Introduction

Proteases are enzymes that can hydrolyze proteins and peptides and can be divided in exoproteases and endoproteases depending on their activity. Based on their mechanisms of catalysis, endoproteases are divided into aspartic proteases, cysteine proteases, metalloproteases, serine proteases, and threonine proteases (Barrett 2001). Metalloproteases are proteases that contain one or two metal ions in their active centers. Most metalloproteases contain Zn2+, while a few contain Mg2+, Ni2+, or Cu2+. The role of the catalytic metal ions in metalloproteases is to activate a water molecule, which serves as a nucleophile in catalysis. The majority of metalloproteases contain one catalytic metal ion, and those containing two catalytic metal ions so far described are all exoproteases (Rawlings and Barrett 2004).

Metalloproteases are produced by all species of plants, animals, and microorganisms. Some metalloproteases function in the cell or on the membrane. Other metalloproteases that are secreted to the periplasm or outside the cell are called extracellular metalloproteases. Extracellular metalloproteases in mammals, matrix metalloproteases, have been widely studied because they are related to many diseases, such as arthritis, sclerosis, and some malignant tumors (Malemud 2006). Heterotrophic bacteria usually secrete proteases outside their cells to degrade environmental proteins for nutrition. Most of the bacterial extracellular proteases (BEMPs) that have been studied are various serine proteases and metalloproteases. Some BEMPs are important virulence factors of pathogenic bacteria (Goguen et al. 1995), and some BEMPs have been applied in biotechnology (Adekoya and Sylte 2009). In recent years, a series of new BEMPs have been explored, and important progress has been made on the characterization and application of BEMPs.

Diversity of BEMPs

In early studies, BEMPs were mainly found in soil bacteria and pathogenic bacteria. Thermolysin-like metalloproteases (TLPs) from Bacillus thermoproteolyticus, Alicyclobacillus acidocaldarius, etc. have been the model to study metalloproteases (Ohta et al. 1966; Tronrud et al. 1987; Eijsink et al. 1995). Many BEMPs are known virulence factors, such as fragilysin from Bacteroides fragilis (Myers et al. 1984), pseudolysin from Pseudomonas aeruginosa (also called P. aeruginosa elastase, P. aeruginosa neutral proteinase) (Morihara 1964), λ-toxin from Clostridium perfringens (Jin et al. 1996), vibriolysin from Vibrio vulnificus (Miyoshi et al. 1987), and flavastacin from Chrysebacterium meningosepticum (Grimwood et al. 1994; Tarentino et al. 1995). The famous anthrax lethal factor, one of the three proteins of anthrax toxin, is an extracellular metalloprotease of Bacillus anthracis (Smith and Stanley 1962; Leppla 2000; Smith 2002). In recent years, with increasing studies on marine bacterial enzymes, many marine bacteria have been found to secrete various extracellular metalloproteases. Zhou et al. (2009) investigated the diversity of extracellular proteases of the cultivable bacteria in the sediments from the South China Sea and showed that various metalloproteases are secreted by many bacterial strains of Pseudoalteromonas, Alteromonas, Vibrio, and Shewanella of γ-proteobacteria. Some extracellular metalloproteases from marine bacteria have been characterized, such as myroilysin from deep-sea bacterium Myroides profundi D25 (Chen et al. 2009), MCP-02 from deep-sea bacterium Pseudoalteromonas sp. SM9913, and E495 from the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495 (Chen et al. 2003; Xie et al. 2009).

