Characteristics and Application of a Novel Cold-Adapted and Salt-Tolerant Protease EK4-1 Produced by an Arctic Bacterium Mesonia algae K4-1

Mesonia algae K4-1 from the Arctic secretes a novel cold-adapted and salt-tolerant protease EK4-1. It has the highest sequence similarity with Stearolysin, an M4 family protease from Geobacillus stearothermophilus, with only 45% sequence identity, and is a novel M4 family protease. Ek4-1 has a low optimal catalytic temperature (40 °C) and is stable at low temperatures. Moreover, EK4-1 is still active in 4 mol/L NaCl solution and is tolerant to surfactants, oxidizing agents and organic solvents; furthermore, it prefers the hydrolysis of peptide bonds at the P1’ position as the hydrophobic residues, such as Leu, Phe and Val, and amino acids with a long side chain, such as Phe and Tyr. Mn2+and Mg2+ significantly promoted enzyme activity, while Fe3+, Co+, Zn2+ and Cu2+ significantly inhibited enzyme activity. Amino acid composition analysis showed that EK4-1 had more small-side-chain amino acids and fewer large-side-chain amino acids. Compared with a thermophilic protease Stearolysin, the cold-adapted protease EK4-1 contains more random coils (48.07%) and a larger active pocket (727.42 Å3). In addition, the acidic amino acid content of protease EK4-1 was higher than that of the basic amino acid, which might be related to the salt tolerance of protease. Compared with the homologous proteases EB62 and E423, the cold-adapted protease EK4-1 was more efficient in the proteolysis of grass carp skin, salmon skin and casein at a low temperature, and produced a large number of antioxidant peptides, with DPPH, ·OH and ROO· scavenging activities. Therefore, cold-adapted and salt-tolerant protease EK4-1 offers wide application prospects in the cosmetic and detergent industries.


Introduction
Proteases are a class of enzymes that hydrolyze protein peptide chains and can be divided into different groups according to their active site and catalytic mechanism [1,2]. To date, there are 273 different families of protein hydrolases in the MEROPS database. The vast majority of these proteins are derived from microorganisms [3]. Microbial proteases not only play an important role in cellular metabolic processes, but are also important sources of industrial proteases. They account for about 2/3 of total enzyme sales worldwide [4], and are highly favored in detergent, food processing, feed production, peptide synthesis, leather processing and biodegradation industries. However, there are still some limitations in the industrial application of commercial proteases, such as low enzyme activity at low temperature, poor salt tolerance, low tolerance to chemical reagents, single cleavage site, high price and so on. Therefore, it remains a challenge to find and develop novel proteases that tolerate one or more extreme conditions. Cold-adapted proteases present a class of enzymes that can be catalyzed at a low temperature (the optimum temperature is <40 • C) with high efficiency, and are usually still

Purification of Protease EK4-1
After fermentation, the highest enzyme activity in the Mesonia algae K4-1 fermentation broth was observed at 96 h. The protease, named EK4-1, was purified via (NH 4 ) 2 SO 4 gradient precipitation, DEAE anion-exchange chromatography and size-exclusion chromatography using a Superdex-75 column and a chromatographic FPLC system (Bio-Rad, Hercules, CA, USA). Under optimum assay conditions, the purified enzyme had a specific activity of 893 U•mg −1 , with a final yield of 7.53% and purification fold of 14 fold ( Table 1). The molecular weight of protease EK4-1 was estimated to be 37 kDa based on SDS-PAGE ( Figure S1).

Enzymatic Properties of EK4-1
The optimum temperature for protease EK4-1 was determined by measuring the efficiency of EK4-1 hydrolysis casein at different temperatures ( Figure 1A). The results showed that the optimal reaction temperature of EK4-1 for casein hydrolysis was about 40 • C, and the enzyme activity could be maintained at 22% at 4 • C. The thermal stability of protease EK4-1 showed that EK4-1 was stable at 30 • C, and the enzyme activity remained at about 93% after 120 min ( Figure 1B). However, the thermal stability of the enzyme at 40 • C was poor, with a half-life of about 35 min. At 50 • C for 5 min, the enzyme activity lost nearly 92%. To assess the thermotropic behavior of protease EK4-1, differential scanning calorimetry (DSC) was used as it allows for the detection of conformational changes and the structural stability of proteins. Protease EK4-1 showed a heat peak at 61.52 • C ( Figure 1C). Compared with E495 of the M4 family secreted by Arctic Sea ice bacteria Pseudoalteromonas sp.SM495, the optimum enzyme activity temperature was 57 • C, and the enzyme activity at 0 • C was only 1.2% of the highest enzyme activity. The optimal enzyme activity temperature of EK4-1 was much lower, and the cold-adapted protease EK4-1 had a better effect on performing a catalytic function at a low temperature. These results indicated that EK4-1 had higher activity at a low temperature [8,13,14]. As show in Figure 1D, EK4-1 had high catalytic activity under neutral conditions, and the optimal pH was 8.0. After mixing EK4-1 in different pH buffer solutions for 4 h, EK4-1 could still maintain more than 70% enzyme activity in the pH range of 7.0-9.0 ( Figure 1E).
