**1. Introduction**

Bioactive oligopeptides are referred to peptides that consist of 2–20 amino acids, which have various bioactivities [1]. In addition to being efficient amino acid sources, bioactive oligopeptides have been reported to possess many physiological functions and attractive

**Citation:** Li, J.; Cheng, J.-H.; Teng, Z.-J.; Zhang, X.; Chen, X.-L.; Sun, M.-L.; Wang, J.-P.; Zhang, Y.-Z.; Ding, J.-M.; Tian, X.-M.; et al. A Novel Gelatinase from Marine *Flocculibacter collagenilyticus* SM1988: Characterization and Potential Application in Collagen Oligopeptide-Rich Hydrolysate Preparation. *Mar. Drugs* **2022**, *20*, 48. https://doi.org/10.3390/ md20010048

Academic Editor: Sik Yoon

Received: 6 December 2021 Accepted: 30 December 2021 Published: 3 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

physic properties in pharmacy (e.g., anticancer, antimicrobial, antihypertensive and antiinflammatory activities, anticoagulant, and immunomodulatory), foods (gelling activity and emulsifying property), cosmetic (antioxidant and water holding capacity), and other functional products (foaming ability and hydrophobicity) [2–5]. In recent years, collagen oligopeptides attract more and more attention due to their various bioactive properties, such as angiotensin I converting enzyme (ACE) inhibitory activity, antioxidant activity, immunomodulatory and antimicrobial activities [6–9], and beneficial effects on human health, including improving skin health, muscle strength, and bone density [10–12], and reducing obesity, joint pain, and blood pressure [13–15]. Collagen oligopeptides have been widely applied in food, cosmetics, healthcare, and pharmaceutical industries [16–18].

Enzymatic hydrolysis is now the common method to prepare collagen bioactive peptides from collagen-rich animal tissues, such as skin, bones, tendons, and ligaments. Nowadays, the common enzymes for preparing collagen bioactive peptides are proteases from plants, animals, and bacteria, such as serine proteases alcalase of the MEROPS S8 family and trypsin and α-chymotrypsin of the MEROPS S1 family, aspartic protease pepsin of the MEROPS A1 family, cysteine protease papain of the MEROPS C1 family, and metalloprotease thermolysin of the MEROPS M4 family [6,19–22]. The S8 family is the second largest family of serine proteases after the S1 family [23]. In the S8 family, many members have activity on gelatin, the denatured form of collagen, and some are collagenolytic proteases, such as the thermostable protease from *Geobacillus collagenovorans* MO-1 [24], MCP-01 from *Pseudoalteromonas* sp. SM9913 [25], myroicolsin from *Myroides profundi* D25 [26], and P57 from *Photobacterium* sp. A5-7 [27]. Due to their activity on natural or denatured collagen, these S8 peptidases may have potentials in collagen oligopeptide preparation. However, only a few S8 peptidases have been used in preparing collagen oligopeptides, or their potentials have been evaluated. In addition to alcalase that are from *Bacillus* and have been used in collagen oligopeptide preparation [28], MCP-01 has also been shown to have a potential in preparing collagen bioactive peptides from codfish skin [7]. It is still necessary to identify more S8 peptidases suitable for preparing collagen bioactive peptides.

Recently, we isolated and identified a novel marine bacterium *Flocculibacter collagenilyticus* SM1988<sup>T</sup> (hereafter SM1988) that has a high collagenase production [29]. According to the genome and secretome analyses of this strain, Aa2\_1884 was the most abundant of the 6 secreted S8 proteases and was predicted to be a potential collagenase [29]. The aim of this study was to characterize Aa2\_1884 and to evaluate its potential in preparing collagen bioactive peptides. In this study, Aa2\_1884 was expressed in *Escherichia coli* and biochemically characterized. The potential of Aa2\_1884 in preparing collagen oligopeptides from bovine bone collagen was further evaluated. The results indicate that Aa2\_1884 is a novel multimodular gelatinase with a good potential in preparing collagen oligopeptide from bovine bone collagen.