The MEROPS database (http://merops.sanger.ac.uk) is a widely used peptidase database, which groups peptidases into families based on sequence homology (Rawlings et al. 2010). A peptidase is classified into a family based on sharing significant similarities in amino acid sequence with the type example or another member of the family. In the current release (Release 9.4) of the MEROPS database, there are a total of 63 metalloprotease families, nine of which include but are not limited to BEMP members. As shown in Table 1, BEMPs are distributed among the metalloprotease families M4, M5, M9, M10, M12, M13, M23, M30, and M34. The proteases in the M4, a big family of metalloproteases, are mostly BEMPs. Thermolysin is the prototype of the M4 family. Many members of M4 are virulence factors, such as the aforementioned pseudolysin, λ-toxin, and vibriolysin. Mycolysin secreted by Streptomyces cacaoi is the only metalloprotease in the M5 family that has been characterized so far (Chang et al. 1990). The M9 family includes some bacterial collagenases secreted by strains of Vibrio (Fukushima et al. 1990) and Clostridium (Bond and Van Wart 1984a, b). In the M10 family, BEMPs belong to subfamilies M10B and M10C. The M10B subfamily includes serralysin (Miyata et al. 1970), serralysin-like proteases (Kim et al. 1997; Kumeta et al. 1999; Delepelaire and Wandersman 1989, 1990; Daborn et al. 2001), and aeruginolysin (or P. aeruginosa alkaline proteinase) (Morihara 1963; Morihara and Tsuzuki 1977). The virulence factor fragilysin from B. fragilis is the only characterized BEMP in the M10C subfamily (Myers et al. 1984). In the M12 family, there are two metalloproteases, flavastacin (Tarentino et al. 1995) and myroilysin (Chen et al. 2009). Interestingly, the M12 family is a big family of metalloproteases, in which most metalloproteases are from animals, whereas flavastacin and myroilysin are the only two members reported from bacteria. Furthermore, PepO (or oligopeptidase O) and PepO-like oligopeptidases secreted by lactic acid bacteria and Streptococcal strains are members of the M13 family (Tan et al. 1993; Monnet 1995). In the M34 family, the anthrax lethal factor is the only BEMP that has been characterized. The M23 family contains many BEMPs, such as β-lytic metalloendopeptidase (or β-lytic protease) (Whitaker et al. 1965), staphylolysin (or LasA endopeptidase, staphylolytic endopeptidase) (Kessler et al. 1993), and lysostaphin (Schindler and Schuhardt 1965), which all have bacteriolytic activity (Li et al. 1998; Trine et al. 2003; Schindler and Schuhardt 1964; Ahmed et al. 2003). Notably, a number of bacteria secrete more than one metalloprotease. For example, Staphylococcus aureus secretes two metalloproteases, aureolysin (M4) (Arvidson 1973) and hyicolysin (M30) (Ayora and Götz 1994). Additionally, P. aeruginosa secretes three metalloproteases, pseudolysin (M4), aeruginolysin (M10), and staphylolysin (M23).

Table 1 Representative bacterial extracellular metalloproteases in each MEROPS family

Structural characteristics of BEMPs

Similar to other secreted enzymes, BEMPs need signal peptides to mediate their translocation to the matrix. These signal peptides are then removed after the enzyme is secreted. In the precursors of BEMPs, immediately following the signal peptides are propeptides that act as intramolecular chaperones (IMC) and assist in the correct folding of the following catalytic domains (Kessler and Ohman 2004; Gao et al. 2010). Some BEMPs also contain C-terminal extensions in their precursors. For example, the precursor of MCP-02 from Pseudoalteromonas sp. SM9913 contains two PPC domains (bacterial Pre-peptidase C-terminal domain) in its C-terminal extension (Gao et al. 2010). During maturation, the propeptide and the C-terminal extension are all cleaved.

TLPs are typical BEMPs. The first crystal structure of a TLP, thermolysin, was reported in 1972 (Matthews et al. 1972). Thermolysin is comprised of 316 amino acid residues and has a molecular mass of 34.2 kDa (Titani et al. 1972). Thermolysin contains two regions, the N-terminal region and the C-terminal region, which form the catalytic domain. The N-terminal region is predominantly β-pleated sheets, and the C-terminal region is predominantly α-helices, which are connected by an α-helix (Fig. 1). The catalytic Zn2+ lies in a deep cleft that is formed by the two regions. This cleft is also the substrate-binding site in catalysis. Zn2+ and its four ligands, His142, His146, Glu166, and an activated water molecule, are bound in an approximate tetrahedral geometry to form the catalytic center (Fig. 2). Additionally, thermolysin contains four Ca2+ ions; two are located near the catalytic center, and the other two are located on two surface loops. These Ca2+ ions stabilize thermolysin by preventing the autolysis (Matthews et al. 1974).