The effect of NaCl on the catalytic activity of protease EK4-1 showed that the activity of EK4-1 increased first and then decreased with an increase in salt concentration. When the concentration of NaCl was 0.5 M, the activity of protease was the best, which was almost twice as high as that without salt, and even at 4.5 M NaCl concentration, 60% of the enzyme activity was still maintained. The results of high-salt-tolerance experiments showed that the relative enzyme activity was 110% after 48 h incubation with 1.0 M NaCl, and the enzyme activity remained at 58% at a salt concentration of 4.0 M NaCl ( Figure 1H). The salt activation and salt tolerance of EK4-1 indicated the potential application of EK4-1 in highsalt environments, such as soy sauce production and fish sauce fermentation. Many studies have found that in addition to having high catalytic activity under a high salt concentration, salt-tolerant enzymes were likely to be stable in the same organic solvent medium with low available water activity, which was a valuable tool for biocatalysts in water-organic media [15]. EK4-1 could maintain more than 50% activity in isopropanol, DMSO, xylene, n-propanol and hexane, while ethanol, methanol and isoamyl alcohol had strong inhibitory effects on enzyme activity ( Figure 1I). The effects of oxidant H 2 O 2 , non-ionic surfactants Tween 80 and Triton x-100 and anionic surfactant SDS on the enzyme activity of protease EK4-1 were further studied ( Figure 1F). The enzyme can maintain more than 50% activity under the conditions of 1% H 2 O 2 , 1% Tween 80 and 1% tritonx-100, compatible with detergent formulation. Protease EK4-1 could be widely used in the detergent industry due to its good properties of being cold-adapted, salt-tolerant and detergent-resistant. Among the metal ions tested, 10 mM Mn 2+ and Mg 2+ can enhance the activity of EK4-1 to 132.3% and 146.6%, respectively. EK4-1 was relatively stable in 2.5 mM and 10 mM K + , Ca 2+ and Ba 2+ , and its activity was more than 75%, while the catalytic activity of EK4-1 could be significantly inhibited by Fe 3+ , Co 2+ , Zn 2+ and Cu 2+ ( Figure 1J). and 10 mM K + , Ca 2+ and Ba 2+ , and its activity was more than 75%, while the catalytic activity of EK4-1 could be significantly inhibited by Fe 3+ , Co 2+ , Zn 2+ and Cu 2+ ( Figure 1J).  40 • C for 10 min and determined using the folinphenol method. (E) pH stability: protease EK4-1 was diluted into buffers which were 20 mM citrate buffer (pH 4.0-7.0), Tris-HCl buffer (pH 7.0-9.0) and glycine buffer (pH 9.0-11.0) and stored at 4 • C for 24 h; then, the residual activity was measured. In (D,E), the highest enzyme activity was set to 100%. (F) Effects of oxidants and surfactants: protease EK4-1 was diluted into oxidants and surfactants at 4 • C for 30 min and then the residual activity was determined. The untreated enzyme was used as a control (100%). (G) Effects of NaCl concentration on EK4-1. The effect of NaCl on protease activity was measured under the same conditions in 20 mM Tris-HCl buffer (pH 8.0) in a range of 0-4.5 M NaCl. (H) Salt tolerance: protease EK4-1 was incubated at different concentrations of NaCl ranging from 0 M to 4.5 M at 4 • C for 48 h, and then the residual activity was determined. In (G,H), the relative activity was calculated by considering the enzyme activity without NaCl as being 100%. (I) Effects of organic reagents: protease EK4-1 was diluted into organic reagents at 4 • C for 30 min and then the residual activity was determined. The relative activities were calculated by considering the enzyme activity without organic reagents as being 100%. (J) Effects of metal ions: the effects of metal ions on enzyme activity were determined in 20 mM Tris-HCl buffer (pH 8.0) in the presence of each metal ion (final concentration of 2.5 mM and 10 mM) at 4 • C for 24 h, and then the residual activity was determined. The relative activity was calculated by considering the enzyme activity without metal ions as being 100%. All of the data shown above are the mean of at least three replicates (** p < 0.01 and *** p < 0.001).