### **2. Results and Discussion**

### *2.1. Aa2\_1884 Is a Novel Multimodular Peptidase of the S8 Family*

The amino acid sequence of protein Aa2\_1884 (WP\_199608745.1) deduced from the genome of strain SM1988 is composed of 1135 amino acid residues, containing a signal peptide with a length of 34 amino acid residues at the N terminus based on the SignalP 5.0 prediction. Aa2\_1884 is annotated as an S8 family serine peptidase by BLASTP through the non-redundant protein database. InterProScan analysis indicated that, in addition to the predicted signal peptide, Aa2\_1884 has five conserved domains (Figure 1), including an inhibitor I9 domain (Tyr72-Thr175, IPR010259), a peptidase S8 domain (Gly209-Lys661, IPR000209), a protease associated (PA) domain (Ser466-Leu545, IPR003137), a fibronectin type-III (FN3) domain (Leu711-Arg790, IPR041469), and a domain of unknown function (DUF11) (Lys817-Val863, IPR001434). The inhibitor I9 domain likely functions as a molecular chaperone to assist the protein folding of Aa2\_1884 [30,31]. The peptidase S8 domain is the catalytic domain, containing the characteristic catalytic triad of the S8 family, namely

Asp218, His287, and Ser622 (Figure 2). The PA domain is an inserted domain in the peptidase S8 domain, which has been shown to play a role in collagen binding in some S8 proteases [27,32]. FN3 domain has been found in several proteases [33,34], whose function in proteases, however, has not been revealed. DUF11 domain is also present in peptidase brachyurin-T of the S1 family from *Caldilinea aerophile* based on InterProScan prediction (Figure 1). However, its function in peptidases such as brachyurin-T and Aa2\_1884 needs further study.

**Figure 1.** Domain architectures of Aa2\_1884 and similar proteases predicted by InterPro. Marked numbers show the corresponding position of predicted domains in the amino acid sequences of the proteases.

Among the characterized peptidases, Aa2\_1884 shares the highest sequence identity (44.05%) with brachyurin-T of the S1 family [35]. It is also most close to brachyurin-T in the phylogenetic tree (Figure 3). However, the domain architectures of these two enzymes are different. Compared to brachyurin-T, Aa2\_1884 lacks the C-terminal Trypsin domain (Figure 1). Among all the characterized peptidases, none were found to have the same domain architecture as Aa2\_1884 (Figure 1). These data sugges<sup>t</sup> that Aa2\_1884 is a novel multimodular protease of the S8 family. In addition, as shown in Figure 2, sequence alignment indicated that Aa2\_1884 contains several motifs that are conserved in reported S8 collagenases, which suggests that Aa2\_1884 may have collagenolytic activity.

**Figure 2.** Sequence alignment of Aa2\_1884 with similar proteases and reported S8 collagenases using the ClustalW program. Similar amino acid residues are boxed and shown in red, conserved amino acid residues are shown with red background. Amino acid residues constituting the catalytic triad of the MEROPS S8 family are marked with blue triangles, and motifs containing these residues are marked with blue underlines. Aa2\_1884 (WP\_199608745) is from *Flocculibacter collagenilyticus* SM1988, brachyurin-T (YP\_005442656) from *Caldilinea aerophile*, At3g14240 (MER0006049) from *Oryza barthii*, P69 peptidase (XP\_002275452) from *Vitis vinifera*, At5g51750 (XP\_009789180) from *Nicotiana sylvestris*, MO-1 (BAF30978) from *Geobacillus* sp. MO-1, and P57 (KT923662) protease from *Photobacterium* sp. A5-7.

**Figure 3.** Neighbor-joining (NJ) phylogenetic tree based on amino acid sequences of Aa2\_1884 and similar proteases. The tree was constructed with MEGA X. Bootstrap values (>50%) based on 1000 replicates were presented at nodes. Bar, 0.20. The reported S8 collagenases are indicated by asterisks.

