INTRODUCTION

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Proteases are the single class of enzymes which occupy a pivotal position with respect to their applications in both physiological and commercial fields. Proteolytic enzymes catalyze the cleavage of peptide bonds in other proteins. Proteases are degradative enzymes which catalyze the total hydrolysis of proteins. Advances in analytical techniques have demonstrated that proteases conduct highly specific and selective modifications of proteins such as activation of zymogenic forms of enzymes by limited proteolysis, blood clotting and lysis of fibrin clots, and processing and transport of secretory proteins across the membranes. The current estimated value of the worldwide sales of industrial enzymes is $1 billion (72). Of the industrial enzymes, 75% are hydrolytic. Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total worldwide sale of enzymes (Fig. 1). Proteases execute a large variety of functions, extending from the cellular level to the organ and organism level, to produce cascade systems such as hemostasis and inflammation. They are responsible for the complex processes involved in the normal physiology of the cell as well as in abnormal pathophysiological conditions. Their involvement in the life cycle of disease-causing organisms has led them to become a potential target for developing therapeutic agents against fatal diseases such as cancer and AIDS. Proteases have a long history of application in the food and detergent industries. Their application in the leather industry for dehairing and bating of hides to substitute currently used toxic chemicals is a relatively new development and has conferred added biotechnological importance (235). The vast diversity of proteases, in contrast to the specificity of their action, has attracted worldwide attention in attempts to exploit their physiological and biotechnological applications (64, 225). The major producers of proteases worldwide are listed in Table 1.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Distribution of enzyme sales. The contribution of different enzymes to the total sale of enzymes is indicated. The shaded portion indicates the total sale of proteases.

Since proteases are enzymes of metabolic as well as commercial importance, there is a vast literature on their biochemical and biotechnological aspects (64, 128, 192, 235, 309). However, the earlier reviews did not deal with the molecular biology of proteases, which offers new possibilities and potentials for their biotechnological applications. This review aims at analyzing the updated information on biochemical and genetic aspects of proteases, with special reference to some of the advances made in these areas. We also attempt to address some of the deficiencies in the earlier reviews and to identify problems, along with possible solutions, for the successful applications of proteases for the benefit of mankind. The genetic engineering approaches are also discussed, from the perspective of making better use of proteases. The reference to plant and animal proteases has been made to complete the overview. However, the major emphasis of the review is on the microbial proteases.

Papain. Papain is a traditional plant protease and has a long history of use (250). It is extracted from the latex of Carica papaya fruits, which are grown in subtropical areas of west and central Africa and India. The crude preparation of the enzyme has a broader specificity due to the presence of several proteinase and peptidase isozymes. The performance of the enzyme depends on the plant source, the climatic conditions for growth, and the methods used for its extraction and purification. The enzyme is active between pH 5 and 9 and is stable up to 80 or 90°C in the presence of substrates. It is extensively used in industry for the preparation of highly soluble and flavored protein hydrolysates.

The most familiar proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin, and rennins (23, 97). These are prepared in pure form in bulk quantities. However, their production depends on the availability of livestock for slaughter, which in turn is governed by political and agricultural policies.

Trypsin. Trypsin (M_r 23,300) is the main intestinal digestive enzyme responsible for the hydrolysis of food proteins. It is a serine protease and hydrolyzes peptide bonds in which the carboxyl groups are contributed by the lysine and arginine residues (Table 2). Based on the ability of protease inhibitors to inhibit the enzyme from the insect gut, this enzyme has received attention as a target for biocontrol of insect pests. Trypsin has limited applications in the food industry, since the protein hydrolysates generated by its action have a highly bitter taste. Trypsin is used in the preparation of bacterial media and in some specialized medical applications.

The inability of the plant and animal proteases to meet current world demands has led to an increased interest in microbial proteases. Microorganisms represent an excellent source of enzymes owing to their broad biochemical diversity and their susceptibility to genetic manipulation. Microbial proteases account for approximately 40% of the total worldwide enzyme sales (72). Proteases from microbial sources are preferred to the enzymes from plant and animal sources since they possess almost all the characteristics desired for their biotechnological applications.

