with 0.05 mol / liter sodium acetate, pH 5.0. Proteases associated with the column were eluted with acetate buffer to which 1 mol / liter NaCl and 10% (v / v) isopropanol were added (J. Appl. Biochem. 1983, pp. 420-428). Active fractions were collected, dialyzed against distilled water and applied to a 20 ml bacitracin-sepharose column, after which they were again equilibrated with acetate buffer. As before, elution was carried out using an acetate buffer to which NaCl and isopropanol were added. Active fractions, i.e. fractions exhibiting the desired activity, were collected, dialyzed against 5 mmol / liter acetate buffer, pH 5.0, and then concentrated by ultrafiltration on an Amicon PM-10 membrane. To obtain a substantially pure protease, the concentrated liquid was chromatographed on a Superdex 75 column equilibrated with 0.05 mol / liter sodium acetate buffer, pH 5.0, to which 0.5 mol / liter NaCl was added. Subsequent experiments using purified enzyme by PAG electrophoresis can confirm whether its molecular weight matches the molecular weight predicted from the available sequence data. Final confirmation can be obtained by analyzing an incomplete, N-terminal amino acid sequence.

Example 6

Properties of a new cysteine protease derived from A.niger

In this example, Aspergillus No. 28 gene was cloned and overexpressed in A.niger as described above. The obtained enzyme was purified in accordance with the procedures described in example 4, and used to destroy the trypsin-inhibiting activity of soybeans under various conditions. Papain and bromelain were used as reference materials. 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 by the method described by Kakade M.L., Rackis, J.J., McGhee, J.E. & Puski, G. (1974): J. Cereal Chemistry 51: 376-382. To measure trypsin activity, the substrate N-benzoyl-L-arginine-p-nitroaniline was split into N-benzoyl-L-arginine and p-nitroaniline. Trypsin was purchased from British Drug Houses Ltd and obtained from bovine pancreas containing more than 0.54 Anson units per gram of product. The marten soy inhibitor was obtained from Sigma.

This trypsin inhibitor was preincubated at a concentration of 2 mg / ml with the aforementioned serine protease enzymes at pH 3 in 50 mM Na-acetate buffer, followed by an assessment of trypsin inhibition. Enzymes were added in relation to the enzyme protein: trypsin inhibitor, 1: 100 (w / w). Albumin served as a negative control for the enzymes. The residual trypsin activity was measured after incubation for 3 hours at 37 ^C. The results are shown in Table 2.

table 2
The effect of various cysteine proteases
for enzymatic inactivation of the marten trypsin inhibitor isolated from soybeans Test enzyme Residual Activity TI (%) Residual TI Activity After Pepsin Treatment Residual TI activity after heat treatment at 75 ° C Residual TI activity after heat treatment at 90 ° C Papain 25 55 78 95 Bromelain thirty 62 86 99 A.niger 26 26 28 35 Albumin (control) one hundred one hundred one hundred one hundred TI: trypsin inhibitor activity

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

Other series of experiments were carried out to evaluate thermal stability. Cysteine proteases were incubated at 75 and 90 ° C for 5 minutes, and then enzymes were added to pre-incubate with trypsin inhibitors. The results are presented in columns 4 and 5.

These results clearly demonstrated the superior activity of the new Aspergillus niger cysteine proteases compared to the already known cysteine proteases used to inactivate trypsin inhibitors in animal feed.

Example 7

Exopeptidases that stimulate the ripening of cheese and the formation of cheese flavor

Aminopetidases encoded by genes No. 20 and 54 (see table 1) were overexpressed in A.niger by the methods described above. The purification of these enzymes was carried out in accordance with the procedures described in example 4. The activity of the samples of the purified enzyme was determined at pH 7.2 in aqueous phosphate buffer (50 mm) containing a paranitroanilide derivative of a number of hydrophobic amino acids (3 mm) as a substrate. The conversion of the substrate by aminopeptidase was evaluated by monitoring the change in absorbance at 400 nm as a result of the conversion of the substrate, and an enzyme-free solution was used as a control. Activity (A) was calculated as the change in OD per minute and expressed, for example, as units of Phe-AP, Leu-AP or Val-AP depending on the substrate used. Normal cheese production milk was inoculated with a starter culture Delvo-tec ^tm DX 31 (DSM Food Specialties Delft, The Netherlands) to produce Gouda type cheese, and coagulation was performed using medium doses of coagulant (50 IMCU per liter of cheese-suitable milk). In addition, 25 Phe units of each exoprotease were added to the two experimental cheeses, while the control did not contain a single exoprotease. The parameters used in the preparation of both cheeses corresponded to the parameters used in the production of semi-hard cheeses. At the same time, differences were observed in the formation of taste and aroma between experimental cheeses and control cheese, namely experimental cheeses acquired most of their organoleptic properties after three (3) weeks, while control cheese acquired similar properties after six (6) weeks. It was shown that in experimental cheeses, the level of free amino acids was three times higher after three weeks, and after six weeks of ripening, it was again comparable. Amino acid analysis was performed by the method of Picotag, Waters (Milford MA, USA).