### *2.2. Aa2\_1884 Is a Gelatinase with High Activity toward Denatured Collagens*

To characterize Aa2\_1884, the gene of Aa2\_1884 was over-expressed in *Escherichia coli* BL21 (DE3) with the vector pET-22b (+) containing a C-terminal His tag. The recombinant Aa2\_1884 protein was purified by affinity chromatography on a His Bind Ni chelating column and gel filtration chromatography on a Sephadex G200 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the purified Aa2\_1884 had an apparent molecular weight of approximately 100,000 Da (Figure 4a). The N-terminal sequence of the purified Aa2\_1884 was determined to T181-D-V-G-P-A186 by N-terminal sequencing. Thus, on the basis of its N-terminal sequence and molecular weight, mature Aa2\_1884 should contain 955 amino acid residues from Thr181 to Lys1135 (Figure 1). The signal peptide and the inhibitor I9 domain are cleaved off during maturation.

**Figure 4.** Purification and characterization of Aa2\_1884: (**a**) SDS-PAGE analysis of purified Aa2\_1884. Lane M, protein mass markers. Lane 1, purified Aa2\_1884. The protein band of Aa2\_1884 is indicated by an arrow; (**b**) Effect of temperature on the activity of Aa2\_1884. The experiment was performed in Tris-HCl buffer (50 mM, pH 9.0) at 40–80 ◦C, and the enzyme activity at 60 ◦C was taken as 100%; (**c**) Effect of pH on the activity of Aa2\_1884. The enzyme activity was measured at 60 ◦C with 40 mM Britton–Robinson buffers ranged from pH 7 to pH 11. The enzyme activity at pH 9.0 was taken as 100%; (**d**) Effect of NaCl concentration on the activity of Aa2\_1884. The activity was measured at 60 ◦C in Tris-HCl buffer (50 mM, pH 9.0) containing different concentrations of NaCl from 0 to 4 M. The enzyme activity in the Tris-HCl buffer with 0 M NaCl was taken as 100%. In (**b**–**d**), bovine bone collagen was used as the substrate in the experiments. The graphs show data from triplicate experiments (mean ± SD).

Sequence alignment implied that Aa2\_1884 may have collagenolytic activity. Indeed, Aa2\_1884 had noticeable activity towards bovine bone collagen at temperatures of 50–70 ◦C, with an optimal temperature at 60 ◦C. However, it had almost no activity towards bovine bone collagen at 40 ◦C (Figure 4b). At 60 ◦C, Aa2\_1884 also had activity towards bovine tendon collagen, gelatin, and casein, but no activity toward elastin-orcein (Table 1). As collagen is denatured at temperatures more than 40 ◦C, these results indicate that Aa2\_1884 can hydrolyze denatured collagen, but not natural collagen. Therefore, Aa2\_1884 is a gelatinase, rather than a collagenase. With bovine bone collagen as the substrate, Aa2\_1884 showed the highest activity at pH 9.0 (Figure 4c), indicating that it is an alkaline protease. In the buffer containing different NaCl (0–4 M), Aa2\_1884 showed the highest activity at 0.5–1 M NaCl (Figure 4d). These characteristics reflect the adaptation of Aa2\_1884 to the marine salty and alkaline environment.

**Table 1.** The substrate specificity of Aa2\_1884 \$.


\$ The activities of Aa2\_1884 towards different substrates were measured in Tris-HCl (50 mM, pH 9.0) at 60 ◦C. The data represent the mean ± SD of three experimental repeats.