Structural Properties of EK4-1
Purified protease EK4-1 was identified using MALDI-TOF/TOF mass spectrometry and then the obtained sequence fragments were searched for in Mesonia algae K4-1 genome data (GenBank: GCA_008017825.1) [11]. The gene-encoding EK4-1 was 2955 bp in length, and it was assigned the accession number WP_147863623.  (Figure 2A). After conducting similarity analysis with various identified homologous proteases in the MEROPS database, it was found that protease EK4-1 had the highest sequence similarity with the identified M4 family Stearolysin from Geobacillus stearothermophilus [17], with a sequence identity of 45%. This result predicted that EK4-1 may be a novel M4 family protease ( Figure 2B) and the activity of protease EK4-1 was inhibited by the metalloprotease inhibitor 1,10-phenanthroline monohydrate (OP) ( Figure S2).
To date, most bacterial extracellular metalloproteases found are endopeptidases containing one zinc ion in the active center, which forms four coordination bonds with three amino acid residues and a water molecule to construct the active center of a tetrahedral structure, usually two His and one Glu [18]. The EK4-1 gene encodes a precursor of 984 residues. The BLAST search of the MEROPS databases using the EK4-1 precursor sequence as a query revealed several sequences with high identities. The metalloprotease Stearolysin identified from Geobacillus stearothermophilus showed the highest similarity (45%) to the EK4-1 precursor. The neutral peptidase identified from Thermoactinomyces sp. 27A showed the second highest similarity (44.3%) to the EK4-1 precursor. Thermolysin identified from the strain Bacillus thermoproteolyticus showed the third highest similarity (44%). The alignment of mature sequences of EK4-1, Stearolysin, neutral peptidase and thermolysin is shown in Figure 2B, and they all had the conserved HEXXH sequence of typical metalloproteases. In contrast to the other three proteases, the third and fourth amino acid in the conserved region of protease EK4-1 are Ile and Gly, respectively ( Figure 2B). To date, most bacterial extracellular metalloproteases found are endopeptidases con- Studies have reported that cold-adapted proteases generally have high structural flexibility and poor thermal stability [19]. Xie et al. studied MCP-02 from a deep-sea bacterium, E495 from Arctic Sea ice bacterium and homolog mesophilic metalloprotease pseudolysin from a terrestrial bacterium, which showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that increased flexibility was a strategy for cold adaptation [20]. Compared with E495, MCP-02 and Stearolysin, EK4-1 had more small-side-chain amino acids, such as Gly, Ser, Thr and Asn, and fewer large-side-chain amino acids, such as Arg, Leu and Lys (Table 2). This result was consistent with the amino acid composition characteristics of cold-adapted proteins that had been reported [21]. It is worth mentioning that the cold-adapted characteristics of EK4-1 were more obvious than those of E495. In addition to the differences in amino acid composition, cold-adapted proteases usually contained higher amounts of random coils to increase the flexibility [22]. The prediction of the secondary structure of the metalloproteases showed that the contents of the random coil in EK4-1, E495, MCP-02 and Stearolysin decreased in the order of 48.07%, 47.12%, 43.47% and 42.88%, respectively. The large number of random coils in EK4-1 helped it to be more flexible in space structure, meaning it was easier to unfold and produced conformational changes. In addition to cold adaptation, salt tolerance was another distinctive characteristic of EK4-1. Moshe's research showed that acidic amino acids made the protein surface have a large number of negative charges, so that the protease could maintain a certain flexibility in the high-salt environment to ensure the catalytic activity of the enzyme [23]. On the other hand, the acidic side chain can bind water and salt ions, which makes it possible for proteins to adapt to the high-salt environment and maintain stability, and also increases the solubility of proteins in the high-salt environment [24]. Compared with the other three homogenous metalloproteases, the amino acid composition of EK4-1 from Arctic Sea water showed that the amount of acidic amino acids was much higher than that of basic