These data suggest that the product was ready for sale three weeks earlier and at the same time it was not inferior in quality to the quality of normal cheese. The organoleptic properties of the experimental cheeses differed from the organoleptic properties of the control cheese in that, in contrast to the control cheese, a slightly bitter aftertaste disappeared in the presence of aminopeptidase in the experimental cheeses. It was found that the consistency of these cheeses was also somewhat more tender.

Example 8

The new specificity of the protease encoded by gene 55

As explained above, some proteins may be resistant to enzymatic hydrolysis, due to the specific amino acid composition or the presence of specific tertiary structures. In such cases, the number of peptides that are present in these protease-resistant proteins and which can be solubilized can be dramatically increased using proteases with new specificities.

Beta casein is a protein with a very limited tertiary structure, but an exceptionally high level of proline residues. Many proteases cannot cleave proline-containing sequences, and therefore hydrolysis of beta-casein with known proteases yields a hydrolyzate relatively enriched in large protease-resistant peptides. These resistant peptides may be responsible for a number of undesirable hydrolyzate properties. For example, it is well known that these larger peptides have a relatively strong effect in terms of allergenicity and have a bitter aftertaste. Moreover, these peptides are resistant to subsequent degradation into free amino acids, and therefore, in some processes, the presence of these large protease-resistant peptides is equivalent to a loss of yield. Consequently, the availability and use of proteases capable of cleaving protease-resistant portions of proteins will significantly contribute to technological progress and economic benefits.

Beta casein is one of the main casein fractions of cow's milk. This protein has been well characterized in terms of amino acid sequence and is commercially available in almost pure form. Essentially, beta-casein is an excellent test substrate used to study the relationship between enzyme cleavage sites and the length of various peptides formed during enzymatic hydrolysis.

This example demonstrates that, despite the wide range of subtilisin endoprotease cleavage, the addition of a highly specific enzyme, such as prolyl endopeptidase encoded by gene 55 (see Table 1), has a huge effect on the size of the formed beta-casein fragments.

Beta casein of cow's milk (lyophilized, substantially salt free milk powder) containing at least 90% beta casein was purchased from Sigma. Subtilisin isolated from B.licheniformis (Delvolase®, 560,000 DU per gram) was purchased from DSM Food Specialties (Seclin, France). The proline-specific endopeptidase encoded by gene 55 was overexpressed in A. niger and purified using the procedures described in example 4.

Beta casein powder was dissolved in a concentration of 10 wt.% Together with 0.1 wt.% Delvolase ^™ powder in 0.1 mol / liter of phosphate buffer pH 7.0. After incubation for 24 hours at 45 ° C in a water bath with shaking, the reaction was stopped by heating the solution for 15 minutes at 90 ° C. To one 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 heat shock at 90 ° C, samples of beta-casein material treated with Delvolase ^™ and Delvolase ^™ + proline-specific protease were analyzed using LC / MS equipment to study the exact size distribution of the peptides in two samples.

LC / MS analysis

The enzyme protein hydrolysates produced using the enzyme mixture of the present invention were characterized by HPLC using an ion trap mass spectrometer (Thermoquest ^TM , Breda, The Netherlands) connected to a P4000 pump (Thermoquest ^TM , Breda, The Netherlands). The resulting peptides were separated on a PEPMAP C18 300A column (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands), eluting 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). This gradient elution was started with 100% solution A, increasing to 70% solution B in 45 minutes, and maintained at this 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 approximately 50 micrograms / milliliter.

Detailed information on individual peptides was obtained using the MS / MS scan-dependent algorithm, which is a characteristic algorithm for an ion trap mass spectrometer. After a complete scanning analysis, a scanning analysis was performed by scaling to determine the charge of the strongest ion within the entire scanned mass. Subsequent MS / MS analysis of the later ion yielded incomplete peptide sequence information that could be used to search the database using the SEQUEST application from Xcalibur Bioworks (Thermoquest ^TM , Breda, The Netherlands). The data banks used were taken from the OWL.fasta data bank, available at the NCBI (National Center for Biotechnological Information) and containing proteins of interest to the application used.

