Marine eukaryotes and prokaryotes produce dissolved and particulate organic nitrogen, which includes a variety of macromolecules, such as insoluble proteins (e.g., collagen, elastin), dissolved proteins and peptides, nucleic acid, and N-acetyl amino polysaccharides (Aluwihare et al. 2005; Hu et al. 2016; Jørgensen 2009). Microbial enzymatic activity leads to the mineralization and cycling of these macromolecules (Arnosti 2011; Arnosti et al. 2014); i.e., microbes secrete extracellular proteases that hydrolyze proteins into small peptides and amino acids that are suitable for uptake. Consequently, protease-producing microorganisms are key players in marine biogeochemical cycling (Lloyd et al. 2013; Thamdrup and Dalsgaard 2008).
Based on catalytic type, proteases are divided into aspartic proteases, cysteine proteases, metalloproteases, serine proteases, proteases of mixed catalytic type, and proteases of unknown type (Rawlings et al. 2018). A variety of marine heterotrophic bacteria can secrete a diverse range of proteases. Laboratory studies suggest that the majority are serine proteases and metalloproteases (Zhang et al. 2015; Zhao et al. 2008, 2012a). Furthermore, the culturable bacteria that secrete proteases are mainly Gammaproteobacteria, of which Pseudoalteromonas strains are usually abundant or dominant, suggesting the importance of this one genus in marine organic nitrogen degradation (Kim et al. 2010; Liu et al. 2018; Olivera et al. 2007; Park et al. 2014; Xiong et al. 2007; Zhang et al. 2019; Zhao et al. 2008).
The strictly marine genus Pseudoalteromonas, Order Alteromonadales, Class Gammaproteobacteria includes 48 species (Table 1), with > 3900 strains deposited in the NCBI database. Species exhibit some diversity (Table 1), but they are all Gram stain negative, heterotrophic, aerobic, motile, non-spore forming, require Na+ ions for growth, and have genomic G + C contents of 38-50 mol% (Ivanova et al. 1998; Navarro-Torre et al. 2020). Strains are also widely distributed in environments from seawater to sediment of a wide range of depths (Zhao et al. 2020; Zhou et al. 2009, 2013). They are often associated with eukaryotes and produce a range of biologically active agents, such as extracellular enzymes, antibiotics, toxins, and polysaccharides (Bowman 2007; Holmström and Kjelleberg 1999; Ivanova et al. 2003; Offret et al. 2016; Zhang et al. 2016b). Many Pseudoalteromonas strains are protease secreting, producing a range of proteases (Olivera et al. 2007; Zhou et al. 2009, 2013), some of which exhibit unique properties: e.g., cold adaptation (Chen et al. 2007a; Kurata et al. 2010; Yan et al. 2009; Yang et al. 2013); salt tolerance (Yan et al. 2009); distinct substrate specificity; and catalytic mechanism (Ran et al. 2013). Moreover, both the ecological roles and potential application of Pseudoalteromonas proteases have been considered. To facilitate our appreciation of and future use of these potentially valuable resources, here we review advances in our understanding of Pseudoalteromonas proteases, with a focus on diversity, characters, ecological significance, and application potentials.