We also analyzed the effects of metal ions and protease inhibitors on the activity of Aa2\_1884. As shown in Table 2, Ca2+, Ba2+, Sr2+, and Mg2+ significantly increased the activity of Aa2\_1884 towards bovine bone collagen, Zn2+ and Fe2+ severely inhibited its activity, and Ni2+, Co2+, and Mn2+ completely inhibited its activity (Table 2). Surprisingly, none of the four tested inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), or o-phenanthroline (*o*-P), had inhibitory effect on the activity of Aa2\_1884 (Figure 5), which is an unusual phenomenon for an S8 peptidase. Among these inhibitors, PSMF is a classical inhibitor to serine proteases, however, there are also some exceptions. Kexin has been reported to be resistant to PMSF [36]. Kexin is a typical S8 peptidase produced by *Saccharomyces cerevisiae*, which contains 814 amino acid residues. Different from Aa2\_1884, Kexin contains only a P-proprotein domain in addition to the Peptidase S8 domain [37]. It still remains elusive why kexin is resistant to PMSF. Thus, the underlying mechanisms of kexin and Aa2\_1884 to resist PMSF need further investigation.

**Table 2.** Effects of metal ions on the activity of Aa2\_1884 a.


a The activity of Aa2\_1884 was measured in Tris-HCl (50 mM, pH 9.0) at 60 ◦C with bovine bone collagen as the substrate. The activity (657.37 U/mL) without any metal ion was used as a control (100%). The data represent the mean ± SD of three experimental repeats. b "–" means that enzyme activity was not detectable.

The S8 family, the second largest family of serine peptidases, contains more than 200 peptidases in the MEROPS database (https://www.ebi.ac.uk/merops/cgi-bin/famsum? family=S8, accessed on 6 December 2021). In this family, only a small number have collagenolytic activity, but many have gelatinolytic activity. Different from those with collagenolytic activity, Aa2\_1884 has no activity towards native collagen. On the other hand, although Aa2\_1884 has gelatinolytic activity, it has a distinct domain architecture and is resistant to PMSF, compared to those with gelatinolytic activity in the S8 family. Therefore, Aa2\_1884 is a new gelatinase of the S8 family. The S8 family includes diverse peptidases produced by bacteria, archaea, and eukaryotes from various environments, and most are secreted endopeptidases. Therefore, both the temperature and pH optima of the S8 peptidases are in a wide range due to the adaptation of the peptidases to their respective environments [38]. For example, assays of subtilisins from *Bacillus* species are typically performed at pH 8.2–8.6 and 25 ◦C [39]. The pH optima of the S8 peptidases from

archaea are usually in the range of 7.5–10.7, and their temperature optima are in a wide range of 55–115 ◦C [40]. The pH optima of the S8 collagenases from bacteria are within the range of 7.1–9.3, and their temperature optima are usually 50–60 ◦C [24,26,41,42]. The optimal temperature and pH of Aa2\_1884 are 60 ◦C and pH 9.0, which fall in the optimal temperature and pH ranges of the S8 peptidases.

**Figure 5.** Effects of protease inhibitors on the activity of Aa2\_1884. Aa2\_1884 and the control enzyme alcalase were incubated at 4 °C for 1 h in 50 mM Tris-HCl (pH 9.0) containing 2 mM of each inhibitor, PMSF, EDTA, EGTA, or *o*-P. After incubation, the residual activity toward bovine bone collagen was measured at 60 ◦C, pH 9.0. The activity of Aa2\_1884 without any inhibitor was taken as 100%. The graphs show data from triplicate experiments (mean ± SD).

### *2.3. Aa2\_1884 Shows High Hydrolytic Efficiency on Bovine Bone Collagen*

As Aa2\_1884 had high activity towards bovine bone collagen at 60 ◦C (Figure 4b), it may have a potential in preparing collagen bioactive peptides from bovine bone collagen. Thus, attempts were made to prepare peptides from bovine bone collagen with Aa2\_1884 as a tool. To determine the optimal hydrolysis conditions, three enzymatic hydrolysis parameters were optimized by single factor experiments, including hydrolysis temperature, hydrolysis time and enzyme-substrate ratio (E/S). On the basis of the residual amount of collagen, the appropriate hydrolysis temperature and time of Aa2\_1884 were determined to be 55–65 ◦C (Figure 6a) and ≥3 h (Figure 6b), respectively. When the E/S was more than 400 U/g, no more obvious decrease in the amount of residual collagen was detected (Figure 6c). Hence, considering the hydrolysis efficiency and economic benefit, the optimal conditions of Aa2\_1884 for the hydrolysis of bovine bone collagen on the laboratory scale were determined to be reaction at 60 ◦C for 3 h with an E/S ratio of 400 U/g. Under these hydrolysis conditions, the maximum hydrolytic efficiency of bovine bone collagen reached 95.3 ± 0.3%, indicating that Aa2\_1884 is a good enzyme for the hydrolysis of bovine bone

