Sample collection and isolation of protease-producing strain
As a source of microorganisms, samples were collected from swine carcass and sawdust composting on the 10d, and immediately transported to the laboratory. The information of composting experiment used for sampling was shown in Additional file 1: Tables S1 and S2. Approximately 10 g of sample was added to a sterilized 100 mL conical flask containing 90 mL of sterilized 0.85% saline and glass beads. The conical flask was shook for 2 h at 37 °C and 120 rpm, then left to stand for 30 min, and 100 µL supernatant was removed, inoculated in casein medium (casein 4.00 g/L, Na2HPO4·12H2O 1.07 g/L, KH2PO4 3.00 g/L and agar 20.00 g/L) and the culture plates incubated at 37 °C for 48 h. Protease-producing strains were screened by measuring the ratio of hydrolysis circle diameter to strain diameter. The strain with the largest ratio of hydrolysis circle diameter to strain diameter was selected as the optimal protease-producing strain, purified on Luria-Bertani (LB) medium, and assessed by Gram staining.
Crude protease production
Pre-culture of protease-producing strain was prepared by seeding a single colony into 5 ml mineral salt medium (CH3COONa·3H2O 1.00 g/L, NH4Cl 1.00 g/L, NaCl 1.00 g/L, KH2PO4 0.50 g/L, K2HPO4 1.50 g/L, and MgSO4.7H2O 0.20 g/L; pH 7 ± 0.2), incubated at 37 °C and 120 rpm onto a rotary shaking incubator until OD600nm was 1.0. Subsequently, 1% (v/v) of the pre-culture was inoculated into a 150 mL conical flask containing 100 mL mineral salt medium and the flask incubated at 37 °C and 120 rpm for 48 h. Then, the culture was centrifuged (7104 g, 4 °C for 20 min) and supernatant collected as a crude protease.
Assay method of protease activity and protein concentration
Protease activity was determined as described , with protease unit (U) defined as the amount (µg) of tyrosine produced from hydrolysis of casein by 1 mL protease solution at 40 °C and pH 7.5 in 1 min . Protein concentration was determined by the BCA method , using a commercial BCA protein assay kit (Thermo Fisher Scientific, Rockford, USA).
Genomic identification was based on 16 S rRNA gene sequencing. Genomic DNA was purified with an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) and used as a template for amplification of 16 S rRNA, using the following primers: forward (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse (5′-GGTTACCTTGTTACGACTT-3′). Initial denaturation at 94 °C for 5 min was followed by 30 cycles at 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min, plus final extension at 72 °C for 10 min. The amplified 16 S rRNA fragment was sliced from the 1% agarose gel, purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme, Nanjing, China) and the resulting fragment submitted to Sangon Biotech (Shanghai, China) for nucleotide sequencing and comparison to the nucleotide database of NCBI using the BLAST nucleotide. A multiple sequence alignment program, CLUSTAL-W in MEGA X software, was used to align nucleotide sequences and to prepare a phylogenetic tree using the Neighbor-Joining tree approach.
Optimization of protease production
To optimize protease production, medium composition and fermentation conditions were gradually optimized by single-factor experiments. In the optimization experiments, each treatment was repeated three times.
Optimum carbon source
Based on the previous production method of crude proteases, sodium citrate, sucrose, glucose, maltose, CMC-Na was used to replace CH3COONa as carbon source, respectively. The activities of crude proteases were determined to determine the optimum carbon source. Adjust the optimal carbon source concentration to 1, 3, 5, 7, 9 g/L respectively, and determine the protease activity to determine the optimal carbon source concentration.
Optimum nitrogen source
Based on the experiment results of optimum carbon source, Urea, (NH4)2SO4, NaNO3, yeast powder, peptone was used to replace NH4Cl as nitrogen source, respectively. The activities of crude proteases were determined to determine the optimum nitrogen source. Adjust the optimal nitrogen source concentration to 1, 3, 5, 7, 9 g/L respectively, and determine the protease activity to determine the optimal nitrogen source concentration.