Species Type strains 16S rRNA gene sequence accession number Growth temperature (℃): range Growth pH: range Growth in NaCl (%): range GC mol% Hydrolysis of proteinaceous substrates References Pseudoalteromonas aestuariivivens DB-2T KT366926 15–40 – 0.5–7 54.9 Casein, gelatin Park et al. (2016) Pseudoalteromonas agarivorans KMM 255T AJ417594 7–35 – – 42.2–43.8 – Romanenko et al. (2003a) Pseudoalteromonas aliena SW19T AY387858 4–29 6–10 0.5–6 41–43 Casein, gelatin Ivanova et al. (2004) Pseudoalteromonas amylolytica JW1T KU535632 20–40 6–10.5 0.5–10 43.3 Gelatin Wu et al. (2017a) Pseudoalteromonas antarctica NF3T X98336 4–30 – – 41–42 Casein, gelatin Bozal et al. (1997) Pseudoalteromonas arabiensis k53T AB576636 6–35 5.5–9.5 0.5–10 43 Casein, gelatin Matsuyama et al. (2013) Pseudoalteromonas arctica A 37–1-2T DQ787199 4–25 6–8 0–9 39 – Khudary et al. (2008) Pseudoalteromonas atlantica ATCC 19262T X82134 5–35 5.5–8.5 – 40.6–41.7 Casein, gelatin Gauthier et al. (1995), Matsushita et al. (1992) Pseudoalteromonas aurantia ATCC 33046T X82135 4–30 7–10 – 38.8 Casein Gauthier and Breittmayer (1979), Gauthier et al. (1995, Pseudoalteromonas byunsanensis FR1199T DQ011289 10–40 5–10 0.5–5 39 – Park et al. (2005) Pseudoalteromonas carrageenovora ATCC 43555T X82136 5–35 5.5–9 – 39.5 Casein, gelatin Gauthier et al. (1995), Matsushita et al. (1992) Pseudoalteromonas citrea ATCC 29719T X82137 4–40 6–12 1.0–11 38.9–44.7 Casein, gelatin Gauthier et al. (1995), Ivanova et al. (1998), Gauthier (1977) Pseudoalteromonas denitrificans ATCC 43337T X82138 4–22 – 1.5–5.5 36.8 – Enger et al. (1987), Gauthier et al. (1995) Pseudoalteromonas distincta KMM 638T AF043742 – – – 43.8 – Ivanova et al. (2000) Pseudoalteromonas donghaensis HJ51T FJ754319 4–45 5.5–9.5 1–13 41.8 casein, gelatin Oh et al. (2011) Pseudoalteromonas elyakovii KMM 162T AF082562 10–37 – – 38.5 Gelatin Sawabe et al. (2000) Pseudoalteromonas espejiana ATCC 29659T X82143 – – – 43 – Chan et al. (1978), Gauthier et al. (1995) Pseudoalteromonas fenneropenaei rzy34T KR709258 20–40 6–10 1–6 45.3 Casein, gelatin Ying et al. (2016) Pseudoalteromonas flavipulchra NCIMB 2033T AF297958 10–44 5–12 0.5–10 41.7 Gelatin Ivanova et al. (2002a) Pseudoalteromonas fuliginea KMM 216T AF529062 5–30 – 1–9 41.5–43.8 – Machado et al. (2016) Pseudoalteromonas gelatinilytica NH153T KT377064 15–45 5.5–9.5 0–10 41.4 Gelatin Yan et al. (2016) Pseudoalteromonas haloplanktis ATCC 14393T AB681739 – – – 41.8–44.4 – Gauthier et al. (1995), Reichelt and Baumann (1973) Pseudoalteromonas issachenkonii KMM 3549T AF316144 4–37 6–10 0.5–15 42.9–43.3 Casein, gelatin Ivanova et al. (2002b) Pseudoalteromonas lipolytica LMEB 39T FJ404721 15–37 5.5–9.5 0.5–15 42.3 Casein, gelatin Xu et al. (2010) Pseudoalteromonas luteoviolacea ATCC 33492T X82144 10–30 – – 40.9–42.2 Casein, gelatin Gauthier (1982), Gauthier et al. (1995) Pseudoalteromonas maricaloris KMM 636T AF144036 10–37 6–10 0.5–10 38.9 Gelatin Ivanova et al. (2002a) Pseudoalteromonas marina mano4T AY563031 4–37 5.3–8.8 3–12 41.2 Casein Nam et al. (2007) Pseudoalteromonas mariniglutinosa NCIMB 1770T AJ507251 5–37 – 1–9 40.3 – Romanenko et al. (2003b) Pseudoalteromonas neustonica PAMC 28425T KU716039 4–30 6–9 1–7 39.7 Casein Hwang et al. (2016) Pseudoalteromonas nigrifaciens ATCC 19375T X82146 4–30 – – 42.9 – Baumann et al. (1984), Gauthier et al. (1995) Pseudoalteromonas paragorgicola KMM 3548T AY040229 4–30 6–10 1–6 41.1 Gelatin Ivanova et al. (2002c) Pseudoalteromonas peptidolytica F12-50-A1T AF007286 15–40 – 1–10 42 – Venkateswaran