Viruses. Viral proteases have gained importance due to their functional involvement in the processing of proteins of viruses that cause certain fatal diseases such as AIDS and cancer. Serine, aspartic, and cysteine peptidases are found in various viruses (236). All of the virus-encoded peptidases are endopeptidases; there are no metallopeptidases. Retroviral aspartyl proteases that are required for viral assembly and replication are homodimers and are expressed as a part of the polyprotein precursor. The mature protease is released by autolysis of the precursor. An extensive literature is available on the expression, purification, and enzymatic analysis of retroviral aspartic protease and its mutants (147). Extensive research has focused on the three-dimensional structure of viral proteases and their interaction with synthetic inhibitors with a view to designing potent inhibitors that can combat the relentlessly spreading and devastating epidemic of AIDS.

According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in subgroup 4 of group 3 (hydrolases) (114a). However, proteases do not comply easily with the general system of enzyme nomenclature due to their huge diversity of action and structure. Currently, proteases are classified on the basis of three major criteria: (i) type of reaction catalyzed, (ii) chemical nature of the catalytic site, and (iii) evolutionary relationship with reference to structure (12).

Proteases are grossly subdivided into two major groups, i.e., exopeptidases and endopeptidases, depending on their site of action. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate. Based on the functional group present at the active site, proteases are further classified into four prominent groups, i.e., serine proteases, aspartic proteases, cysteine proteases, and metalloproteases (85). There are a few miscellaneous proteases which do not precisely fit into the standard classification, e.g., ATP-dependent proteases which require ATP for activity (183). Based on their amino acid sequences, proteases are classified into different families (5) and further subdivided into "clans" to accommodate sets of peptidases that have diverged from a common ancestor (236). Each family of peptidases has been assigned a code letter denoting the type of catalysis, i.e., S, C, A, M, or U for serine, cysteine, aspartic, metallo-, or unknown type, respectively.

Aminopeptidases. Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide, or a tripeptide (Table 3). They are known to remove the N-terminal Met that may be found in heterologously expressed proteins but not in many naturally occurring mature proteins. Aminopeptidases occur in a wide variety of microbial species including bacteria and fungi (310). In general, aminopeptidases are intracellular enzymes, but there has been a single report on an extracellular aminopeptidase produced by A. oryzae (150). The substrate specificities of the enzymes from bacteria and fungi are distinctly different in that the organisms can be differentiated on the basis of the profiles of the products of hydrolysis (31). Aminopeptidase I from Escherichia coli is a large protease (400,000 Da). It has a broad pH optimum of 7.5 to 10.5 and requires Mg²⁺ or Mn²⁺ for optimal activity (48). The Bacillus licheniformis aminopeptidase has a molecular weight of 34,000. It contains 1 g-atom of Zn²⁺ per mol, and its activity is enhanced by Co²⁺ ions. On the other hand, aminopeptidase II from B. stearothermophilus is a dimer with a molecular weight of 80,000 to 100,000 (272) and is activated by Zn²⁺, Mn²⁺, or Co²⁺ ions.

Carboxypeptidases. The carboxypeptidases act at C terminals of the polypeptide chain and liberate a single amino acid or a dipeptide. Carboxypeptidases can be divided into three major groups, serine carboxypeptidases, metallocarboxypeptidases, and cysteine carboxypeptidases, based on the nature of the amino acid residues at the active site of the enzymes. The serine carboxypeptidases isolated from Penicillium spp., Saccharomyces spp., and Aspergillus spp. are similar in their substrate specificities but differ slightly in other properties such as pH optimum, stability, molecular weight, and effect of inhibitors. Metallocarboxypeptidases from Saccharomyces spp. (61) and Pseudomonas spp. (174) require Zn²⁺ or Co²⁺ for their activity. The enzymes can also hydrolyze the peptides in which the peptidyl group is replaced by a pteroyl moiety or by acyl groups.

Endopeptidases are characterized by their preferential action at the peptide bonds in the inner regions of the polypeptide chain away from the N and C termini. The presence of the free amino or carboxyl group has a negative influence on enzyme activity. The endopeptidases are divided into four subgroups based on their catalytic mechanism, (i) serine proteases, (ii) aspartic proteases, (iii) cysteine proteases, and (iv) metalloproteases. To facilitate quick and unambiguous reference to a particular family of peptidases, Rawlings and Barrett have assigned a code letter denoting the catalytic type, i.e., S, C, A, M, or U (see above) followed by an artibrarily assigned number (236).