Optimum metal ion
Based on the experiment results of optimum nitrogen source, metal ions (Cu2+, Mn2+, Mg2+, Zn2+, Fe3+, Fe2+, Ca2+) were added to increase protease production, respectively. The activities of crude proteases were determined to determine the optimum metal ion. Adjust the optimal metal concentration to 0.5, 1.0, 1.5, 2.0, 2.5 mmol/L respectively, and determine the protease activity to determine the optimal metal ion concentration.
Optimum incubation temperature
Based on the experiment results of optimum metal ion, incubation temperatures were set to 20, 25, 30, 35, 40, 45 °C. The activities of crude proteases were determined to determine the optimum incubation temperature.
Optimum initial pH-value
Based on the experiment results of optimum incubation temperature, initial pH-values were set to 4, 5, 6, 7, 8, 9. The activities of crude proteases were determined to determine the optimum initial pH-value.
Optimum inoculation amount
Based on the experiment results of initial pH-value, inoculation amounts were set to 1%, 3%, 5%, 7%, 9%. The activities of crude proteases were determined to determine the optimum inoculation amount.
Optimum fermentation time
Based on the experiment results of inoculation amount, fermentation times were set to 12, 24, 36, 48, 60, 72 h. The activities of crude proteases were determined to determine the optimum fermentation time.
Ammonium sulfate precipitation
Crude protease (20 mL) was put in a beaker that was placed in an ice bath, and ammonium sulfate powder slowly added to reach 20% saturation. Thereafter, this step was repeated, making the concentration of ammonium sulfate reach 30%, 40%, 50%, 60%, 70%, and 80% respectively. After that, the beakers were placed overnight in the refrigerator (4 °C), and then the contents centrifuged at 12,800 g for 30 min at 4 °C. Supernatants were retained, and precipitations were resuspended with 5 mL citric acid-sodium hydrogen phosphate buffer (pH 7.5). Protease activity and protein concentration in supernatant and precipitate were measured. The specific activity was determined to identify the optimum salting-out interval and to create a salting-out curve.
According to the optimum salting-out interval, 200 mL crude protease was precipitated by ammonium sulfate. The precipitation was resuspended in 100 mL buffer solution and the salt precipitation sample was dialyzed overnight using a 14 kDa MW membrane to remove ammonium sulfate. Therefore, it was concentrated to 40 mL by polyethylene glycol (PEG) 20,000.
Sephadex G-75 gel filtration chromatography
The protease was further purified by gel filtration chromatography, using a Sephadex G-75 chromatography column (20 × 1.6 cm). The column was pre-equilibrated with citric acid-sodium hydrogen phosphate buffer (pH 7.5). The elution was adjusted to a flow rate of 0.5 mL/min, with each 3 mL collected as a fraction. Thereafter, protein concentration and protease activities of the fractions were determined. Fractions with the highest specific activities were combined and used for subsequent studies.
Molecular weight determination
At successive stages of purification, molecular weights were estimated by SDS-PAGE . Proteins were separated with 12% (W/V) acrylamide, and a protein marker mixture (Thermo Fisher, Shanghai, China) was applied to assess molecular weights. Gels were stained with 0.25% Coomassie blue (R-250) and de-stained in 1% acetic acid.
Amino acid sequence determination
The protein region in SDS-PAGE was excised and washed three times with 50% ACN/100 mm NH4HCO3 (pH 8.0) solution, vibrated for 10 min, and then the washing solution discarded. This step was repeated three times. Thereafter, the gel was cleaned with 100% ACN, and dried in a vacuum. Then, 10 mM dithiothreitol (DTT)/50 mM NH4HCO3 (pH 8.0) solution was added to the gel and incubated at 56 °C for 1 h. Thereafter, 55 mM iodoacetamide/50 mM NH4HCO3 (pH 8.0) solution was added into the gel and incubated in dark for 30 min. The gel was washed in 100% ACN, an appropriate amount of trypsin added and completely covered with 50 mM NH4HCO3 solution. The reaction system was incubated overnight at 37 °C for enzyme digestion and protein was extracted twice with 60% ACN/5% formic acid. Extracts were combined, desalted with a C18 small column and the sample frozen (-20 °C).