Fig. 1
figure 1

A comparison of the structures of thermolysin (M4), serralysin (M10), lysostaphin (M23), and anthrax lethal factor (M34). All crystallized BEMPs in the four families are α/β fold proteins with one catalytic Zn2+ coordinated in the active site. Thermolysin and serralysin both harbor several Ca2+ ions that are important for structural stabilization. In serralysin, a C-terminal extension mainly composed of β-sheets is shown in orange. In lysostaphin, the propeptide is shown in purple

Fig. 2
figure 2

The active site of thermolysin. Zn2+ in the catalytic cleft is coordinated by an activated water molecule and three residue ligands, two histidines, and one glutamic acid (shown in purple). The water molecule acts as a nucleophile, which is important for substrate hydrolysis. Three other strictly conserved residues, glutamic acid, histidine, and tyrosine, are also shown in red. These three residues are required to stabilize the enzyme-substrate transition state

The crystal structures of a number of BEMPs have been solved (Table 1). Similar to the primary sequence diversity of BEMPs, the three-dimensional structures of BEMPs also vary significantly (Fig. 1). Some BEMPs contain more than one catalytic domain, such as the anthrax lethal factor that has four domains with a molecular mass (Mr) up to 90.2 kDa in its mature enzyme (Smith and Stanley 1962). However, most BEMPs only contain one catalytic domain and the Mr ranging from 20 to 35 kDa. Despite the difference in their sequences and size, all BEMPs contain a catalytic Zn2+ at their active center. Deprivation of the catalytic Zn2+ by a chelator can lead to loss of the activity of a metalloprotease. The four ligands of the catalytic Zn2+ are an activated water molecule and three amino acid residues, two His and one Glu in a majority of BEMPs; therefore, these proteases contain a typical HEXXH motif. However, in the metalloproteases of the M23 family, this motif is replaced by HXH (Rawlings and Barrett 2004).

Maturation mechanism of BEMPs

The precursor of a BEMP is synthesized as a polypeptide in the cell, which undergoes a complicated process to form an active enzyme. BEMPs generally mature via two ways. Some enzymes, such as TLPs, mature by autocatalysis, and their propeptides are cleaved and degraded by the enzyme during maturation. In contrast, other enzymes cannot mature by autocatalysis. The cleavage and degradation of the propeptides of these enzymes depend on the help of other enzymes, such as staphylolysin from P. aeruginosa.

The maturation process of the TLP, pseudolysin, has been studied in detail. Prepropseudolysin synthesized in the cell is secreted into the periplasm by the Sec machinery, and the signal peptide is removed in this process. In the periplasm, the resultant proenzyme folds into an unautoprocessed zymogen mediated by its propeptide that serves as an IMC (Kessler and Ohman 2004). Then, the propeptide domain is rapidly cleaved off by autocatalysis, which is still noncovalently bound to the catalytic domain, leading to the formation of the autoprocessed complex of pseudolysin (Kessler and Safrin 1988a, b). In the periplasm, two disulfide bonds also form in the zymogen and the complex, respectively (Malhotra et al. 2000; Braun et al. 2001). The autoprocessed complex is translocated through the outer membrane by the Xcp export machinery (Filloux et al. 1998; Braun et al. 2000). Outside the cell, the propeptide in the complex is dissociated and degraded by other active pseudolysin, resulting in the formation of an active pseudolysin (Braun et al. 1998; Kessler et al. 1998).