Using this technique as a scanning method, only peptides having a weight in the range of about 400-2000 Da were considered as peptides suitable for subsequent analysis by MS sequencing.

Angiotensin (M = 1295.6) was used to adjust the optimal sensitivity in the MS mode and for optimal fragmentation in the MS / MS mode, with continuous infusion of 60 μg / ml, which mainly led to the production of doubly charged and triply charged ions in the MS mode and an optimal collision energy of approximately 35% in MS / MS mode.

In a Delvolase-only digested sample, LC / MS / MS analysis identified 40 peptides spanning various parts of the beta-casein molecule. Together, these peptides account for 79% of the total beta casein sequence. It was found that peptides ranging in length from 2 to 23 amino acid residues have different retention times on the C18 column. It was found that in total <15% of the peptides were less than 6 amino acids in size. In the sample digested with Delvolase ^™ and a proline-specific protease, a large number of peptides identified in beta-casein were also formed. Together, these peptides covered> 50% of the entire beta-casein protein sequence. In the indicated sample, the size distribution of the peptides was quite homogeneous, since these peptides only had a length of 2 to 6 residues. The results showed that the hydrolyzate obtained using a proline-specific protease contained a large fraction of peptides having 2-6 amino acids, which indicated a clearly positive effect of co-incubation with an endoprotease with unusual specificity. It is also apparent from these experiments that the endoprotease encoded by gene 55 is an endoprotease that cleaves the peptide chain at the carboxy-terminus of the proline residue.

Example 9

Selective release of specific amino acids to stimulate the formation of taste

Free amino acids, such as leucine and phenylalanine, not only participate in Maillard reactions, but are also precursors that form the necessary odors during the fermentation of various foods. To stimulate the formation of such odors during the fermentation of food products or during the heating, frying or baking of food products, it may be preferable to introduce a protein hydrolyzate into these products that contains relatively high levels of these specific free-form amino acids. In this example, the authors describe the preparation of yeast extracts selectively enriched in leucine and phenylalanine. Such enrichment is achieved by combining an endoprotease, which has a preference for cleaving a selected series of amino acid residues, with an exoprotease, which has a preference for the release of a similar series of amino acid residues. Such a preference for said endoprotease should correspond to the preference for the exoprotease used. For example, the authors found that aminopeptidases encoded by genes 20 and 54 (see table 1) have a definite preference for the release of leucine and phenylalanine residues, which coincides with the preference for thermolysin in its hydrolyzing action. Carboxypeptidases encoded by genes 23 and 24 have a preference for the release of arginine and lysine residues, which coincides with the preference for trypsin in its hydrolyzing action. The carboxypeptidase encoded by gene 5 has a highly unusual preference for glycine release and can be combined with some endoproteases present in papain. The carboxypeptidase encoded by gene 51 is capable of removing glutamate residues, and this ability coincides with the ability of the glutamate-specific protease encoded by gene 43.

The thermolysin endoprotease (commercially available under the name of thermoase) C180, available from Daiwa Kasei KK (Osaka, Japan), is known to cleave peptide bonds from the amino terminus of bulky hydrophobic amino acids such as Leu and Phe. The authors used aminopeptidases encoded by genes Nos. 20 and 54 to release the amino acids thus treated from the already formed peptides (see Table 1). These genes were overexpressed in A.niger by previously described methods, and the purification of these enzymes was carried out in accordance with the procedures described in example 4.

Obviously, for the release of leucine and phenylalanine and, as far as possible, without the concomitant release of undesirable amino acids, using this combination of enzymes, it is obviously necessary to correctly select the conditions used in the process of enzymatic hydrolysis. In addition, endogenous (and probably non-specific) proteases present in yeast must be inactivated. After a series of test incubations, a protocol was developed that utilized these two new enzymes and which unexpectedly detected selective and effective release of leucine and phenylalanine from yeast proteins.