amino acids, which was suitable for the sequence characteristics of salt-tolerant proteases ( Table 2). volume of cold-adapted protease EK4-1 was 727.42 Å 3 ( Figure 3A) and that of thermophilic protease Stearolysin was 538.43 Å 3 ( Figure 3B). The active pocket of cold-adapted protease EK4-1 was much larger than that of Stearolysin. The larger catalytic cavity might allow the cold-adapted protease EK4-1 easier binding of substrates. This might also be an adaptation of the protease EK4-1 to the low-temperature environment. Cold-adapted protease EK4-1 and thermophilic protease Stearolysin were modeled via the Swiss-Model website and then the active pocket size of the protease catalytic domain was calculated using Proteins Plus. The calculated results showed that the catalytic pocket volume of cold-adapted protease EK4-1 was 727.42 Å 3 ( Figure 3A) and that of thermophilic protease Stearolysin was 538.43 Å 3 ( Figure 3B). The active pocket of cold-adapted protease EK4-1 was much larger than that of Stearolysin. The larger catalytic cavity might allow the cold-adapted protease EK4-1 easier binding of substrates. This might also be an adaptation of the protease EK4-1 to the low-temperature environment. To analyze the cleavage sites of protease EK4-1, digestion was performed using insulin B chain as the substrate. The peptide fragments obtained after hydrolysis were then analyzed using HPLC and mass spectrometry ( Figure 4A). The molecular weight of 14 peptides with a high abundance was determined, the amino acid composition of these 14 peptides was matched in the FindPept database (Table 3) and cleavage sites were further analyzed ( Figure 4B,C). The insulin B chain was hydrolyzed to produce multiple cleavage sites, which were, respectively, His + Leu, Leu + Cys, Gly + Ser, Glu + Ala, Ala + Leu, Leu + Tyr, Tyr + Leu, Leu + Val, Arg + Gly and Gly + Phe. These results indicated that most of the P1' positions were hydrophobic amino acids such as Leu, Ala and Val, and amino acids with a large side chain, such as Phe and Tyr. The substrate specificity of EK4-1 is similar to other proteases of the M4 family, such as aureolysin and thermolysin, and the specific hydrolyzed P1' position is a large hydrophobic amino acid [25]. Compared with the M4 family protease MCP-02, four cleavage sites of EK4-1 were the same as those of MCP-02 (Leu + Cys, Ala + Leu, Leu + Tyr and Arg + Gly). In addition, protease EK4-1 also had a certain preference for hydrophobic amino acids and long-side-chain amino acids at the P1 position. However, MCP-02 had a weak preference for these amino acids. In addition, protease EK4-1 displayed more cleavage sites for the B chain of insulin. More cleavage To analyze the cleavage sites of protease EK4-1, digestion was performed using insulin B chain as the substrate. The peptide fragments obtained after hydrolysis were then analyzed using HPLC and mass spectrometry ( Figure 4A). The molecular weight of 14 peptides with a high abundance was determined, the amino acid composition of these 14 peptides was matched in the FindPept database (Table 3) and cleavage sites were further analyzed ( Figure 4B,C). The insulin B chain was hydrolyzed to produce multiple cleavage sites, which were, respectively, His + Leu, Leu + Cys, Gly + Ser, Glu + Ala, Ala + Leu, Leu + Tyr, Tyr + Leu, Leu + Val, Arg + Gly and Gly + Phe. These results indicated that most of the P1' positions were hydrophobic amino acids such as Leu, Ala and Val, and amino acids with a large side chain, such as Phe and Tyr. The substrate specificity of EK4-1 is similar to other proteases of the M4 family, such as aureolysin and thermolysin, and the specific hydrolyzed P1' position is a large hydrophobic amino acid [25]. Compared with the M4 family protease MCP-02, four cleavage sites of EK4-1 were the same as those of MCP-02 (Leu + Cys, Ala + Leu, Leu + Tyr and Arg + Gly). In addition, protease EK4-1 also had a certain preference for hydrophobic amino acids and long-side-chain amino acids at the P1 position. However, MCP-02 had a weak preference for these amino acids. In addition, protease EK4-1 displayed more cleavage sites for the B chain of insulin. More cleavage sites could help Mesonia algae K4-1 to better degrade extracellular protein particles for nutrients, which might be an adaptation of Arctic bacteria to extreme environments.