TLPs are known to mature by an autocatalytic process, and their propeptides also play a key role in maturation (Marie-Claire et al. 1998; Nickerson et al. 2008). However, the molecular mechanism of TLP autoprocessing and the accurate role of the propeptide have remained unclear due to the lack of structures of the unautoprocessed zymogen and the autoprocessed complex of TLPs. Recently, Gao et al. (2010) reported the structure of the autoprocessed complex of the TLP, MCP-02. Using MCP-02 as a model, Gao et al. clarified the structure and role of the TLP propeptide in maturation. Furthermore, additional insight was gained on the maturation mechanism of TLPs. The results of this study demonstrated that the unautoprocessed zymogen is in an unstable state with high energy and basal protease activity, which leads to the prompt initiation of the first autocleavage of the peptide bond between the propeptide and the catalytic domain. After break of this bond, a large conformational change occurs in the catalytic domain, and a relatively stable autoprocessed complex of the propeptide and the catalytic domain with lower energy forms. In the autoprocessed complex, the C-terminus of the propeptide inserts into the cleft between the two regions of the catalytic domain to act as an inhibitor. The last residue, His, of the propeptide replaces the activated water molecule in the mature enzyme, acting as a monodentate ligand to zinc, inhibiting the enzyme activity. Then, the propeptide in the complex is degraded into small peptide pieces and is released from the catalytic domain. When the C-terminus of the propeptide is released from the catalytic cleft, a water molecule occupies the fourth coordinating site of the catalytic Zn2+, converting the catalytic domain into an active enzyme.

As for the BEMPs that do not undergo autocatalytic maturation, the maturation mechanism of staphylolysin from P. aeruginosa has been well studied. Staphylolysin is encoded by the lasA gene on the chromosome of P. aeruginosa and is positively regulated by genes lasR and rhlR (Shortridge et al. 1991; Toder et al. 1991; Brint and Ohman 1995). The signal peptide in the precursor of staphylolysin is removed in the cell. The resultant 42 kDa proenzyme is secreted via the Xcp (type II) export machinery. Outside the cell, a 14-kDa fragment of the propeptide in the proenzyme is cleaved, and a 28-kDa proenzyme intermediate is formed. Then, another part (8 kDa) of the propeptide is cleaved from the intermediate, which results in the final 20 kDa active enzyme (Gustin et al. 1996; Braun et al. 1998; Kessler et al. 1998). This maturation process involves other proteases from P. aeruginosa. It is now known that the protease pseudolysin and the lysine-specific protease from P. aeruginosa are involved in staphylolysin maturation (Kessler et al. 1998).

Properties and physiological role of BEMPs

Proteins around a bacterial cell must be degraded into amino acids and oligopeptides before they are utilized by the bacterial cell for nutrition. For this reason, heterotrophic bacteria secrete proteases outside the cell, including metalloproteases and other kinds of extracellular proteases, which are responsible for the degradation of the surrounding proteins into amino acids and oligopeptides for bacterial utilization (Zhou et al. 2009). To date, BEMPs that have been reported are endoproteases, which coordinate with other extracellular proteases to degrade the proteins outside the bacterial cell. In addition to the role of BEMPs in nutrition, some BEMPs may play a role in bacterial defense. For example, the BEMPs of the M23 family can lyse the cell wall of other bacteria, leading to cell death (Li et al. 1998; Trine et al. 2003; Schindler and Schuhardt 1964; Ahmed et al. 2003).

Similar to other metalloproteases, the activity of BEMPs can be inhibited by metal chelators due to the deprivation of catalytic Zn2+. 1,10-Phenanthroline is a common inhibitor of metalloproteases, which can inhibit the activity of most metalloproteases (Chen et al. 2003, 2009; Kessler and Ohman 2004). The other metal chelators, such as EDTA and EGTA, can inhibit all or some of the activity of metalloproteases. Since these proteases function outside the cell, BEMPs must be adapted to the environment around the cell to maintain high activity for efficient protein degradation. As a result, some characteristics of BEMPs, such as the optimum temperature and pH, thermostability, pH stability, and isoelectric point, are closely related to the environment these enzymes are in. Because sea water is alkaline, the metalloproteases secreted by marine bacteria usually are alkaline proteases, which have alkaline optimum pH and are stable in an alkaline range (Chen et al. 2003, 2009; Xie et al. 2009). The BEMPs from the bacteria isolated from saline environments have high salt tolerance (Rohban et al. 2009; Sánchez-Porro et al. 2003; Karbalaei-Heidari et al. 2007; Sánchez-Porro et al. 2009). The BEMPs from thermophilic bacteria isolated from hot environments usually have high thermostability and high optimal temperatures (Ohta et al. 1966; Sookkheo et al. 2000). In contrast, most of the BEMPs from psychrophilic bacteria have cold-adapted characteristics such as high activity at low temperatures and low thermostability at moderate and high temperatures (Chen et al. 2009; Xie et al. 2009). Even BEMPs of the same family from different environments have significant differences in their characteristics. For example, Xie et al. (2009) studied the thermostability and the cold-adapted mechanism of three BEMPs of the M4 family, E495, MCP-02, and pseudoalterin, all of which have similar structures but are from different environments. E495 is from an Arctic sea-ice bacterium, MCP-02 from a deep-sea sediment bacterium, and pseudoalterin from a land soil bacterium. The results of Xie et al. demonstrated that the thermostability of E495, MCP-02, and pseudoalterin increases orderly, whereas their cold-adapted ability decreased orderly, suggesting that the environment mainly determines these characteristics of BEMPs.