To inactivate yeast endogenous proteases, the yeast suspension was kept for 5 minutes at 95 ° C. Then this suspension was quickly cooled to the desired temperature and the pH was adjusted to 7.0 using 4n NaOH. Yeast, thermolysin, and one of the aminopeptidases were incubated simultaneously under the conditions described below. After heat shock, the pH of 2000 milliliters of yeast suspension was adjusted to 7.0, after which 680 milligrams of thermoase was added and, after stirring, purified aminopeptidase was added. This mixture was incubated with stirring at 50 ° C. for 3 hours and centrifuged. To suppress all enzymatic activities, the pH of the supernatant was adjusted to 4 and subjected to additional heat treatment for 45 minutes at 95 ° C. After additional centrifugation, a sample was taken from the supernatant for amino acid analysis. Precipitated or undissolved material was removed by centrifugation for 15 minutes at 3500 rpm in a Hereaus Megafuge 2.0R mega-centrifuge. The supernatant was removed and frozen at -20 ° C. Immediately after thawing, supernatant samples were analyzed for amino acid content by the method of Picotag, Waters (Milford MA, USA).

In these amino acid analyzes, the values for Trp and Cys were omitted, and the values for Asp and Asn were summed to give a single value. In accordance with these data, the ratio of alanine to leucine (21.3: 11.7) in the obtained hydrolyzate was 1: 0.5. Commercially available yeast hydrolysates typically had an alanine to leucine ratio of 1: 0.3.

In a second experiment, a yeast extract was obtained that was enriched with free glutamate. To achieve this, an endoprotease (encoded by gene No. 43 according to table 1) was used, which has a preference for cleavage at the C-terminus of glutamate residues, and carboxypeptidase (encoded by gene No. 51 according to table 1), capable of removing the glutamate residues treated with it. The endoprotease encoded by gene 43 and the carboxypeptidase encoded by gene 51 (see table 1) were overexpressed in A. niger by the methods described previously. Purification of these enzymes was carried out in accordance with the procedures described in example 4.

The main role of free glutamate in a number of odor-forming processes is well described, and it is known that MSG, the glutamic acid sodium salt, is the single and most important ingredient in enhancing taste.

In this example, a pH of 200 ml of the heat-shocked yeast suspension was adjusted to 8.0, and then the purified enzyme product encoded by gene 43 was added and the mixture was incubated for 4 hours at 50 ° C. Then the pH was lowered to 5.0 and the suspension was centrifuged. The purified gene product of gene 51 was added to 100 milliliters of the supernatant. Incubation with this carboxypeptidase was carried out for 30 minutes at 50 ° C and with constant pH correction. After the incubation of the enzyme was stopped by heat treatment for 5 minutes at 95 ° C, the obtained material was again centrifuged (see above) and a sample was taken for amino acid analysis.

In accordance with the data obtained for amino acids, in the hydrolyzate produced, the ratio of alanine to glutamate (30.0: 48.7) was 1: 1.6. Commercially available yeast hydrolysates typically have a 1: 1 ratio of alanine to glutamate.

Example 10

Organoleptic evaluation of yeast hydrolysates enriched in specific amino acids

To confirm that the protein hydrolyzate of the present invention enriched with specific amino acids can generate specific odors, a series of experiments were performed using the yeast hydrolysates described in the previous example. For this purpose, larger fragments of these hydrolysates were obtained and lyophilized. The properties of the obtained powders were compared with the properties of a commercially available yeast extract (Gistex LS, supplied by DSM Food Specialties, Delft, The Netherlands) in a standardized mixture under several reaction conditions. The standardized mixture contained one of the hydrolysates, a mixture of bases and water.

The base mixture contained 22 grams of powder (Maxarome Plus Powder) (a specialized natural high nucleotide yeast extract, also available from DSM Food Specialties), 29.2 grams of glucose, 9 grams of REFEL-F fat (hydrogenated soybean oil supplied by Barentz, Hoofddorp, The Netherlands) and 0.2 grams of calcium stearoyl lactylate (emulsifier supplied by Abitec, Northampton, UK), thoroughly mixed in a mortar.

All standardized mixtures contained 5 grams of powdered yeast hydrolyzate (i.e., material enriched with either leucine, or glutamate, or a commercially available yeast extract), 3 grams of a base mixture, and 3 grams of water. After thorough mixing, these three suspensions were subjected to heat treatment in various modes, that is, they were either kept at 90-95 ° С for 65 minutes in a reaction vessel (liquid reaction) or dried under a pressure of 20 mbar at 120 ° С in a vacuum oven (reaction vacuum frying), or heated in an open reaction vessel at 120 ° C for 10 minutes after evaporation of all water (frying reaction).

After heat treatment, all three products were evaluated for color in the range from dark brown to almost black. In the case of the vacuum frying reaction, only slightly colored upper layers were used. The taste of the heated product was evaluated by grinding the blackened layers to obtain fine powders and dissolving these powders to a concentration of 2% (w / w) in water containing 0.6% (w / w) NaCl. Tasting observations are presented in table 3.