To further confirm the digestion characteristics of EK4-1, six peptides were synthesized and hydrolyzed via protease EK4-1. EK4-1 could degrade the dipeptide substrates FA-Gly-Leu-NH 2 and FA-Gly-Phe-NH 2 with large side chains and strong hydrophobicity at the P1' position. The hydrolysis rate of FA-Gly-Phe-NH 2 with the aromatic amino acid Phe at the P1' position was the fastest, which was 1.20 times higher than that of FA-Gly-Leu-NH 2 as the hydrolysis substrate (Table 4, Figure 5). However, unlike the protease MCP-02, EK4-1 could not degrade FA-Gly-Val-NH2, suggesting that it might also select amino acids at the P position of the substrate. sites could help Mesonia algae K4-1 to better degrade extracellular protein particles for nutrients, which might be an adaptation of Arctic bacteria to extreme environments.     To further confirm the digestion characteristics of EK4-1, six peptides were synthesized and hydrolyzed via protease EK4-1. EK4-1 could degrade the dipeptide substrates FA-Gly-Leu-NH2 and FA-Gly-Phe-NH2 with large side chains and strong hydrophobicity at the P1' position. The hydrolysis rate of FA-Gly-Phe-NH2 with the aromatic amino acid Phe at the P1' position was the fastest, which was 1.20 times higher than that of FA-Gly-Leu-NH2 as the hydrolysis substrate (Table 4, Figure 5). However, unlike the protease MCP-02, EK4-1 could not degrade FA-Gly-Val-NH2, suggesting that it might also select amino acids at the P position of the substrate.

EK4-1 Efficiently Degrades Low-Value Protein Substrates at Low Temperature
The aquatic product processing industry leads to the production of a large number of waste products (skin, bone, head, viscera and scales). However, in addition to being used as fertilizer or feed, most of these low-value fish products and their by-products are treated as garbage, resulting in a huge waste of resources and environmental pollution, and are a source of low-value protein. Recent studies have shown that the hydrolysis of these protein-rich fish by-products with proteases can produce a variety of bioactive peptides which can be used as additives in functional foods and have high economic value that enhance the added value of the protein.
Our results showed that the cold-adapted protease EK4-1 has high catalytic efficiency at a low temperature and multiple cleavage sites. In order to further explore the possibility and superiority of cold-adapted protease EK4-1 in industrial applications, we used protease EK4-1 to proteolyze three protein substrates (grass carp skin, salmon skin and casein) at different temperatures and compared the catalytic efficiency of the homologous metalloproteases E423 and EB62. The results showed that the content of -NH 2 in the reaction system increased significantly after protease digestion, and the content of free -NH 2 could be used to indicate the degree of hydrolysis of the substrate protein. The hydrolysis degree (DH) of different substrates treated with the three proteases at 40 • C was defined as being 100%. The DH of three enzymes EK4-1, E423 and EB62 for different substrates decreased as the reaction temperature decreased. However, it was easy to see from Figure 6A,D,G that EK4-1 still maintained a high DH for the three substrates at lower temperatures (20 • C), retaining 80% of DH at 40 • C. The lower the temperature, the more obvious the advantage of EK4-1. Additionally, at 20 • C, the DH of the E423-digested substrate samples was only about 65% of that at 40 • C, while the DH of the EB62-treated sample only remained about 35% of that at 40 • C. These results indicated that with a decrease in temperature, the enzyme activity of protease E423 and EB62 decreased significantly, while protease EK4-1 could still maintain high proteolytic activity at a low temperature. These results indicated that the high catalytic efficiency of EK4-1 at a low temperature was superior to other homologous proteases and had good prospects for application. A high degree of hydrolysis predicted that the protein substrates were digested into more oligopeptides and amino acids. More and more studies have shown that many hy- A high degree of hydrolysis predicted that the protein substrates were digested into more oligopeptides and amino acids. More and more studies have shown that many hydrolysate oligopeptides possess a variety of biological activities, such as antioxidant, hypotensive, antibacterial and immune-promoting. Therefore, the hydrolysate products of grass fish skin and salmon skin were further studied for antioxidant analysis (Figure 6B,E). The results showed that both salmon skin and grass carp skin produced high levels of antioxidant peptides after proteolysis, and the hydrolysate product of protease EK4-1 had the highest antioxidant activity. The maximum DPPH and· OH scavenging activities of grass carp skin hydrolysis products via protease EK4-1 reached 68.05 ± 1.08% and 47.62 ± 1.52%, respectively, which were much higher than those of protease E423 and protease EB62. Similarly, the highest scavenging activity of DPPH and the ·OH of salmon skin hydrolysis products via protease EK4-1 was 47.38 ± 1.61% and 53.84 ± 3.01%, respectively.