Applications and prospects of BEMPs

Microbial proteases are important industrial enzymes among hydrolytic enzymes and account for over a half of the total enzyme sales in the world market. Many industrial microbial proteases are BEMPs, such as Thermoase PC10F (Amano Enzyme Inc., Japan), Neutrase (Novo Nordisk, Denmark), Protin PC10F (Amano Enzyme Inc., Japan), and the highly stable TLP-ste variant Boilysin (Groningen, The Netherlands). These proteases are widely used in the industries of food, medicine, brewing, leather, film, and baking. Additionally, some BEMPs for laboratory use, such as thermolysin, can be provided by several companies like Sigma, Merck.

Applications in food industry

TLPs are widely used in enzymatic peptide synthesis. Since 1979, thermolysin has been studied and used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (Z-Asp-Phe-OMe), the precursor to the artificial sweetener aspartame (Isowa et al. 1979; Ooshima et al. 1985; Ager et al. 1998). During the past decades, many procedures for the synthesis of Z-Asp-Phe-OMe have been developed, including aqueous systems (Inouye 1992; Murakami et al. 1996), systems with water-miscible organic solvents (Lee et al. 1992; Kühn et al. 2002), biphasic systems (Hirata et al. 1997; Murakami and Hirata 1997; Murakami et al. 1998; Miyanaga et al. 2000), solid-to-solid synthesis (Erbeldinger et al. 1998a; Erbeldinger et al. 1998b; Erbeldinger et al. 2001), and low-water solvent systems (Nakanishi et al. 1985) and membrane systems (Iacobucci et al. 1994). New TLPs have also been explored for peptides synthesis. The metalloprotease vimelysin from Vibrio sp. T1800 was shown to be more appropriate than thermolysin in the synthesis of aspartame because of its lower optimal temperatures and higher yield (Kunugi et al. 1997). In 1995 and 1997, Neutrase from B. subtilis began to be applied in the synthesis of Celite-545 and Polyamide-PA6 in industry, respectively (Clapes et al. 1995, 1997). Pseudolysin secreted by P. aeruginosa may also be developed for peptide synthesis, which has been demonstrated to be a suitable catalyst for peptide bond formation through reverse proteolysis (Rival et al. 2000).

Some BEMPs, mostly in combination with other proteases, are also used to hydrolyze food proteins to produce flavor-enhancing peptides in food industry. Flavor development via a cocktail of proteases, including Neutrase, has been used to accelerate the ripening of dry fermented sausages (Fernandez et al. 2000). Soy and wheat hydrolysates are used as the flavor enhancer of soups and sauces, and milk protein hydrolysates are preferred for the refinement of cheese products (Mansfeld 2007). Additionally, some BEMPs, mostly thermolysin and Neutrase, are often applied to hydrolyze proteins to produce novel peptides with various bioactivities, such as antioxidant activity (Shen et al. 2010; Song et al. 2008; Qian et al. 2008) and angiotensin-I converting enzyme inhibitory activity (Cheung et al. 2009).