Serine proteases. Serine proteases are characterized by the presence of a serine group in their active site. They are numerous and widespread among viruses, bacteria, and eukaryotes, suggesting that they are vital to the organisms. Serine proteases are found in the exopeptidase, endopeptidase, oligopeptidase, and omega peptidase groups. Based on their structural similarities, serine proteases have been grouped into 20 families, which have been further subdivided into about six clans with common ancestors (12). The primary structures of the members of four clans, chymotrypsin (SA), subtilisin (SB), carboxypeptidase C (SC), and Escherichia D-Ala-D-Ala peptidase A (SE) are totally unrelated, suggesting that there are at least four separate evolutionary origins for serine proteases. Clans SA, SB, and SC have a common reaction mechanism consisting of a common catalytic triad of the three amino acids, serine (nucleophile), aspartate (electrophile), and histidine (base). Although the geometric orientations of these residues are similar, the protein folds are quite different, forming a typical example of a convergent evolution. The catalytic mechanisms of clans SE and SF (repressor LexA) are distinctly different from those of clans SA, SB, and SE, since they lack the classical Ser-His-Asp triad. Another interesting feature of the serine proteases is the conservation of glycine residues in the vicinity of the catalytic serine residue to form the motif Gly-Xaa-Ser-Yaa-Gly (25).

(i) Serine alkaline proteases. Serine alkaline proteases are produced by several bacteria, molds, yeasts, and fungi. They are inhibited by DFP or a potato protease inhibitor but not by tosyl-L-phenylalanine chloromethyl ketone (TPCK) or TLCK. Their substrate specificity is similar to but less stringent than that of chymotrypsin. They hydrolyze a peptide bond which has tyrosine, phenylalanine, or leucine at the carboxyl side of the splitting bond. The optimal pH of alkaline proteases is around pH 10, and their isoelectric point is around pH 9. Their molecular masses are in the range of 15 to 30 kDa. Although alkaline serine proteases are produced by several bacteria such as Arthrobacter, Streptomyces, and Flavobacterium spp. (21), subtilisins produced by Bacillus spp. are the best known. Alkaline proteases are also produced by S. cerevisiae (189) and filamentous fungi such as Conidiobolus spp. (219) and Aspergillus and Neurospora spp. (165).

(ii) Subtilisins. Subtilisins of Bacillus origin represent the second largest family of serine proteases. Two different types of alkaline proteases, subtilisin Carlsberg and subtilisin Novo or bacterial protease Nagase (BPN'), have been identified. Subtilisin Carlsberg produced by Bacillus licheniformis was discovered in 1947 by Linderstrom, Lang, and Ottesen at the Carlsberg laboratory. Subtilisin Novo or BPN' is produced by Bacillus amyloliquefaciens. Subtilisin Carlsberg is widely used in detergents. Its annual production amounts to about 500 tons of pure enzyme protein. Subtilisin BPN' is less commercially important. Both subtilisins have a molecular mass of 27.5 kDa but differ from each other by 58 amino acids. They have similar properties such as an optimal temperature of 60°C and an optimal pH of 10. Both enzymes exhibit a broad substrate specificity and have an active-site triad made up of Ser221, His64 and Asp32. The Carlsberg enzyme has a broader substrate specificity and does not depend on Ca²⁺ for its stability. The active-site conformation of subtilisins is similar to that of trypsin and chymotrypsin despite the dissimilarity in their overall molecular arrangements. The serine alkaline protease from the fungus Conidiobolus coronatus was shown to possess a distinctly different structure from subtilisin Carlsberg in spite of their functional similarities (218).

Aspartic proteases. Aspartic acid proteases, commonly known as acidic proteases, are the endopeptidases that depend on aspartic acid residues for their catalytic activity. Acidic proteases have been grouped into three families, namely, pepsin (A1), retropepsin (A2), and enzymes from pararetroviruses (A3) (13), and have been placed in clan AA. The members of families A1 and A2 are known to be related to each other, while those of family A3 show some relatedness to A1 and A2. Most aspartic proteases show maximal activity at low pH (pH 3 to 4) and have isoelectric points in the range of pH 3 to 4.5. Their molecular masses are in the range of 30 to 45 kDa. The members of the pepsin family have a bilobal structure with the active-site cleft located between the lobes (259). The active-site aspartic acid residue is situated within the motif Asp-Xaa-Gly, in which Xaa can be Ser or Thr. The aspartic proteases are inhibited by pepstatin (63). They are also sensitive to diazoketone compounds such as diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the presence of copper ions. Microbial acid proteases exhibit specificity against aromatic or bulky amino acid residues on both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than that of pepsin. Microbial aspartic proteases can be broadly divided into two groups, (i) pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) rennin-like enzymes produced by Endothia and Mucor spp.