and Dohmoto (2000) Pseudoalteromonas phenolica O-BC30T AF332880 18–37 6.5–9.5 1–5 39.9–40.6 – Isnansetyo and Kamei (2003) Pseudoalteromonas piratica OCN003T KF042038 14–39 5.5–10 1–6 40.0 – Beurmann et al. (2017) Pseudoalteromonas piscicida ATCC 15057T X82215 – – – – – Bein (1954), Gauthier et al. (1995) Pseudoalteromonas profundi TP162T KT900238 10–40 6–9 0.5–9 46.7 Casein, gelatin Zhang et al. (2016a) Pseudoalteromonas prydzensis ACAM 620T U85855 0–30 – 0.5–15 38–39 Casein, gelatin Bowman (1998) Pseudoalteromonas rhizosphaerae RA15T KU588400 4–32 5–9 0–15 40.4 Casein, gelatin Navarro-Torre et al. (2020) Pseudoalteromonas rubra ATCC 29570T X82147 – – – – – Gauthier (1976), Gauthier et al. (1995) Pseudoalteromonas ruthenica KMM 300T AF316891 10–35 6–10 1–9 48.4–48.9 Elastin, gelatin Ivanova et al. (2002d) Pseudoalteromonas shioyasakiensis SE3T AB720724 5–40 5.5–9.5 0.5–12 46.9 Casein, gelatin Matsuyama et al. (2014) Pseudoalteromonas spongiae UST010723-006T AY769918 12–44 5.0–10 2–6 40.6 – Lau et al. (2005) Pseudoalteromonas tetraodonis IAM 14160T X82139 4–35 5.5–9.5 1–10 41.5 – Ivanova et al. (2001), Simidu et al. (1990) Pseudoalteromonas translucida KMM 520T AY040230 4–30 6–10 1–8 46.3 Elastin, gelatin Ivanova et al. (2002c) Pseudoalteromonas tunicata D2T Z25522 – – – 42.2 Gelatin Holmström et al. (1998) Pseudoalteromonas ulvae UL12T AF172987 – 5.5–12 – – Gelatin Egan et al. (2001) Pseudoalteromonas undina ATCC 29660T AB681919 – – – – – Chan et al. (1978), Gauthier et al. (1995) Pseudoalteromonas xiamenensis Y2T JN188399 10–40 5–10 0.5–6 45.1 Casein, gelatin Zhao et al. (2014a)
Table 1. Characteristics of the described species in the genus Pseudoalteromonas
Although many protease-producing Pseudoalteromonas strains have been identified, only a few of the proteases have been characterized (Table 2). Below we summarize what is known.
Strain Protease Family Molecular weight Structure Optimal temperature Optimal pH Thermostability Halotolerance Substrate specificity References SM9913 MCP-01 S8 65.8 + 35 (casein)
Cold-adapted - Casein,
Chen et al.(2003a, 2007a)
Zhao et al. (2008)
SM9913 MCP-03 S8 58 – 45 8.0 Cold-adapted Halophilic Casein Yan et al. (2009) 129–1 Alkaline protease (serine) 35 – 50–60 8.0 High Halophilic Casein,
Wu et al. (2015) PAMC 21,717 Pro21717 S8 37 + 30 9.0 Cold-adapted – Azocasein,
Park et al. (2018) A28 Protease I (serine) 50 – 30 8.8 – – Algicidal activity Lee et al. (2000) AS-11 Apa1 S8 45 + – – Cold-adapted – - Dong et al. (2005) SM9913 MCP-02 M4 36 + 55 8.0 Moderate – Casein Chen et al. (2003a) CF6-2 Pseudoalterin M23 19.3 + 25 9.5 Cold-adapted – Elastin, peptidoglycan Zhao et al. (2012a) NW4327 Collagenase U32 114.6 – – – – – Gelatin,
Bhattacharya et al. (2018) NW4327 Collagenase U32 52.5 – – – – – Gelatin,
Bhattacharya et al. (2019) A28 EmpI M4 38 – 50 8.6 – – Casein,
Lee et al. (2002) CP76 CP1 M4 38 – 55 8.5 Moderate Moderate Casein Sánchez-Porro et al. (2003) SM495 E495 M4 55/43.9/33.4 – – – Cold-adapted – Casein,
He et al. (2012) KCTC32086 Pph_Pro1 – – – 80–90 8.5–9.0 High Halophilic Casein,
algal waste proteins
Johnson et al. (2018) P96-47 Neutral metalloproteases – – – 45 7–9 Cold-adapted – Azocasein Vázquez et al. (2008)
Table 2. Characters of Pseudoalteromonas proteases