Table 3 The control Leucine Glutamate Liquid Broth, easy frying Iced tea, floral scent, yeast flavor The taste of strong broth, meat taste, yeast taste Vacuum frying Burning, french fries Tartness, Bean Flavor, Yeast Flavor Burning, broth, yeast flavor Frying Strong roasting, broth, umami Weak frying, floral smell, umami Frying, the taste of strong broth, a more pronounced taste by the minds

Example 11

Non-allergenic whey protein hydrolysates formed by tripeptidyl peptidases

The 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), were overproduced as described above, and they can be purified by methods, described in example 4. After purification, the optimal pH and thermal stability of each individual enzyme can be established by any available methods known to specialists. In addition, the specificity of each individual enzyme can be determined by the methods described in example 1. The selectivity of tripeptidyl peptidases is illustrated in the following experiment.

The enzyme encoded by gene 12 was overproduced in the Aspergillus niger host cell and purified in accordance with the procedures described in Example 4. The enzyme thus obtained was incubated at pH 5 and at 50 ° C. with various synthetic chromogenic substrates, that is, Ala-Ala- Phe-pNA and Ala-Phe-pNA (both supplied by Bachem, Switserland). Incubation with Ala-Ala-Phe-pNA substrate led to a significant increase in optical density at 410 nm, while incubation with Ala-Phe-pNA did not give such an effect. This observation clearly demonstrated that tripeptidyl peptidases cleave tripeptides and do not exhibit aminopeptidase activity, which can lead to an undesirable increase in the number of free amino acids.

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

In a conventional method for producing a high tripeptide hydrolyzate, whey protein (WPC 75) can be dissolved / suspended at a concentration of 100 grams of protein / liter in an aqueous medium having a pH of 8.5. The first enzyme was incubated with subtilisin, a broad spectrum endoprotease (Delvolase®, 560,000 DU per gram, from DSM). After preliminary splitting of the whey with this enzyme, taken at a concentration of 0.5% of the enzyme per gram of protein, for 2 hours at 60 ° C, the mixture was subjected to heat treatment to inactivate the endoprotease used. Then, the temperature was brought to 50 ° C, tripeptidyl peptidase was added, and the whole mixture was incubated to obtain the desired level of tripeptides. The subsequent processing steps of the hydrolyzate thus obtained will depend on the purpose of the particular application, but they may also include microfiltration or centrifugation, followed by evaporation and spray drying.

Claims (13)

1. The selected polynucleotide encoding aminopeptidase, hybridizing under high stringency conditions with a sequence selected from the group consisting of SEQ ID NO: 57 and 114. 2. The selected polynucleotide according to claim 1, obtained from filamentous fungi. 3. The selected polynucleotide according to claim 2, obtained from A.niger. 4. The selected polynucleotide encoding an aminopeptidase containing the amino acid sequence corresponding to the sequence of SEQ ID NO: 171. 5. An isolated polynucleotide encoding an aminopeptidase containing a nucleotide sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 57 and 114. 6. The expression vector containing the polynucleotide sequence according to any one of claims 1 to 5. 7. The expression vector according to claim 6, characterized in that the polynucleotide sequence according to any one of claims 1 to 5, is functionally linked to regulatory sequences suitable for expression of the specified polynucleotide sequence in a suitable host cell. 8. The expression vector according to claim 7, characterized in that said suitable host cell are filamentous fungi. 9. The selected polypeptide, which is an aminopeptidase, corresponding to the sequence of SEQ ID NO: 171, which can be obtained by expression of the polynucleotide according to claims 1-5 or the vector according to claims 6-8 in the corresponding host cell. 10. The selected polypeptide according to claim 9, obtained from Aspergillus niger. 11. A method of producing a polypeptide according to any one of claims 9 or 10, comprising the steps of transforming a suitable host cell with an isolated polynucleotide according to any one of claims 1-5 or a vector according to any one of claims 6-8, culturing said cell under conditions suitable for expressing said polynucleotide; and optionally purifying the polypeptide from said cell or culture medium. 12. A recombinant host cell expressing a polypeptide according to any one of claims 9 or 10, containing a polynucleotide according to any one of claims 1 to 5, or a vector according to any one of claims 6 to 8. 13. A method for diagnosing Aspergillus infection in the body, comprising the steps of:
a. isolating a biological sample from said organism, which is believed to be infected with Aspergillus,
b. isolation of nucleic acid from the specified sample,
c. determining whether said isolated nucleic acid contains polynucleotides hybridizing with the polynucleotide according to any one of claims 1 to 5.

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