*3.1. Materials*

Bovine tendon collagen was purchased from Worthington (Lakewood, NJ, USA), and bovine bone collagen from Kinry Biotech Co., Ltd. (Jinan, China). Casein (from bovine milk), gelatin (from cold water fish skin), elastin-orcein, EGTA, *o*-P, aprotinin, and cytochrome C were purchased from Sigma (St. Louis, MO, USA). Alcalase was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). PMSF was purchased from BBI (Shanghai, China). EDTA was purchased from HUSHI (Shanghai, China). Bacitracin was purchased from Aladdin (Shanghai, China). Tripeptide GGG and tetrapeptide GGYR were synthesized by Qiangyao Biotechnology Co., Ltd. (Shanghai, China). DPPH• was purchased from Tokyo Chemical Industry (Tokyo, Japan). HA was purchased from Shandong Freda Bioeng Co., Ltd. (Jinan, China). Other chemicals were of analytical grade and commercially available.

### *3.2. Sequence Analysis*

The domains of Aa2\_1884 (WP\_199608745) from *Flocculibacter collagenilyticus* SM1988 (CP05988) and of the other proteases shown in Figure 1 were predicted by InterPro (https: //www.ebi.ac.uk/interpro/; 6 December 2021) [55]. The signal peptide of Aa2\_1884 was predicted by the SignalP 5.0 server (https://services.healthtech.dtu.dk/service.php? SignalP-5.0; 6 December 2021) [56]. For sequence alignment, previously reported MCP-01 (ABD14413) from *Pseudoalteromonas* sp. SM9913, the collagenolytic protease (BAF30978) from *Geobacillus* sp. MO-1, myroicolsin (AEC33275) from *Myroides profundi* D25, and P57 (KT923662) from *Photobacterium* sp. A5-7 were selected to align with Aa2\_1884 by ClustalW with bootstrap of 1000 [57]. The sequence alignment was displayed using ESPript 3.0 [58]. The conserved sites were predicted by MEME (https://meme-suite.org/meme/; 6 December 2021) [59]. The phylogenetic tree was constructed via MEGA X [60].

### *3.3. Protein Expression and Purification*

The genome DNA of strain SM1988 was extracted with bacterial genomic DNA isolation kit (BioTeke, Beijing, China) according to the manufacturer's instructions. The gene sequence of Aa2\_1884 was amplified by PCR using the genome DNA of strain SM1988 with primers 1884-F (5-AAGAAGGAGATATACATATGATGAAAATAGAACATAGT-3) and 1884-R (5-TGGTGGTGGTGGTGCTCGAGTTTATTGTCACACGTGGTT-3). The primers were synthesized by Tsingke Biotechnology Co., Ltd. (Qingdao, China). The PCR product

was then cloned into vector pET-22b (+) (Vazyme) with a C-terminal His tag. The constructed plasmid carrying the gene sequence of Aa2\_1884 was verified by sequencing and then transformed into Fe2 BL21 (DE3). Recombinant *E. coli* cells were cultured in Lysogeny broth (LB) medium with 100 μg/mL ampicillin at 37 ◦C, 180 rpm to an OD600 of 0.8–1.0. Then, 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the cells were further incubated at 15 ◦C, 120 rpm for 5 days [26]. After incubation, cells were lysed by a high-pressure cracker and centrifuged at 4656× *g*, 4 ◦C for 1 h, and the supernatant was collected. The recombinant Aa2\_1884 protein was extracted from the supernatant by an His binding Ni chelating column, and then purified on a Sephadex G200 gel filtration column (GE, Boston, MA, USA) using fast protein liquid chromatography (FPLC) on AKTA purifier (GE, Boston, MA, USA) [61]. The purified Aa2\_1884 was analyzed by 12.5% SDS-PAGE. Protein concentration was determined by a BCA protein assay kit (Thermo, Waltham, MA, USA) with bovine serum albumin (BSA) as the standard according to the manufacturer's instructions.