Most serine proteases from Pseudoalteromonas are subtilisin-like proteases of the S8 family. Among them, MCP-01 has been studied in detail. MCP-01 is the most abundant protease secreted by Pseudoalteromonas sp. SM9913 isolated from a deep-sea sediment (Chen et al. 2003a). It is a novel multidomain S8 protease deseasin containing a S8 catalytic domain, a PKD domain, and a long linker between them (Chen et al. 2007a). MCP-01 is a cold-adapted protease with an optimal temperature of 35 ℃ for casein hydrolysis, and it is highly thermolabile and tends to autolysis (Chen et al. 2003a, b; 2007b). Substrate specificity analysis shows that MCP-01 is a collagenolytic serine protease, having broad specificity toward bovine collagens of types Ⅰ, Ⅱ and Ⅳ and much higher activity toward fish insoluble collagen than that of the collagenase from Clostridium histolyticum (Zhao et al. 2008). Its C-terminal PKD domain is a collagen-binding domain (CBD) that binds on collagen fibers and swells collagen fibers to expose the triple helix monomers, but it does not unwind the collagen triple helix (Wang et al. 2010). The exposed collagen monomers are hydrolyzed by the catalytic domain. The structure of the catalytic domain of MCP-01 was solved (Ran et al. 2013) (Fig. 1). Structural and biochemical analyses show that, compared to the S8 prototype subtilisin Carlsberg that has no collagenolytic activity, MCP-01 has an enlarged substrate-binding pocket, mainly composed of loops 7, 9, and 11. The acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment, which is necessary for collagen recognition. MCP-01 displays a non-strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1 site in collagen. His211 is a key residue for the P1-basic-residue preference of MCP-01 (Ran et al. 2013). MCP-01 is the first reported collagenolytic protease from marine bacteria, and the first serine collagenolytic protease to be studied in detail. Four tripeptides, SPP, RYP, RQF, and FRQ, derived from casein have significant inducing effect on the expression of the gene encoding extracellular protease MCP-01 in P. sp. SM9913 (Chen et al. 2019).
In addition to MCP-01, the structures of two other subtilisin-like proteases of the S8 family from Pseudoalteromonas were also solved (Fig. 1): the psychrophilic alkaline serine protease Apa1 from the Antarctic psychrotroph Pseudoalteromonas sp. AS-11 (Dong et al. 2005) and the cold-active protease Pro21717 from the psychrophilic bacterium, P. arctica PAMC 21717 (Park et al. 2018). The structure of Pro21717 was analyzed for its cold-adapted properties. The catalytic domain of Pro21717 shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Pro21717 has a wide substrate pocket size, an abundant active-site loop content, and a flexible structure that provide potential explanations for the cold-adapted properties of Pro21717 (Park et al. 2018). As subtilisin-like proteases, the structures of the catalytic domains of MCP-01, Apa1, and Pro21717 are similar (Fig. 1).
MCP-03 is another characterized S8 protease from P. sp. SM9913. MCP-03 is a cold-adapted multidomain protease, containing a catalytic domain and two PPC domains. Compared to mesophilic subtilisin Carlsberg, MCP-03 has higher activity at low temperatures, lower optimum temperature, and higher thermolability. MCP-03 has good halophilic ability, showing maximal activity at 3 mol/L NaCl/KCl and good stability in 3 mol/L NaCl. These characters reflect the adaptation of MCP-03 to the cold and salty deep-sea environment. In addition, the function of the C-terminal PPC domains was studied by deletion mutagenesis, which indicated that the C-terminal PPC domains are essential for MCP-03 thermostability, unnecessary for enzyme secretion, and have an inhibitory effect on MCP-03 catalytic efficiency (Yan et al. 2009).