Cysteine/thiol proteases. Cysteine proteases occur in both prokaryotes and eukaryotes. About 20 families of cysteine proteases have been recognized. The activity of all cysteine proteases depends on a catalytic dyad consisting of cysteine and histidine. The order of Cys and His (Cys-His or His-Cys) residues differs among the families (12). Generally, cysteine proteases are active only in the presence of reducing agents such as HCN or cysteine. Based on their side chain specificity, they are broadly divided into four groups: (i) papain-like, (ii) trypsin-like with preference for cleavage at the arginine residue, (iii) specific to glutamic acid, and (iv) others. Papain is the best-known cysteine protease. Cysteine proteases have neutral pH optima, although a few of them, e.g., lysosomal proteases, are maximally active at acidic pH. They are susceptible to sulfhydryl agents such as PCMB but are unaffected by DFP and metal-chelating agents. Clostripain, produced by the anaerobic bacterium Clostridium histolyticum, exhibits a stringent specificity for arginyl residues at the carboxyl side of the splitting bond and differs from papain in its obligate requirement for calcium. Streptopain, the cysteine protease produced by Streptococcus spp., shows a broader specificity, including oxidized insulin B chain and other synthetic substrates. Clostripain has an isoelectric point of pH 4.9 and a molecular mass of 50 kDa, whereas the isoelectric point and molecular mass of streptopain are pH 8.4 and 32 kDa, respectively.

Metalloproteases. Metalloproteases are the most diverse of the catalytic types of proteases (13). They are characterized by the requirement for a divalent metal ion for their activity. They include enzymes from a variety of origins such as collagenases from higher organisms, hemorrhagic toxins from snake venoms, and thermolysin from bacteria (92, 210, 253, 311, 314). About 30 families of metalloproteases have been recognized, of which 17 contain only endopeptidases, 12 contain only exopeptidases, and 1 (M3) contains both endo- and exopeptidases. Families of metalloproteases have been grouped into different clans based on the nature of the amino acid that completes the metal-binding site; e.g., clan MA has the sequence HEXXH-E and clan MB corresponds to the motif HEXXH-H. In one of the groups, the metal atom binds at a motif other than the usual motif.

The mechanism of action of proteases has been a subject of great interest to researchers. Purification of proteases to homogeneity is a prerequisite for studying their mechanism of action. Vast numbers of purification procedures for proteases, involving affinity chromatography, ion-exchange chromatography, and gel filtration techniques, have been well documented. Preparative polyacrylamide gel electrophoresis has been used for the purification of proteases from Conidiobolus coronatus (220). Purification of staphylocoagulase to homogeneity was carried out from culture filtrates of Staphylococcus aureus by affinity chromatography with a bovine prothrombin-Sepharose 4B column (109) and gel filtration (335). A number of peptide hydrolases have been isolated and purified from E. coli by DEAE-cellulose chromatography (217).

The catalytic site of proteases is flanked on one or both sides by specificity subsites, each able to accommodate the side chain of a single amino acid residue from the substrate. These sites are numbered from the catalytic site S1 through Sn toward the N terminus of the structure and Sl' through Sn' toward the C terminus. The residues which they accommodate from the substrate are numbered Pl through Pn and P1' through Pn', respectively (Fig. 2).

View larger version (5K): [in this window] [in a new window]   FIG. 2.   Active sites of proteases. The catalytic site of proteases is indicated by * and the scissile bond is indicated by   ; S1 through Sn and S1' through Sn' are the specificity subsites on the enzyme, while P1 through Pn and P1' through Pn' are the residues on the substrate accommodated by the subsites on the enzyme.

Serine proteases usually follow a two-step reaction for hydrolysis in which a covalently linked enzyme-peptide intermediate is formed with the loss of the amino acid or peptide fragment (60). This acylation step is followed by a deacylation process which occurs by a nucleophilic attack on the intermediate by water, resulting in hydrolysis of the peptide. Serine endopeptidases can be classified into three groups based mainly on their primary substrate preference: (i) trypsin-like, which cleave after positively charged residues; (ii) chymotrypsin-like, which cleave after large hydrophobic residues; and (iii) elastase-like, which cleave after small hydrophobic residues. The Pl residue exclusively dictates the site of peptide bond cleavage. The primary specificity is affected only by the Pl residues; the residues at other positions affect the rate of cleavage. The subsite interactions are localized to specific amino acids around the Pl residue to a unique set of sequences on the enzyme. Some of the serine peptidases from Achromobacter spp. are lysine-specific enzymes (179), whereas those from Clostridium spp. are arginine specific (clostripain) (71) and those from Flavobacterium spp. are post proline-specific (329). Endopeptidases that are specific to glutamic acid and aspartic acid residues have also been found in B. licheniformis and S. aureus (52).