### *3.4. Enzyme Assay*

The activities of Aa2\_1884 toward bovine bone collagen, bovine tendon collagen, and gelatin at 60 ◦C were measured by the method provided by Worthington Biochemical Co. (Lakewood, NJ, USA) [26]. For collagen, a mixture of 5 mg substrate and 1 mL enzyme solution was incubated at 60 ◦C in Tris-HCl buffer for 0.5 h with continuous stirring. For gelatin, 100 μL enzyme solution was incubated with 100 μL of 2% (*w/v*) gelatin at 60 ◦C for 10 min. The reaction was stopped by the addition of 10 μL of 1.25 M trichloroacetic acid. The released amino acids were quantified using the colorimetric ninhydrin method [62] with L-leucine as the standard. One unit of enzyme activity was defined as the amount of enzyme that released 1 nmol of L-leucine per hour from collagen or gelatin [26]. The caseinolytic activity was determined at 60 ◦C using the method described by He et al. [63]. A reaction mixture containing 100 μL enzyme solution and 100 μL of 2% (*w/v*) casein was incubated at 60 ◦C for 10 min, and then the reaction was terminated by 200 μL trichloroacetic acid (0.4 M). The mixture was centrifuged at 17,935× *g* for 10 min, and 100 μL of the supernatant was incubated with 500 μL of sodium carbonate solution (0.4 M) and 100 μL of the Folin-phenol reagen<sup>t</sup> at 40 ◦C for 20 min. After the reaction, the OD660 of the mixture was measured. One unit of enzyme activity was defined as the amount of enzyme that liberated 1 mg tyrosine per minute [63]. The elastolytic activity at 60 ◦C was determined using the method described by Chen [64]. A mixture of 250 μL enzyme solution and 5 mg elastin-orcein was incubated at 60 ◦C for 1 h. After the reaction, the residual elastin-orcein was removed by centrifugation. The OD590 of the supernatant was recorded. One unit of enzyme activity was defined as the amount of enzyme that caused an increase of 0.01 in OD590 per minute [64].

### *3.5. Enzyme Characterization*

The optimal temperature was determined by measuring the activity of Aa2\_1884 toward bovine bone collagen in Tris-HCl buffer (50 mM, pH 9.0) at 40, 50, 60, 70, and 80 ◦C. The optimal pH was determined by measuring the activity of Aa2\_1884 toward bovine bone collagen at 60 ◦C in 40 mM Britton–Robinson buffers from pH 7.0 to pH 11.0. The effect of NaCl concentration on the activity of Aa2\_1884 was determined by measuring the activity of Aa2\_1884 toward bovine bone collagen in Tris-HCl buffer (50 mM, pH 9.0) containing NaCl of different concentrations (0–4 M) at 60 ◦C. To evaluate the effect of metal ions (Li+, K+, Ca2+, Mg2+, Cu2+, Ni2+, Mn2+, Ba2+, Fe2+, Zn2+, Co2+, Sn2+, Sr2+) on the enzymatic activity, Aa2\_1884 was incubated in Tris-HCl buffer (50 mM, pH 9.0) containing each metal ion (2 mM or 4 mM) at 4 ◦C for 1 h, and the enzymatic activity toward bovine bone collagen was then measured at 60 ◦C. For the inhibitory experiment, Aa2\_1884 and alcalase were incubated at 4 ◦C for 1 h with 2 mM of an inhibitor, PMSF, EDTA, EGTA, or *o*-P. After incubation, the residue activity toward bovine bone collagen was measured at 60 ◦C and pH 9.0.