Wu et al. (2015) purified an extracellular alkaline protease from marine Pseudoalteromonas sp. 129-1, isolated from the seawater of South China Sea, and characterized this enzyme. This protease shows considerable activity and stability at a wide temperature range of 10-60 ℃ and pH range of 6-11. The serine protease inhibitor, PMSF, greatly inactivated this protease, suggesting it is a serine protease. This enzyme is highly stable in the presence of surfactants (SDS, Tween 80, and Triton X-100), the oxidizing agent H2O2, and commercial detergents, and is tolerant to most of the tested organic solvents. These characteristics indicate that this protease may have valuable features as an additive in laundry detergent (Wu et al. 2015). Moreover, this protease can destroy the biofilm of Pseudomonas aeruginosa PAO1, suggesting its potential as an anti-biofilm agent (Wu et al. 2015).
Several metalloproteases secreted by Pseudoalteromonas have been characterized (Table 2). Among them, the M4 metalloprotease MCP-02 from Pseudoalteromonas sp. SM9913, the M23 metalloprotease pseudoalterin from Pseudoalteromonas sp. CF6-2, and two collagenases from P. agarivorans NW4327 have been studied in detail.
Metalloproteases of the M4 family are the most common metalloproteases secreted by Pseudoalteromonas. Several M4 metalloproteases have been characterized, such as CP1 from Pseudoalteromonas sp. CP76 (Sánchez-Porro et al. 2003), E495 from Pseudoalteromonas sp. SM495 (He et al. 2012), and MCP-02 from Pseudoalteromonas sp. SM9913 (Chen et al. 2003a; Gao et al. 2010; Xie et al. 2009). CP1 from Pseudoalteromonas sp. CP76 is the first reported M4 metalloprotease from Pseudoalteromonas, which is a haloprotease secreted by the moderately halophilic bacterium P. ruthenica CP76 by the type Ⅱ secretion pathway (Sánchez-Porro et al. 2003, 2009). E495 is the most abundant protease secreted by the Arctic sea-ice bacterium Pseudoalteromonas sp. SM495. E495 has multiple active forms in the culture of strain SM495. E495-M (containing only the catalytic domain) and E495-M-C1 (containing the catalytic domain and one PPC domain) were two stable mature forms, and E495-M-C1-C2 (containing the catalytic domain and two PPC domains) might be an intermediate. The domain PPC1 in E495-M-C1 could be helpful in binding protein substrate, and therefore, improving the catalytic efficiency of E495 (He et al. 2012). MCP-02 secreted by Pseudoalteromonas sp. SM9913 is the most studied M4 metalloprotease from Pseudoalteromonas. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa (Chen et al. 2003a). Gao et al. (2010) solved the structure of the mature form of MCP-02 and two crystal structures of the autoprocessed complexes of MCP-02 during maturation (Fig. 2), which are the first reported autoprocessed complex structures of the M4 metalloproteases. Based on these structures and a simulated structure of the unautoprocessed zymogen of MCP-02, together with biochemical analysis, the authors reveal the structural basis of MCP-02 maturation, shedding light on the molecular mechanism of maturation of the M4 metalloproteases (Gao et al. 2010). In addition, Xie et al. (2009) studied the cold adaptation of the M4 metalloproteases E495 from an Arctic sea-ice bacterium and MCP-02 from a deep-sea sedimentary bacterium by comparing them with the mesophilic homolog pseudolysin from a terrestrial bacterium. They found that the optimization of hydrogen-bonding dynamics is a strategy for cold adaptation of these M4 enzymes, providing new insights into the structural basis underlying conformational flexibility.
