Isolation and characterization of S. marcescens AD-W2
Serratia marcescens AD-W2, used in the present study was isolated from soil, collected from NW-Himalayas at an altitude of about 1000ft by serial dilution method (James and Natalie 2014), and was maintained on tryptone soya agar (TSA). The culture was identified by 16S rRNA gene sequencing and the sequence was submitted in NCBI GenBank. The phylogenetic tree was made using the minimum evolution method (Rzhetsky and Nei 1992), using MEGA X (Kumar et al. 2018). The culture has been deposited in Sir Col. R. N. Chopra Microbial Resource Centre, Jammu, India, with accession number MRCJ-997.
Serratiopeptidase production from S. marcescens AD-W2
The isolated microorganism was subjected to growth in a 500 mL Erlenmeyer flask containing 100 mL tryptone soya broth in a rotary shaker at 200 RPM at 30 °C. In order to evaluate the production of enzyme, several production media were used for enzyme production followed by evaluation of protease activity up to 72 h. Based on the screening of various production media, soyabean meal and casein based medium (constituents in g/L: Soyabean meal—20.0, Casein—15.0, dihydrogen ammonium phosphate—7.5, soya oil—5.0, NaCl—0.5, KCl—0.1, MgSO4—0.1, ZnSO4—0.1, dextrose anhydrous-20.0, pH 7.5) was selected for protease production for further studies.
Extracellular protease assay and protein estimation
The qualitative protease assay of the culture supernatant was performed on the milk agar plate (James and Natalie 2014), while the quantitative assay was performed according to Cupp-Enyard (2008) with slight modifications. One Unit of Protease (serratiopeptidase) activity was defined as micrograms of tyrosine released per minute on hydrolysis of casein under standard assay conditions (pH 8.0, 37 °C). Protein estimation was performed by the Bradford method (Bradford 1976).
Purification of serratiopeptidase from S. marcescens AD-W2
The culture supernatant having the desired specific activity was partially purified by ammonium sulphate up to 30–80% saturation. The precipitates were dissolved in 0.05 M buffer (pH 8.0), dialyzed against the same buffer for enzyme activity or protein content evaluation, and further subjected to Ion exchange chromatography using the MonoQ5/50 GL column (GE make) installed with Bio-Rad DuoFlow Chromatography system. 40 mg of partially purified protein was loaded onto the column, and elution was performed using NaCl gradient in phosphate buffer (pH 6.0). 0.5 mL fractions were collected and evaluated for protease (serratiopeptidase) activity.
Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography
The crude, partially purified and purified enzymes were subjected to 10% SDS-PAGE according to Laemmli discontinuous system (Laemmli 1970). Zymography was performed as per protocol given by Lantz and Ciborowski (1994) with few modifications.
Characterization of serratiopeptidase from S. marcescens AD-W2
In order to evaluate the optimum activity, the assay was carried out, keeping all the assay conditions the same, except the variables (pH, temperature, substrate concentration, or inhibitors). The relative activities were calculated, considering the initial activity as 100%. The effect of temperature on serratiopeptidase activity was studied at 30–60 °C, while the effect of pH on the serratiopeptidase activity was evaluated at varied pH range, i.e., pH 6.0–7.0 (phosphate buffer), 8.0–9.0 (Tris–HCl buffer), pH 10.0 (Glycine NaOH), and pH 11.0–12.0 (Sodium bicarbonate buffer). The effect of inhibitors was studied with 2 mM inhibitors (Olajuyigbe and Falade 2014). The kinetic studies of purified serratiopeptidase were performed by activity assay with varying concentrations of casein under standard assay conditions to determine Vmax. Km values were determined by the double reciprocal Lineweaver–Burk plot. The critical temperature of the purified serratiopeptidase and activation energies below and above critical temperatures were determined by the Arrhenius plot generated from the temperature-activity dataset.
Identification of purified serratiopeptidase
The purified protein band from the SDS-PAGE gel was trypsin digested by the procedure discribed in Sigma Proteoprep kit, while reduction and alkylation steps were omitted for rapid processing (Shevchenko et al. 2006). The mass fingerprints of the peptides were obtained on Bruker UltrafleXtreme MALDI TOF/TOF using multiple laser shots. 2 µL of the tryptic digest was mixed with α-cyano-4-hydroxycinnamic acid (10 mg/mL CHCA in 50% acetonitrile and 0.1% TFA) in a 1:1 ratio, transferred on the target MALDI plate and was allowed to dry at room temperature. Bovine serum albumin was used as a positive control. The mass spectra were searched in the Swiss-prot database along with the contaminant database on the MASCOT server for identification of the protein (Perkins et al. 1999). The sequence obtained using the MASCOT software after MALDI-TOF analysis was subjected to BLAST in the UNIPROT database. The top six results were used for multiple sequence alignment in SEAVIEW software (Galtier et al. 1996). The three-dimensional structure of serratiopeptidase was built using the SWISS-MODEL server (Schwede et al. 2003) using serralysin protein from Serratia sp. (PDB entry 1srp) as the template.