collagen. We then prepared bovine bone collagen hydrolysate with Aa2\_1884 under the determined hydrolysis conditions.

**Figure 6.** Effects of temperature, time and E/S on the hydrolysis efficiency of bovine bone collagen: (**a**) Effect of hydrolysis temperature determined at the hydrolysis time of 3 h and the hydrolysis E/S of 400 U/g; (**b**) Effect of hydrolysis time determined at the hydrolysis temperature of 60 ◦C and the hydrolysis E/S of 400 U/g; (**c**) Effect of hydrolysis E/S determined at the hydrolysis temperature of 60 ◦C and the hydrolysis time of 3 h. The graphs show data from triplicate experiments (mean ± SD).

### *2.4. The Collagen Hydrolysate Prepared with Aa2\_1884 Is Rich in Collagen Oligopeptides*

To evaluate the quality of the prepared hydrolysate, we analyzed the contents of amino acids and peptides, amino acid composition, and molecular weight distribution of peptides in the hydrolysate. According to the ninhydrin method, there were 2.2 ± 0.1% free amino acids and 97.8 ± 0.1% peptides in the hydrolysate. Analysis of the composition of free amino acids in the hydrolysate by automatic amino acid analyzer also showed that there was only a small amount of free amino acids in the hydrolysate (Table 3). Thus, the hydrolysate is rich in peptides. In the hydrolysate, glycine is the most abundant (17.2%), followed by proline (10.1%). In addition, there were 1.0% hydroxylysine and 8.2% hydroxyproline in the peptides in the hydrolysate (Table 3). As hydroxylysine and hydroxyproline are two unique amino acids in collagen, our results indicated that the hydrolysate is rich in collagen peptides. The molecular weight distribution of peptides in the hydrolysate was analyzed by high performance liquid chromatography (HPLC) (Figure 7). The results showed that peptides with a molecular weight lower than 3000 Da, 1000 Da, and 500 Da accounted for approximately 71.6 ± 0.2%, 55.1 ± 0.2%, and 39.5 ± 0.2%, respectively (Table 4), indicating that the hydrolysate is rich in collagen oligopeptides.

**Figure 7.** Size exclusion chromatography analysis of the molecular weight distribution of peptides in the hydrolysate. The hydrolysate dissolved in deionized water was analyzed by HPLC with a TSK gel G2000 SWXL column.


**Table 3.** Composition and content of free and total amino acids in the hydrolysate a.

a Composition and content of free and total amino acids in the hydrolysate were analyzed by using an amino acid analyzer. The data represent the mean ± SD of three experimental repeats; b Trp was not detectable because it was destroyed in the process of acid hydrolysis.



**a** The content of each range of peptides in the hydrolysate were calculated based on the percentage of the area of corresponding molecular weight range in the total chromatograph area of the hydrolysate in the HPLC chromatogram.

### *2.5. Antioxidant Activity of Bovine Bone Collagen Hydrolysate*

The antioxidant activity of the hydrolysate was further evaluated by measuring its free radical scavenging activity towards 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) with hyaluronic acid (HA) as a control. The scavenging ratio of the hydrolysate to DPPH• increased with the hydrolysate concentration, which reached 32.8 ± 1.1% at the concentration of 10 mg/mL (Figure 8). In addition, as shown in Figure 8, the DPPH• scavenging ratio of the hydrolysate was obviously higher than that of HA, especially at high concentrations. A comparison of the DPPH• scavenging ratio of the hydrolysate with those of some reported collagen hydrolysates are shown in Table 5. The differences in the DPPH• scavenging ratios among the hydrolysates are likely attributed to the differences in the collagen sources, the preparation methods, and the enzymes used.