Pseudoalterin is a M23 metalloprotease secreted by Pseudoalteromonas sp. CF6-2, isolated from deep-sea sediments of the South China Sea, and represents the first marine-derived M23 metalloprotease (Zhao et al. 2012a; Zhou et al. 2009). Pseudoalterin has high specific activity towards elastin. Differing from terrestrial M23 proteases that only cleave glycyl bonds in elastin, pseudoalterin not only cleaves glycyl bonds but also peptide bonds involved in cross-linking in elastin (Ala-Ala, Ala-Lys, Lys-Ala), representing a new elastolytic mechanism (Zhao et al. 2012a). Moreover, pseudoalterin is active toward the peptide chain of peptidoglycan of Gram-positive bacteria. The structure of pseudoalterin contains a catalytic domain and a C-terminal domain (Fig. 2). Structural and biochemical analyses suggest that, during peptidoglycan degradation, the C-terminal domain binds on the glycan chain of peptidoglycan, and the catalytic domain degrades the peptide chain by attacking most of the peptide bonds on the peptide chain, producing amino acids and small peptides, thereby providing nutrients for strain CF6-2 (Tang et al. 2020).
Pseudoalteromonas agarivorans NW4327, the primary pathogen of the disease affecting the Great Barrier Reef sponge Rhopaloeides odorabile, secretes two collagenases (both are metalloproteases) that degrade R. odorabile skeletal fibers (Bhattacharya et al. 2018, 2019; Choudhury et al. 2015). One collagenase, that has a molecular mass of 116.25 kDa and requires Ca2+ and Zn2+ as cofactors, is a TonB-dependent receptor, having a carboxypeptidase regulatory-like domain in the N-terminus along with an outer membrane channel superfamily domain (Bhattacharya et al. 2018). The other collagenase, a U32 metalloprotease, has a molecular mass of 52.51 kDa, contains two Ca2+, and has only 13% identity with known hydrolases. Molecular docking shows that two interacting loops of the collagenase bind collagen triple helices and two Ca2+ between the loops (Bhattacharya et al. 2019). These properties of these two collagenases are different from their homologs from thermophilic bacteria and terrestrial pathogens.
As described above, Pseudoalteromonas strains are often an abundant or dominant group of culturable protease-producing bacteria and produce considerable amounts of protease. Moreover, each strain usually secretes a variety of proteases; e.g., Pseudoalteromonas sp. SM9913 produces more than ten proteases (Yang et al. 2019), three of which (the serine proteases MCP-01 and MCP-03 and the metalloprotease MCP-02), have been characterized (Gao et al. 2010; Ran et al. 2013; Yan et al. 2009). Pseudoalteromonas agarivorans NW4327 contains 30 genes encoding proteases with a putative signal peptide; two of these (both collagenases) have been characterized (Bhattacharya et al. 2018, 2019; Choudhury et al. 2015). The secreted Pseudoalteromonas proteases include a variety of serine proteases and metalloproteases of different families, and the substrate specificity of these proteases is diverse. For instance, they can degrade collagen, elastin, and bacterial peptidoglycan (Table 2). Considering the widespread nature of Pseudoalteromonas, these findings suggest that the protease-producing strains are likely a key group involved in marine organic nitrogen degradation.
Pseudoalteromonas proteases facilitate their interactions with a range of organisms, including planktonic microalgae, metazoa, and bacteria (Table 3). We outline some of these below.
Protease Protease-producing strain Interacted organism Action mode References A serine protease,
P. sp. A28 Skeletonema costatum Algicidal activity Lee et al.(2000, 2002) Two collagenases P. agarivorans NW4327 Rhopaloeides odorabile Degrading skeletal fibers Bhattacharya et al.(2018, 2019) Pseudoalterin P. sp. CF6-2 Gram-positve bacteria Degrading peptidoglycan in cell wall Tang et al. (2020) Multiple proteases P. piscicida Vibrio, Photobacterium, Shewanella – Richards et al. (2017) Multiple proteases P. sp. strain Nephrops norvegicus Degrading muscle proteins Ridgway et al. (2008)
Table 3. Proteases involved in the interactions of Pseudoalteromonas with other marine organisms
Laboratory study showed that strain A28 isolated from seawater from the Ariake Sea of Japan can lyse the marine algae Skeletonema costatum, Thalassiosira sp., Eucampia zodiacs, and Chattonella antiqua (Lee et al. 2000), suggesting that this species may have algicidal activity in the ocean. Further studies show that both a serine protease and a metalloprotease secreted by this strain are algicidal (Lee et al. 2000, 2002). However, there are no further reports on the algicidal activity of Pseudoalteromonas proteases, indicating that this is an avenue for future research.