Mass spectrometry was done with a Thermo Q Exactive Plus system. The sample was separated by a liquid phase UltiMate 3000 RSLCnano system with nano-lift flow rate. The peptide sample was dissolved in sample buffer, loaded with an automatic injector, then combined with the C18 capture column (3 μm, 120 Ω, 100 μm × 20 mm), and eluted to the analytical column (2 μm, 120 Ω, 75 μm × 150 mm) for separation. Two mobile phases (mobile phase A: 3% dimethyl sulfoxide (DMSO), 0.1% formic acid, and 97% H2O; and mobile phase B: 3% DMSO, 0.1% formic acid, and 97% ACN) were used to establish the analytical gradient. The flow rate of liquid phase was \300 nL/min. In MS DDA mode analysis, each scan cycle contained one MS full scan (R = 70 K, AGC = 3e6, maxIT = 20 ms, scan range = 350–1800 m/z) and subsequent 15 MS/MS scans (R = 17.5 K, AGC = 2e5, maxIT = 100 ms). The HCD collision energy was 28, filter window for the four lever was 1.6 Da, and dynamic exclusion time for repeated ion collection was 35 s.
Mass spectrometry data generated by Q Exactive Plus were retrieved by ProteinPilot (V4.5) and the Paragon database retrieval algorithm. The screening standard of retrieval results was Unused ≥ 1.3. After deleting contaminated protein, the remaining identification information was used for subsequent analyses.
Homology modelling and substrate docking studies
The three-dimensional structure of actin protein was downloaded from previous models in the repository of the SWISS-MODEL Web Server (https://swissmodel.expasy.org/) . Amino acid sequences of purified protease were selected as templates to build a homology model via the SWISS-MODEL Web Serve, with Ramachandran plots used to evaluate model quality. All protein structures were processed in the Molecular Operating Environment (MOE 2019.1) platform, including removal of water and ions, protonation, addition of missing atoms and completion of missing groups, and protein energy minimization. Molecular docking used HDOCK software to obtain the purified protease-actin complex structure, with the purified protease and actin defined as the receptor and ligand, respectively. The protein was set to rigid, the docking contact site was set to the full surface, and the conformation generated after docking was set to 100, with the most negative energy conformation selected by the scoring function.
Characterization of the purified protease
Influence of temperature on protease activity and stability
The characterization method of the influence of temperature on the activity and stability had referred to the report of Ghafoori et al. , and was appropriately modified in this study.
To determine the optimum temperature for protease function, a protease activity analysis was done from 30 to 80 °C. Protease activity at 40 °C was defined as 100%, and relative activity calculated. Thermal stability of the protease was investigated by pre-incubating the protease at various temperatures for 2 h, and the enzymatic reaction was conducted under standard conditions. Residual protease activity, calculated on the basis of the activity of protease without incubation, was defined as 100%.
Influence of pH on protease activity and stability
The characterization method of the influence of pH value on the activity and stability had referred to the report of Tito et al. and Dwivedi et al. [29, 30], and was appropriately modified in this study.
The optimal pH for protease was determined by performing the enzyme reaction in various buffers within a pH range of 3–10. The protease activity at pH 7.5 was defined as 100%, and relative activity calculated. Protease was pre-incubated in various pH values (range of 3–10) for 2 h. Subsequently, protease activity was analyzed following standard assay conditions and then residual activity was evaluated, with protease activity without incubation defined as 100%.
Effect of metal ions on protease activity
The characterization method of the effect of metal ions on protease activity had referred to the report of Papagianni et al. , and was appropriately modified in this study.
To investigate effects of metal ions on protease activity, the purified protease was pre-incubated in 1 or 5 mM of Fe3+, Li+, Fe2+, Mg2+, Ag+, Cu2+, Co2+, Mn2+, Ca2+and Hg2+ for 1 h. Protease activity was analyzed following standard assay conditions and residual activities calculated on the basis of activity of protease without metal ions being defined as 100%.
Effect of compounds on protease activity
The characterization method of the effect of metal ions on protease activity had referred to the report of Ghafoori et al. , and was appropriately modified in this study.
The influence of various compounds on protease activity was determined by treating the protease with 1 or 5 mM chemical reagents for 1 h and then measuring protease activity. Residual activity was calculated according to activity of protease without chemical reagents being defined as 100%. Compounds selected in this experiment included phenylmethylsulfonyl fluoride (PMSF), DTT, ß-mercaptoethanol, DMSO, and ethylenediaminetetraacetic acid (EDTA).