While the substrate was the skin of salmon, the peroxide radical scavenging activity in the hydrolysis products of the three proteases was 0.79 ± 0.02 mM·TE/g, 0.59 ± 0.03 mM·TE/g and 0.36 ± 0.02 mM·TE/g, respectively ( Figure 6F). When casein was the substrate, the hydrolyzed products of the three proteases had peroxyradical scavenging activity, and EK4-1 had the highest scavenging activity (0.31 ± 0.02 mM·TE/g), followed by EB62 (0.3 ± 0.01 mM·TE/g) and E423 (0.26 ± 0.01 mM·TE/g). These results suggested that the cold-adapted protease EK4-1 had important potential for the high-value modification of low-value protein resources at low temperatures.

Discussion
Mesonia belongs to the Flavobacteriaceae of Bacteroidetes, which was first proposed by Nedashkovskaya et al. in 2003. The discovery time was short, and the functions of a large number of genes have not been clarified [26]. At present, the situation of proteases in this genus has not been reported. This paper is the first systematic study of proteases in this genus, which is of great significance for people to understand and explore bacteria of the genus Mesonia. In this study, a novel M4 family metalloprotease EK4-1 with coldadapted and salt-tolerant properties from an Arctic cryophilic bacterium Mesonia algae K4-1 was investigated. The protease EK4-1 had a low optimum proteolytic temperature, stable protein structure and high catalytic activity at a low temperature. It was found to be a typical cold-adapted protease. In addition, the high tolerance of the protease EK4-1 to NaCl, organic solvents, surfactants and oxidants has the potential to make it a bioenzyme additive compatible with detergent.
In the MEROPS database, the protease EK4-1 showed the highest sequence similarity with the identified Stearolysin from the M4 family of Geobacillus stearothermophilus, with a sequence identity of only 45%, indicating that the enzyme was relatively new in the amino acid sequence. EK4-1, as a metalloprotease with a typical HEXXH-conserved sequence, could specifically hydrolyze the substrate of the large hydrophobic and long-side-chain amino acids at the P1' position, which is consistent with the characteristics of most M4 family proteases. Moreover, EK4-1 had more small-side-chain amino acids, fewer largeside-chain amino acids, more random coils and a larger activity pocket. These structural properties confirmed the flexibility of EK4-1, which might be an important reason for its high catalytic activity at low temperatures. In addition, the number of acidic amino acids in protease EK4-1 was far more than that of basic amino acids, which might be related to the salt tolerance of EK4-1.
By hydrolyzing a variety of low-value proteins at different temperatures, the superiority of cold-adapted protease EK4-1 for its high catalytic efficiency at low temperatures was demonstrated. In addition, the high antioxidant activity of these proteolytic products also provided a new reference for the application of cold-adapted protease EK4-1. This made it possible for the application of protease EK4-1 in the field of the high-value conversion of low-value proteins. In conclusion, the multi-tolerance and efficient proteolysis abilities at a low temperature of the cold-adapted protease EK4-1 indicate that EK4-1 has great potential for application in the field of low-temperature proteolysis.

Protease Activity Assay and Protein Quantification
The protease activity of the culture supernatant on casein was detected using the Folin phenol method [10]. One unit of protease activity was determined as the amount of enzyme that catalyzed the formation of 1 µg tyrosine per min. The protein contained in the supernatant was quantified using the BCA protein quantification kit (Biosharp, Anhui, China), and bovine serum albumin (Biosharp, Anhui, China) at concentrations of 0, 0.2, 0.4, 0.6 and 1 mg/mL was used as the standard for the determination of the protein content [28].