The recent studies based on the three-dimensional structures of proteases and comparisons of amino acid sequences near the primary substrate-binding site in trypsin-like proteases of viral and bacterial origin suggest a putative general substrate binding scheme for proteases with specificity towards glutamic acid involving a histidine residue and a hydroxyl function. However, a few other serine proteases such as peptidase A from E. coli and the repressor LexA show distinctly different mechanism of action without the classic Ser-His-Asp triad (12). Some of the glycine residues are conserved in the vicinity of the catalytic serine residue, but their exact positions are variable (25).

The chymotrypsin-like enzymes are confined almost entirely to animals, the exceptions being trypsin-like enzymes from actinomycetes and Saccharopolyspora spp. and from the fungus Fusarium oxysporum.

A few of the serine proteases belonging to the subtilisin family show a catalytic triad composed of the same residues as in the chymotrypsin family; however, the residues occur in a different order (Asp-His-Ser). Some members of the subtilisin family from the yeasts Tritirachium and Metarhizium spp. require thiol for their activity. The thiol dependance is attributable to Cys173 near the active-site histidine (122).

The carboxypeptidases are unusual among the serine-dependent enzymes in that they are maximally active at acidic pH. These enzymes are known to possess a Glu residue preceding the catalytic Ser, which is believed to be responsible for their acidic pH optimum. Although the majority of the serine proteases contain the catalytic triad Ser-His-Asp, a few use the Ser-base catalytic dyad. The Glu-specific proteases display a pronounced preference for Glu-Xaa bonds over Asp-Xaa bonds (8).

Aspartic endopeptidases depend on the aspartic acid residues for their catalytic activity. A general base catalytic mechanism has been proposed for the hydrolysis of proteins by aspartic proteases such as penicillopepsin (121) and endothiapepsin (215). Crystallographic studies have shown that the enzymes of the pepsin family are bilobed molecules with the active-site cleft located between the lobes and each lobe contributing one of the pair of aspartic acid residues that is essential for the catalytic activity (20, 259). The lobes are homologous to one another, having arisen by gene duplication. The retropepsin molecule has only one lobe, which carries only one aspartic residue, and the activity requires the formation of a noncovalent homodimer (184). In most of the enzymes from the pepsin family, the catalytic Asp residues are contained in an Asp-Thr-Gly-Xaa motif in both the N- and C-terminal lobes of the enzyme, where Xaa is Ser or Thr, whose side chains can hydrogen bond to Asp. However, Xaa is Ala in most of the retropepsins. A marked conservation of cysteine residue is also evident in aspartic proteases. The pepsins and the majority of other members of the family show specificity for the cleavage of bonds in peptides of at least six residues with hydrophobic amino acids in both the Pl and Pl' positions (132).

The specificity of the catalysis has been explained on the basis of available crystal structures (166). The structural and kinetic studies also have suggested that the mechanism involves general acid-base catalysis with lytic water molecule that directly participates in the reaction (Fig. 3A). This is supported by the crystal structures of various aspartic protease-inhibitor complexes and by the thiol inhibitors mimicking a tetrahedral intermediate formed after the attack by the lytic water molecule (120).

View larger version (9K): [in this window] [in a new window]   View larger version (8K): [in this window] [in a new window]   FIG. 3.   Mechanism of action of proteases. (A) Aspartic proteases. (B) Cysteine proteases. Im and +HIm refer to the imidazole and protonated imidazole, respectively.

The mechanism of action of metalloproteases is slightly different from that of the above-described proteases. These enzymes depend on the presence of bound divalent cations and can be inactivated by dialysis or by the addition of chelating agents. For thermolysin, based on the X-ray studies of the complex with a hydroxamic acid inhibitor, it has been proposed that Glu143 assists the nucleophilic attack of a water molecule on the carbonyl carbon of the scissile peptide bond, which is polarized by the Zn²⁺ ion (98). Most of the metalloproteases are enzymes containing the His-Glu-Xaa-Xaa-His (HEXXH) motif, which has been shown by X-ray crystallography to form a part of the site for binding of the metal, usually zinc.