Pseudoalteromonas strains are also often isolated from marine animals. Pseudoalteromonas agarivorans NW4327 is a pathogen of the Great Barrier Reef Sponge Rhopaloeides odorabile. Two collagenases secreted by strain NW4327 can degrade R. odorabile skeletal fibers (Bhattacharya et al. 2018, 2019). Pseudoalteromonas sp. strain N10 was isolated from the Norway lobster Nephrops norvegicus that was suffering from bacterial infections (Ridgway et al. 2008). Further study showed that the extracellular products secreted by strain N10 contain multiple proteases and selectively degrade the myosin heavy chain, troponin-T, troponin-Ⅰ, paramyosin, and several unidentified muscle proteins approximately 110 kDa in size, suggesting that these proteases are involved in the spoilage of host muscle tissue (Ridgway et al. 2008).
Finally, proteases are also involved in the bacteriolytic activity of Pseudoalteromonas. Three P. piscicida strains isolated from seawater can inhibit the growth of Vibrio vulnificus, V. parahaemolyticus, V. cholerae, Photobacterium damselae, and Shewanella algae, and this is likely related to the secretion of multiple proteases (Richards et al. 2017). Pseudoalteromonas sp. CF6-2 isolated from marine sediment can kill a variety of marine Gram-positve bacteria via secreting the M23 protease, pseudoalterin, which attacks peptidoglycan in the bacterial cell wall (Tang et al. 2020). Furthermore, glycine and its peptides, such as GG, GGG, and GGGG, derived from the peptidoglycan of Gram-positive bacteria can induce the biosynthesis of pseudoalterin in Pseudoalteromonas sp. CF6-2, which then is secreted by the type Ⅱ secretion system (Tang et al. 2020).
Organic nitrogen degradation and recycling
Protease is the most widely used enzyme in industrial, medical, and biotechnological fields. Although only a few Pseudoalteromonas proteases are characterized, the potential of several has been considered (Table 4).
Protease Protease-producing strain Potential Work meterial References Extracellular protease P. sp. SM9913 Bioactive peptide preparation Codfish skin Chen et al. (2017) Collagenases P. sp. SJN2 Bioactive peptide preparation Collagens from Spanish mackerel bone, seabream scale and octopus flesh Yang et al. (2017) Extracellular proteases P. sp. SQN1 Bioactive peptide preparation Muscle proteins from a salmon skin Wu et al. (2017b) Extracellular protease P. sp. SM9913 Meat tast improvement Refrigerated meats He et al. (2004) MCP-01 P. sp. SM9913 Meat tenderization Beef meat Zhao et al. (2012b)
Table 4. Potentials of Pseudoalteromonas proteases
The potentials of Pseudoalteromonas proteases in the preparation of bioactive peptides from the by-products of seafoods have been evaluated. MCP-01, the most abundant protease secreted by Pseudoalteromonas sp. SM9913, is highly efficient in the hydrolysis of fish collagen (Zhao et al. 2008). With SM9913 extracellular protease as a tool, a process to prepare collagen oligopeptide-rich hydrolysate from codfish skin was established and was further scaled up to pilot (100 L) and plant levels (2000 L), with yields > 66%. The hydrolysate contains ~ 95% peptides with molecular weights lower than 3 kDa and ~ 60% lower than 1 kDa. Bioactivity analyses showed that the hydrolysate has moisture-retention ability, antioxidant activity, and promoting effects on cell viability of human dermal fibroblasts. Safety evaluation indicated that the hydrolysate is nontoxic and nonirritating to skin. These data encourage the potential use of SM9913 extracellular protease in preparing bioactive oligopeptides from fish skin (Chen et al. 2017). The collagenases produced by Pseudoalteromonas sp. SJN2 can hydrolyze marine collagens extracted from fish by-products, Spanish mackerel bone, seabream scale, and octopus flesh. The resulting hydrolysates have remarkable antioxidant capacities shown by high DPPH radical scavenging rate and oxygen radical absorbance capacity (Yang et al. 2017). The extracellular proteases secreted by Pseudoalteromonas sp. SQN1 was used to hydrolyze the muscle proteins from a salmon skin by-product and the hydrolysate displays a strong activity of antioxidant in DPPH and hydroxyl radical scavenging. A purified fraction U2-S2-I from the hydrolysate possesses the capability to protect plasmid DNA against the damage of hydroxyl radical (Wu et al. 2017b).