**Figure 8.** Antioxidant activity towards DPPH• of the hydrolysate and hyaluronic acid (HA).



a DPPH• scavenging ratio, b SWH, subcritical water hydrolysis.

As oligopeptides from various proteins have been demonstrated as beneficial compounds for skin protection or against diseases such as hypertension, hypercholesterolemia, and atherosclerosis, oligopeptides have been widely prepared from a variety of proteins, including proteins from various plant fruits and seeds, and proteins from skins and meats of various marine and terrestrial animals [4,47,48]. Protein hydrolysates containing oligopeptides have been prepared with both commercial and non-commercial proteases. For example, a loach protein hydrolysate prepared with papain contained approximately 30% oligopeptides with a molecular weight lower than 500 Da, and exhibited good hydroxyl radical scavenging and antioxidant activities [49]. A salmon skin hydrolysate prepared with alcalase and papain contained approximately 90% oligopeptides with a molecular weight lower than 1000 Da, and showed the ACE inhibitory effect from different fractions collected by reversed-phase HPLC [19]. A 1301 Da peptide from the cod fish skin hydrolysate prepared with pepsin, trypsin, and α-chymotrypsin exhibited potent ACE inhibitory and antioxidant activities [20]. A shrimp hydrolysate prepared with the crude enzyme from *Bacillus* sp. SM98011 contained approximately 41% oligopeptides with molecular mass lower than 3000 Da, and exhibited good hydroxyl radical scavenger and antioxidant activities [50]. A codfish skin hydrolysate prepared with the collagenolytic protease MCP-01 from *Pseudoalteromonas* sp. SM9913 contained 60% oligopeptides with a molecular weight lower than 1000 Da, and exhibited good hydroxyl radical scavenging activity and promoted an effect on cell viability of human dermal fibroblasts [7]. A bovine bone collagen hydrolysate prepared with the thermolysin-like protease A69 from *Anoxybacillus caldiproteolyticus* 1A02591 contained 21.1% oligopeptides with a molecular

weight lower than 1000 Da, and exhibited good moisture-retention ability and antioxidant activity [43]. The hydrolysate prepared from Bigeye tuna skin collagen contained peptides with molecular weights of 300–425 Da and had DPPH• scavenging activity [51]. It has been demonstrated that di-/tripeptides can be absorbed in their intact forms in human intestine without further hydrolysis [5]. Thus, protein hydrolysates containing more oligo-peptides with a molecular weight of <1000 Da or even <500 Da are preferred in cosmetics, functional food, and nutraceuticals [52].

Collagen used in collagen oligopeptides preparation have been extracted from the bones and skins of various animals, such as fish skins and bones [7,44,53], and goa<sup>t</sup> skin [54]. Bovine bone is a by-product of beef processing industry and its annual production is huge due to the large number of global slaughtered cattle. Bovine bone is rich in collagen and therefore, is a cheap and good source for collagen preparation. However, bovine bone collagen has rarely been used in collagen oligopeptide preparation to our knowledge. In this study, we used Aa2\_1884 to prepare collagen oligopeptides from bovine bone collagen. Aa2\_1884 showed a high hydrolysis efficiency (95%) on bovine bone collagen and the resultant hydrolysate contained a high proportion of collagen oligopeptides, 55.1% peptides with a molecular weight lower than 1000 Da, and 39.5% peptides with a molecular weight lower than 500 Da. These data sugges<sup>t</sup> that Aa2\_1884 has a promising potential in preparing collagen oligopeptides from bovine bone collagen, which may provide a feasible way for the high-value utilization of bovine bone collagen.

### **3. Materials and Methods**