Applications in the pharmaceutical industry

Clostridium collagenases have uses in tissue dissociation in the isolation of hepatocytes, adipocytes, and other cells, and in preparation of vascular endothelial cells for seeding vascular prostheses (Seglen 1976; Sharefkin et al. 1987). These enzymes also have potential in the isolation of pancreatic islets for transplantation and in the treatment of herniated discs (Hedtmann et al. 1992; Wolters et al. 1995). Recently, safe and efficacious doses of Clostridium collagenases have been investigated to treat Dupuytren disease in adult patients. These doses have shown significant short- and mid-term effects (Thomas and Bayat 2010). Furthermore, collagenases from Vibrio strains have potential in tissue cell dispersion, the removal of necrotic tissue from burns, ulcers, and decubitus ulcers (Fukushima and Okuda 2004).

Peptidases of the M23 family can lyse the cell walls of other bacteria, indicating that these enzymes may potentially serve as antimicrobial agents (Kessler et al. 1993; Trine et al. 2003; Ahmed et al. 2003). For example, lysostaphin has been tested as a novel antistaphylococcal agent (Dajcs et al. 2000; Patron et al. 1999; Jaspal 2008). Staphylolysin has also been evaluated in the treatment of endophthalmitis caused by methicillin-resistant S. aureus in a rat model (Barequet et al. 2009).

The BEMPs that are virulence factors of pathogens can also be used as targets in drug and vaccine development (Adekoya and Sylte 2009; Goguen et al. 1995). For example, Staphylococcus hyicus infects pigs, cattle, and chickens, and is also associated with exudative dermatitis, necrosis of the tips of the ears, and tail-biting in pigs. The metalloprotease secreted by S. hyicus is hypothesized to have a role in pathology and to be of agricultural and veterinary importance (Takeuchi et al. 2000). The lethal toxin including lethal factor and protective antigen can be used to selectively block MEK-dependent pathways in intact cells, which can provide a tool to evaluate the role of MEKs (mitogen-activated protein kinase kinases) in a given physiological pathway. Because MEKs are essential for the growth of certain tumors, lethal toxin has been tested as an anticancer drug and shows promising results in mouse models (Koo et al. 2002).

Applications in other fields

In the baking industry, some BEMPs have applications for reducing gluten–protein cohesiveness to produce the desired dough in cake baking and for the modification of proteins in bread manufacturing (Bélafi-Bakó 2007). In the brewing industry, some BEMPs have applications in beer brewing to improve the yield by increasing the available nitrogen for the metabolism of yeast in fermentation (Priest 1992). Additionally, Neutrase can be used in the brewing industry because of its insensitivity to natural plant protease inhibitors (Rao et al. 1998). In the leather processing industry, BEMPs can be used to de-hair animal skins and remove nonfibrillar proteins (Patil and Chaudhari 2009; Thanikaivelan et al. 2004). Furthermore, in the film industry, BEMPs have potential X-ray film processing and waste treatment (Patil and Chaudhari 2009).

Prospects

Many of the BEMPs used in industry are of mesophilic and/or neutrophilic origin. However, BEMPs from extremophilic bacteria may have tremendous utility in many biotechnological areas. The exploration of new BEMPs with novel catalytic properties and/or other special characteristics is in high demand. In recent years, an increasing number of studies have focused on BEMPs from extreme environments because of the special characteristics that these BEMPs may have, such as cold adaptation, hot tolerance, organic solvent tolerance, and alkaline/acid tolerance, which may have the most use in industry. Thermophilic BEMPs could be applied where a cooling step is uneconomical or where high temperatures are required to increase the bioavailability and/or solubility of substrates to reduce both viscosity and the risk of contamination. Acidophilic and alkaliphilic BEMPs would be beneficial in processes where extreme pH conditions are needed. Cold-adapted BEMPs would be valuable to processes where heating is economically counterproductive or where low temperatures are required to avoid alteration of ingredient and/or product quality, to avoid microbial development and to avoid product denaturation. Additionally, the overexpression of valuable BEMPs and the improvement in enzyme activity and/or other properties of some promising BEMPs by gene engineering and protein engineering are essential for their application in industry.