### *3.6. Optimization of the Enzymatic Hydrolysis Parameters*

Three parameters, hydrolysis temperature, hydrolysis time, and E/S, which influence the efficiency of the enzymatic hydrolysis, were optimized via single-factor experiments, in which enzymatic hydrolysis of 10 mg bovine bone collagen in 1 mL 50 mM Tris-HCl (pH 9.0) was performed at 180 rpm in a shaking bath. Each parameter was determined under the optimum conditions of the other two parameters. To determine the hydrolysis temperature, the enzymatic hydrolysis was performed at different hydrolysis temperature (40, 45, 50, 55, 60, 65, 70 ◦C). To determine the hydrolysis time, the enzymatic hydrolysis was performed for different time (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 h). To determine the E/S, the enzymatic hydrolysis was performed with different E/S (0, 4, 8, 40, 80, 160, 400, 600, 800 U/g collagen). After the hydrolysis, the reaction system was heated at 90 ◦C for 15 min to terminate the reaction, and then centrifuged at 4 ◦C for 20 min. The precipitate was freeze-dried and weighted, which was taken as the residual amount of collagen.

### *3.7. Preparation and Evaluation of Collagen Hydrolysate*

To prepare peptides from bovine bone collagen, 10 mg bovine bone collagen was hydrolyzed under the determined parameters (at 60 ◦C for 3 h with an E/S ratio of 400 U/g). After hydrolysis, the reaction system was heated at 90 ◦C for 15 min and then centrifuged at 4 ◦C for 20 min. The supernatant was collected, freeze-dried, and weighted, which was the prepared hydrolysate.

Ten milligrams of the hydrolysate were dissolved in 1 mL deionized water. With L-leucine as the standard, the content of free amino acids in the hydrolysate solution was determined by the ninhydrin method [62]. The content of peptides in the hydrolysate was calculated by subtracting the content of free amino acids from that of the hydrolysate in the solution [43]. The compositions of free and total amino acids of the hydrolysate were analyzed by using an amino acid analyzer HITACHI 835 (Tokyo, Japan). The molecular mass distribution of peptides in the hydrolysates were analyzed by the method described by [7]. Briefly, the hydrolysate was dissolved with deionized water, and then analyzed by HPLC (LC-20AD, SHIMADZU, Tokyo, Japan) equipped with a TSK gel G2000 SWXL column (300 × 7.8 mm; range, <150,000 Da; void volume, 5.7 mL; Tosoh, Japan) that was eluted with the buffer containing 45% acetonitrile and 1% trifluoroacetic acid in deionized water at a flow rate of 0.5 mL/min HPLC under 220 nm monitoring. The calibration standards for molecular mass were tripeptide Gly-Gly-Gly (GGG, *Mr* 189), tetrapeptide Gly-Gly-Tyr-Arg (GGYR, *Mr* 451), bacitracin (*Mr* 1422), aprotinin (*Mr* 6511), and cytochrome C (*Mr* 12400). Based on the calibration standards, the chromatogram of the hydrolysate was separated into several fractions (<500 Da, 500–1000 Da, 1000–3000 Da, 3000–5000 Da, 5000–10,000 Da, and >10,000 Da), and the content of each fraction was determined by its relative peak area.

### *3.8. Analysis of the Antioxidant Activity of the Collagen Hydrolysate*

The antioxidant activity of the prepared hydrolysate was analyzed by measuring its free radical scavenging activity towards 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) according to the method described by Sun [65]. HA was used as a positive control due to its widespread application in scavenging free radical. To determine the DPPH• scavenging activity, 1 mL hydrolysate samples in incremental concentrations (0.1, 0.25, 0.5, 1, 2, 3, 5, 7.5, 10 mg/mL) were reacted with 2 mL of 100 μM DPPH• (dissolved in ethanol solution) for 40 min at room temperature (25 ◦C) in dark, and then the absorbance of the reaction solution was detected at 525 nm. DPPH solution was replaced with ethanol solution to obtain the result of background of sample, and the hydrolysate sample was replaced with water to obtain the result of blank control.

The free radical scavenging activity ( *D*) was calculated as follows:

$$D(\%) = \left[1 - \left(A\_i - A\_j\right) / A\_0\right] \,\,\,\,\,100\,\tag{1}$$

where *Ai* was the absorbance of the sample, *Aj* was the background absorbance of the sample, and *A*0 was the absorbance of the blank control.