Protease Purification and Mass Spectrum Identification
An appropriate amount of the fermentation broth was centrifuged at 12,000 rpm and the supernatant was collected. The 40% (m/v) ammonium sulfate powder was slowly added under an ice bath condition. The precipitate was collected via centrifugation, dissolved in 20 mM Tris-HCl (pH 8) and dialyzed. The samples were loaded onto a 5 mL HiTrapTM DEAE column (GE Healthcare) previously equilibrated with 20 mM Tris-HCl (pH 8.0) and then eluted with a NaCl gradient (0-1.0 M) in the same buffer at a flow rate of 1 mL/min. Active fractions were collected, concentrated via ultrafiltration (10 kDa MW cut-off membrane, Millipore) and then subjected to gel filtration on a Superdex 75 (10 × 300 column, 24 mL) with a flow rate 1 mL/min. The purity of protease EK4-1 was determined via SDS-PAGE as described previously. Substrate immersing zymography was modified to detect the proteolytic activities according to the method developed by Liu [29]. The intact target bands were cut into a centrifuge tube and sent to Sangon Biotech for NanoLC-ESI-MS/MS mass spectrometry identification. The identified sequences were aligned in the whole protein sequence encoded by Mesonia algae K4-1 and Espript (http://espript.ibcp.fr/ESPript/ESPript/ (accessed on 1 March 2023)) [19] was used for amino acid sequence alignment. The amino acid composition and domain of protease EK4-1 were analyzed using the ExPASy ProtParam (https://web.expasy.org/protparam/ (accessed on 1 March 2023)) tool [30] and InterPro database (https://www.ebi.ac.uk/interpro/ (accessed on 1 March 2023)) [31], respectively. The protease was mixed with 2% casein in a ratio of 1:1 (v/v, mL/mL), incubated at different temperatures (4-60 • C) in 20 mM Tris-HCl buffer (pH 8.0) for 10 min. The optimal reaction temperature was determined by measuring the enzyme activity. The highest enzyme activity was set to 100%.
The thermal stability of the protease was determined by pre-incubating the protease in 20 mM Tris-HCl (pH 8.0) under 30-50 • C for 20 min, 40 min, 60 min, 90 min and 120 min, respectively [32]. The non-heated enzymes were taken as being 100% and the residual enzyme activity was measured at 40 • C in 20 mM Tris-HCl (pH 8.0) for 10 min with 2% (w/v, g/mL) casein.
The thermal properties of protease EK4-1 were analyzed using nano differential scanning calorimetry (DSC, TA Instruments, New Castle, DE, USA). Protease EK4-1 was diluted to approximately 2.5 mg/mL with 20mM Tris-HCl (pH 8.0). Experimental group: 500 µL of protease EK4-1 was added to the sample cell of DSC, and the same volume of 20 mM Tris-HCl (pH 8.0) was added to the reference cell. Blank group: both the reference and sample cells were filled with 500 µL of 20 mM Tris-HCl (pH 8.0). The heating rate was 1 • C/min from 20 • C to 90 • C. Data were recorded automatically and subsequently analyzed using the NanoAnalyze Software provided by the manufacturer.

Optimum pH, Acid-Base Stability and Salt Tolerance
The effect of pH on protease activity was studied by measuring its activity in the pH range of 4.0-11.0 using 2% (w/v, g/mL) casein as a substrate at 40 • C. The pH effect was investigated using one of the following buffers (0.02 M): citrate buffer (pH 4.0-7.0), Tris-HCl (pH 7.0-9.0) or glycine-NaOH (pH 9.0-11.0). The highest enzyme activity was set to 100%.
pH stability studies: The enzyme was mixed with pH buffers which included citrate buffer (pH 4.0-7.0), Tris-HCl (pH 7.0-9.0) and glycine-NaOH (pH 9.0-11.0) and was preincubated at 4 • C for 24 h. The residual activity was measured at 40 • C in 20 mM Tris-HCl (pH 8.0) with 2% (w/v, g/mL) casein for 10 min. The highest enzyme activity was set to 100%.
The effect of NaCl on the protease was investigated by measuring the protease activity at various NaCl concentrations (0-4.5 M) in 20 mM Tris-HCl buffer (pH 8.0). The protease activity of the purified enzyme was measured at 40 • C for 10 min with 2% (w/v, g/mL) casein. The relative activity was calculated by considering the enzyme activity without NaCl as being 100%.
Salt The diluted protease solution was mixed with various ionic solutions in a ratio of 1:1 (v/v, mL/mL), so that the final concentrations of various ions were 2.5 mM and 10.0 mM, respectively, and were stored at 4 • C for 24 h. The protease residual activity was measured at 40 • C in 20 mM Tris-HCl (pH 8.0) with 2% (w/v, g/mL) casein for 10 min. The relative activity was calculated by considering the enzyme activity without metal ions as being 100%.