A striking similarity is also observed in the reaction mechanism for several peptidases of different evolutionary origins. The plant peptidase papain can be considered the archetype of cysteine peptidases and constitutes a good model for this family of enzymes. They catalyze the hydrolysis of peptide, amide ester, thiol ester, and thiono ester bonds (226). The initial step in the catalytic process (Fig. 3B) involves the noncovalent binding of the free enzyme (structure a) and the substrate to form the complex (structure b). This is followed by the acylation of the enzyme (structure c), with the formation and release of the first product, the amine R'-NH₂. In the next deacylation step, the acyl-enzyme reacts with a water molecule to release the second product, with the regeneration of free enzyme.

The enzyme papain consists of a single protein chain folded to form two domains containing a cleft for the substrate to bind. The crystal structure of papain confirmed the Cys25-His159 pairing (11). The presence of a conserved aspargine residue (Asn175) in the proximity of catalytic histidine (His159) creating a Cys-His-Asn triad in cysteine peptidases is considered analogous to the Ser-His-Asp arrangement found in serine proteases.

Studies of the mechanism of action of proteases have revealed that they exhibit different types of mechanism based on their active-site configuration. The serine proteases contain a Ser-His-Asp catalytic triad, and the hydrolysis of the peptide bond involves an acylation step followed by a deacylation step. Aspartic proteases are characterized by an Asp-Thr-Gly motif in their active site and by an acid-base catalysis as their mechanisms of action. The activity of metalloproteases depends on the binding of a divalent metal ion to a His-Glu-Xaa-Xaa-His motif. Cysteine proteases adopt a hydrolysis mechanism involving a general acid-base formation followed by hydrolysis of an acyl-thiol intermediate.

All living cells maintain a particular rate of protein turnover by continuous, albeit balanced, degradation and synthesis of proteins. Catabolism of proteins provides a ready pool of amino acids as precursors of the synthesis of proteins. Intracellular proteases are known to participate in executing the proper protein turnover for the cell. In E. coli, ATP-dependent protease La, the lon gene product, is responsible for hydrolysis of abnormal proteins (38). The turnover of intracellular proteins in eukaryotes is also affected by a pathway involving ATP-dependent proteases (91). Evidence for the participation of proteolytic activity in controlling the protein turnover was demonstrated by the lack of proper turnover in protease-deficient mutants.

The formation of spores in bacteria (142), ascospores in yeasts (58), fruiting bodies in slime molds (205) and conidial discharge in fungi (221) all involve intensive protein turnover. The requirement of a protease for sporulation has been demonstrated by the use of protease inhibitors (41). Ascospore formation in yeast diploids was shown to be related to the increase in protease A activity (58). Extensive protein degradation accompanied the formation of a fruiting body and its differentiation to a stalk in slime molds. The alkaline serine protease of Conidiobolus coronatus was shown to be involved in forcible conidial discharge by isolation of a mutant with less conidial formation (221). Formation of the less active protease by autoproteolysis represents a novel means of physiological regulation of protease activity in C. coronatus (219).

The dormant spores lack the amino acids required for germination. Degradation of proteins in dormant spores by serine endoproteinases makes amino acids and nitrogen available for the biosynthesis of new proteins and nucleotides. These proteases are specific only for storage proteins and do not affect other spore proteins. Their activity is rapidly lost on germination of the spores (227). Microconidal germination and hyphal fusion also involve the participation of a specific alkaline serine protease (159). Extracellular acid proteases are believed to be involved in the breakage of cell wall polypeptide linkages during germination of Dictyostelium discoideum spores (118) and Polysphondylium pallidum microcysts (206).

Activation of the zymogenic precursor forms of enzymes and proteins by specific proteases represents an important step in the physiological regulation of many rate-controlling processes such as generation of protein hormones, assembly of fibrils and viruses, blood coagulation, and fertilization of ova by sperm. Activation of zymogenic forms of chitin synthase by limited proteolysis has been observed in Candida albicans, Mucor rouxii, and Aspergillus nidulans. Kex-2 protease (kexin; EC 3.4.21.61), originally discovered in yeast, has emerged as a prototype of a family of eukaryotic precursor processing enzymes. It catalyzes the hydrolysis of prohormones and of integral membrane proteins of the secretory pathway by specific cleavage at the carboxyl side of pairs of basic residues such as Lys-Arg or Arg-Arg (12). Furin (EC 3.4.21.5) is a mammalian homolog of the Kex-2 protease that was discovered serendipitously and has been shown to catalyze the hydrolysis of a wide variety of precursor proteins at Arg-X-Lys or Arg-Arg sites within the constitutive secretory pathway (266). Pepsin, trypsin, and chymotrypsin occur as their inactive zymogenic forms, which are activated by the action of proteases.

Proteolytic inactivation of enzymes, leading to irreversible loss of in vivo catalytic activity, is also a physiologically significant event. Several enzymes are known to be inactivated in response to physiological or developmental changes or after a metabolic shift. Proteinases A and B from yeast inactivate several enzymes in a two-step process involving covalent modification of proteins as a marking mechanism for proteolysis.

Proteolytic modification of enzymes is known to result in a protein with altered physiological function; e.g., leucyl-L-RNA synthetase from E. coli is converted into an enzyme which catalyzes leucine-dependent pyrophosphate exchange by removal of a small peptide from the native enzyme.

Modulation of gene expression mediated by protease has been demonstrated (241). Proteolysis of a repressor by an ATP-requiring protease resulted in a derepression of the gene. A change in the transcriptional specificity of the B subunit of Bacillus thuringiensis RNA polymerase was correlated with its proteolytic modification (154). Modification of ribosomal proteins by proteases has been suggested to be responsible for the regulation of translation (128).

Besides the general functions described so far, the proteases also mediate the degradation of a variety of regulatory proteins that control the heat shock response, the SOS response to DNA damage, the life cycle of bacteriophage (75), and programmed bacterial cell death (303). Recently, a new physiological function has been attributed to the ATP-dependent proteases conserved between bacteria and eukaryotes. It is believed that they act as chaperones and mediate not only proteolysis but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes (275). In addition to the multitude of activities that are already assigned to proteases, many more new functions are likely to emerge in the near future.

All detergent proteases currently used in the market are serine proteases produced by Bacillus strains. Fungal alkaline proteases are advantageous due to the ease of downstream processing to prepare a microbe-free enzyme. An alkaline protease from Conidiobolus coronatus was found to be compatible with commercial detergents used in India (219) and retained 43% of its activity at 50°C for 50 min in the presence of Ca²⁺ (25 mM) and glycine (1 M) (16).

Dairy industry. The major application of proteases in the dairy industry is in the manufacture of cheese. The milk-coagulating enzymes fall into three main categories, (i) animal rennets, (ii) microbial milk coagulants, and (iii) genetically engineered chymosin. Both animal and microbial milk-coagulating proteases belong to a class of acid aspartate proteases and have molecular weights between 30,000 to 40,000. Rennet extracted from the fourth stomach of unweaned calves contains the highest ratio of chymosin (EC 3.4.23.4) to pepsin activity. A world shortage of calf rennet due to the increased demand for cheese production has intensified the search for alternative microbial milk coagulants. The microbial enzymes exhibited two major drawbacks, i.e., (i) the presence of high levels of nonspecific and heat-stable proteases, which led to the development of bitterness in cheese after storage; and (ii) a poor yield. Extensive research in this area has resulted in the production of enzymes that are completely inactivated at normal pasteurization temperatures and contain very low levels of nonspecific proteases. In cheesemaking, the primary function of proteases is to hydrolyze the specific peptide bond (the Phe105-Met106 bond) to generate para--casein and macropeptides. Chymosin is preferred due to its high specificity for casein, which is responsible for its excellent performance in cheesemaking. The proteases produced by GRAS (genetically regarded as safe)-cleared microbes such as Mucor michei, Bacillus subtilis, and Endothia parasitica are gradually replacing chymosin in cheesemaking. In 1988, chymosin produced through recombinant DNA technology was first introduced to cheesemakers for evaluation. Genencor International increased the production of chymosin in Aspergillus niger var. awamori to commercial levels. At present, their three recombinant chymosin products are available and are awaiting legislative approval for their use in cheesemaking (72).

The wide diversity and specificity of proteases are used to great advantage in developing effective therapeutic agents. Oral administration of proteases from Aspergillus oryzae (Luizym and Nortase) has been used as a digestive aid to correct certain lytic enzyme deficiency syndromes. Clostridial collagenase or subtilisin is used in combination with broad-spectrum antibiotics in the treatment of burns and wounds. An asparginase isolated from E. coli is used to eliminate aspargine from the bloodstream in the various forms of lymphocytic leukemia. Alkaline protease from Conidiobolus coronatus was found to be able to replace trypsin in animal cell cultures (36).

Gene cloning is a rapidly progressing technology that has been instrumental in improving our understanding of the structure-function relationship of genetic systems. It provides an excellent method for the manipulation and control of genes. More than 50% of the industrially important enzymes are now produced from genetically engineered microorganisms (96). Several reports have been published in the past decade (Table 4) on the isolation and manipulation of microbial protease genes with the aim of (i) enzyme overproduction by the gene dosage effect, (ii) studying the primary structure of the protein and its role in the pathogenicity of the secreting microorganism, and (iii) protein engineering to locate the active-site residues and/or to alter the enzyme properties to suit its commercial applications. Protease genes from bacteria, fungi, and viruses have been cloned and sequenced (Table 4).

(i) B. subtilis as a host for cloning of protease genes from Bacillus spp. The ability of B. subtilis to secrete various proteins into the culture medium and its lack of pathogenicity make it a potential host for the production of foreign polypeptides by recombinant DNA technology. Several Bacillus spp. secrete two major types of protease, a subtilisin or alkaline protease and a metalloprotease or neutral protease, which are of industrial importance. Studies of these extracellular proteases are significant not only from the point of view of overproduction but also for understanding their mechanism of secretion. Table 5 describes the cloning of genes for several neutral (npr) and alkaline (apr) proteases from various bacilli into B. subtilis.

(ii) B. subtilis. B. subtilis 168 secretes at least six extracellular proteases into the culture medium at the end of the exponential phase. The structural genes encoding the alkaline protease (apr) or subtilisin (270), neutral protease A and B (nprA and nprB) (90, 297, 323), minor extracellular protease (epr) (27, 263), bacillopeptidase F (bpr) (265), and metalloprotease (mpr) (264) have been cloned and characterized. These proteases are synthesized in the form of a "prepro" enzyme. To increase the expression of subtilisin and neutral proteases, Henner et al. replaced the natural promoters of apr and npr genes with the amylase promoter from B. amyloliquefaciens and the neutral protease promoter from B. subtilis, respectively (90). To understand the regulation of npr A gene expression, Toma et al. cloned the genes from B. subtilis 168 (normal producer) and Basc 1A341 (overproducer) (295). The two genes were found to be highly homologous except for a stretch of 66 bp close to the promoter region, which is absent in the Basc 1A341 gene. The epr gene shows partial homology to the apr gene and to the major intracellular serine protease (Isp-1) gene of B. subtilis (138). The epr gene was mapped at a locus different from the apr and npr loci on the B. subtilis chromosome and was shown not to be required for growth or sporulation, similar to apr or npr genes. Deletion of 240 amino acids (aa) from the C-terminal region of the epr gene product did not abolish the enzyme activity (27, 263). The deduced amino acid sequence of the mature bpr gene product is similar to those of other serine proteases of B. subtilis, i.e., subtilisin, Isp-1, and Epr. B. subtilis strains containing mutations in five extracellular protease genes (apr, npr, epr, mpr, and bpr) have been constructed (264) with the aim of expressing heterologous gene products in B. subtilis. The total amino acid sequence of B. subtilis Isp-1 deduced from the nucleotide sequence showed considerable homology (45%) to subtilisin. Highly conserved sequences are present around the essential amino acids, Ser, His, and Asp, indicating that the genes for both the intra- and extracellular serine proteases have a common ancestor.

(iii) Alkalophilic Bacillus spp. Bacillus proteases with an extremely alkaline pH optimum are generally used in detergent powders and are preferred over the subtilisins (optimal pH, 8.5 to 10.0). The information on these enzymes is helpful in designing new subtilisins. Kaneko et al. cloned and sequenced the ale gene, encoding alkaline elastase YaB, a new subtilisin from an alkalophilic Bacillus strain (129). The deduced amino acid sequence showed 55% homology to subtilisin BPN'. Almost all the positively charged residues have been predicted to be present on the surface of the alkaline elastase YaB molecule, facilitating its binding to elastin. The deduced amino acid sequence of the highly alkaline serine protease from another alkalophilic strain, B. alcalophilus PB92, showed considerable homology to YaB (300). The cloned gene was further used to increase the production level of the protease by gene amplification through chromosomal integration. Increased enzyme production and gene stabilization was observed when nontandem duplication occurred.

(iv) Other bacilli. A gene encoding the highly thermostable neutral proteinase (Npr) from Bacillus sp. strain EA1 was shown to be closely related to an npr gene from B. caldolyticus YP-T, except for a single-amino-acid change in the gene product (249). The enzyme from Bacillus sp. strain EA1 was more thermostable than the enzyme from B. caldolyticus YP-T; this can be attributed to the single-amino-acid change.