The potential of the extracellular proteases of strain SM9913 to be used as a cold-adapted flavour enzyme to improve the taste of refrigerated meats has been evaluated. Common flavour enzymes are mesophilic proteases, which exhibit very low efficiency of proteolysis at 0 ℃. In contrast, the extracellular proteases of strain SM9913 retain more than 10% activity at 0 ℃. The refrigerated meat samples treated with the cold-adapted protease of SM9913 released more taste amino acids and essential amino acids than those treated with mesophilic protease, indicating that the cold-adapted protease of SM9913 has potential in improving the taste of refrigerated meat (He et al. 2004).
As a cold-adapted collagenolytic protease, the tenderization effect of MCP-01 on beef meat at low temperature has also been investigated. The enzymes currently used to increase meat tenderness are all mesophilic or thermophilic proteases. Compared to the commercially used tenderizers papain and bromelain, MCP-01 has better tenderization effect on beef meat at 4 ℃. Moreover, MCP-01 shows a unique tenderization mechanism. It has a strong selectivity for degrading collagen at 4 ℃, shows a distinct digestion pattern on the myofibrillar proteins, and has a different disruption pattern on the muscle fibers. The results suggest that the cold-adapted collagenolytic protease MCP-01 may be promising for use as a meat tenderizer at low and moderate temperatures (Zhao et al. 2012b).
As mentioned above, two proteases for strain A28 have algicidal activity to diatom Skeletonema costatum (Lee et al. 2000, 2002), and thus may have potential in the treatment of red tide caused by this alga. The M23 protease pseudoalterin produced by Pseudoalteromonas sp. CF6-2 has lytic activity to a variety of Gram-positive bacteria (Tang et al. 2020), and, therefore, may have potential in the treatment of infection caused by Gram-positive bacteria in human and animals. The P. piscicida strains that secrete multiple proteases and can inhibit the growth of Vibrio and other bacterial strains may be applied in marine aquaculture industry (Richards et al. 2017). The alkaline protease secreted by Pseudoalteromonas sp. 129-1, in the concentration of 1 mg/ml, completely removes the biofilm of Pseudomonas aeruginosa PAO1, suggesting its potential as an anti-biofilm agent (Wu et al. 2015).
The protease production of some Pseudoalteromonas strains has been improved by optimization of fermentation conditions or by strain mutagenesis. The production of extracellular protease of P. arctica PAMC 21717 was enhanced 15-fold via statistical optimization of mineral components and fed-batch fermentation (Han et al. 2016). Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation to improve the yield of its extracellular protease E423, and a mutant strain with a 5.1-fold higher protease yield was obtained (Wu et al. 2016). The production of protease pseudoalterin produced by Pseudoalteromonas sp. CF6-2 is only induced by elastin. To lower the production cost, researchers used bovine artery powder instead of elastin as a cheap and efficient inducer; this led to improved enzyme production by more than twofold via optimizing the fermentation conditions (Yang et al. 2016; Zhao et al. 2014b).
Because proteases of Pseudoalteromonas strains from deep sea and polar regions are usually cold active and thermolabile, compound stabilizers are developed to improve their thermostability. A compound stabilizer for pseudoalterin was developed using medically safe sugars and polyols, which shows a significant protective effect for pseudoalterin against enzymatic thermal denaturation (Yang et al. 2016). Trimethylamine N-oxide can improve both the activity of the cold-adapted protease MCP-01 at low temperature and its thermostability at high temperature, suggesting that it may be used as an ideal stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability (He et al. 2009). These studies lay a solid foundation for the industrial production of Pseudoalteromonas proteases and the development of their biomedical and biotechnological potentials.