Different concentrations of H 2 O 2 , Tween80, Triton X-100 and SDS were mixed with the same volume of protease at 4 • C for 30 min, respectively. The protease residual activity was measured at 40 • C with 2% (w/v, g/mL) casein for 10 min, and the untreated protease EK4-1 was used as a control (%).
The protease EK4-1 was preincubated with organic solvents ethanol, isopropanol, methanol, isoamyl alcohol, DMSO, xylene, propyl alcohol, n-hexane and n-butyl alcohol in a ratio of 1:3 (v/v, mL/mL) for 12 h at 4 • C. Then, the protease residual activity was measured at 40 • C with 2% (w/v, g/mL) casein for 10 min. The relative activities were calculated by considering the enzyme activity without organic reagents as being 100%.
Different concentrations of OP and protease EK4-1 were mixed in a ratio of 1:1 (v/v, mL: mL) to make the final concentrations of OP 0 mM, 2.5 mM and 10mM, respectively, and were stored at 4 • C for 24 h. Then, the protease residual activity was measured at 40 • C. The protease activity in the absence of an inhibitor was defined as being 100% (control). Inhibitor-substrate immersion enzyme zymography was based on Olena's method, with slight modifications [33].

Enzyme Cleavage Site Analysis of Protease EK4-1
Insulin box substrate (5 mg/mL), protease solution (0.2 mg/mL) and 20 mM Tris-HCl (pH 8.0) were mixed at the ratio of 20:1:79 (V:V:V) and incubated for 1 h at 37 • C, and the hydrolysis products were analyzed using LC-MS/MS to determine the molecular weight of each peptide in the mixed peptide samples. The tool FindPept (https://web.expasy.org/ findpept/ (accessed on 1 February 2023)) [37] was used to search the 30 amino acids of the insulin box and the molecular weight of the enzymatic peptides to obtain the possible amino acid composition of each peptide. Six dipeptide substrates (FA-Gly-Leu-NH 2 , FA-Gly-Phe-NH 2 , FA-Gly-Val-NH 2 , FA-Lys-Gly-OH, FA-Glu-Glu-OH and FA-Ala-Arg-OH) were synthesized by China Peptides Co., Ltd. (If these synthetic dipeptides are hydrolyzed via proteases, it leads to a decrease in the absorption value of 345 nm. EK4-1 was mixed with the dipeptide substrate (1 mM) at a ratio of 1:1; then, the absorbance of the mixture was detected with a wavelength of 345 nm at 25 • C for 600 s.

Degradation of Low-Value Protein via Cold-Adapted Protease EK4-1
Grass carp skin was sliced and sterilized at 121 • C for 20 min and then was digested with different proteases with same catalytic activity units (35 U/mL) at an enzyme to substrate ratio of 1:125 (v/w, mL/mg) and temperatures of 20 • C, 30 • C and 40 • C. The preparation method of salmon skin collagen was conducted according to Wu's method [2]. Salmon skin was digested at an enzyme to substrate ratio of 1:10 (v/w, mL/mg). The 2% casein was prepared using Tris-HCl (20 mM, pH 8.0) and digested at an enzyme to substrate ratio of 1:10 (v/w, mL/mg). After the reaction for 10 h, the centrifuge tubes were transferred to a 90 • C water bath and inactivated for 10 min to stop the reaction. The hydrolysis degree at various treatment temperatures was analyzed using the ninhydrin coloration method. The standard curve was determined using leucine at concentrations of 0, 1, 1.5, 2.5 and 3 mM [2]. The DPPH free radical scavenging activity (DPPH RSA) assay, hydroxyl free radical scavenging activity (OH RSA) assay and oxygen radical absorbance capacity (ORAC) assay were conducted according to Liu's method, with minor modifications [38]. The protease E423 used in this experiment was produced by Pseudoalteromonas sp. 423, and the protease EB62 was produced by Pseudoalteromonas sp. B62, whereby both of which are laboratorypreserved strains.

Statistical Analysis
The statistical analysis was performed using SPSS software (version 23, SPSS Inc. Chicago, IL, USA) and the data were presented as mean ± standard deviation (SD) of three replicates. The statistics between the two groups were analyzed using analysis of variance (ANOVA) and Duncan's means comparison test using a significance level of p < 0.05. Charts were developed with the use of origin 2018 software (Northampton, MA, USA).

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy.