Design of the study
The new strain Streptomyces hydrogenans var. MGS13 isolated from Koringa mangrove soil was used in the present study for the production of serratiopeptidase by solid-state fermentation. The enzyme was purified by employing ammonium sulfate precipitation, followed by dialysis and gel filtration. Further characterization was done by performing stability and inhibitor studies. Finally, a combination of 2D gel electrophoresis, mass spectrometry, and bioinformatic tools was used for structural elucidation of purified serratiopeptidase.
Serratiopeptidase production
Serratiopeptidase was obtained from S. hydrogenans var. MGS13 using optimized medium composed of horse gram (4.8 g; particle size 600–250 μm), soya bean 1.1%, initial moisture content 42% and sterilized at 121 °C at 15 lbs pressure for 20 min. After cooling, 1.2 mL of inoculum was added and incubated at 28 °C for 4 days then the crude enzyme was obtained by extracting with sodium borate buffer (pH 9.0).
Determination of serratiopeptidase activity and protein content
Quantitative estimation of serratiopeptidase activity was determined according to IP 2010 [18]. One unit of serratiopeptidase was defined as the amount of enzyme required to liberate 1 μm of free tyrosine per min under standard assay conditions. Protein content was quantified according to the Lowry method [19] using bovine serum albumin as a protein standard.
Purification of serratiopeptidase produced from S. hydrogenans var. MGS13
An attempt was made to decide the optimal concentration required for the precipitation of the enzyme. For this purpose, various concentrations of ammonium sulfate were supplemented to the supernatant to attain 40–80% saturation. The precipitated proteins were redissolved in minimum quantity of sodium borate hydrochloric acid buffer pH 9.0 and dialyzed using dialysis membrane-50 (HiMedia—cutoff value 12 kDa) against the same borate buffer overnight at 4 °C. Then, the sample was concentrated, desalted, and loaded on the Sephadex G-100 column (30 × 1.8 cm) for purification. The enzyme fractions were eluted using sodium borate buffer pH 9.0 with a flow rate of 0.3 mL/min. The fractions eluted from the gel filtration column were tested for serratiopeptidase activity and total protein content. The fractions having enzyme activity were pooled, concentrated, and used as a purified sample for all the characterization studies. The recovery of enzyme and purification fold was calculated in terms of specific activity.
Electrophoretic analysis
The molecular weight of the purified protease was analyzed by SDS-PAGE according to the Laemmli method [20] using 15% polyacrylamide resolving gel. The mass was ascertained by comparing with pre-stained known molecular weight markers ranging from 10 to 245 kDa, HiMedia.
Casein zymography of purified serratiopeptidase was performed according to the Garcia-Carreno method [21] using casein (0.2% w/v) as a substrate in 15% resolving gel. The sample was dissolved in non-reducing SDS-loading buffer without heating. Subsequent to electrophoresis, the gel was kept for 1 h for incubation in 100 mL of sodium borate buffer pH 9.0 containing 2.5% (v/v) Triton X-100 to remove SDS and thoroughly rinsed with double distilled water to remove Triton X-100. Then, the gel was incubated in a buffer for an hour and later stained with 0.025% Coomassie Brilliant Blue R-250 and de-stained.
Effect of pH on serratiopeptidase stability and activity
The influence of pH on serratiopeptidase stability was determined by exposing the purified serratiopeptidase to various pH buffers of 50 mM concentration at 37 °C for 1 h, and the residual serratiopeptidase activity was measured. The buffers used for this study were phosphate buffer pH 4.0, 5.0, 6.0, 7.0, and 8.0; borate buffer pH 9.0 and 10.0; and glycine buffer 11.0. Optimum pH for serratiopeptidase activity was determined by preparing the substrate (casein) in the above buffers, and serratiopeptidase activity was measured as mentioned earlier.
Effect of temperature on serratiopeptidase stability and activity
The influence of temperature on serratiopeptidase stability was determined by incubating the purified protease at various temperatures 20 °C, 28 °C, 37 °C, 50 °C, and 60 °C for 1 h in sodium borate buffer at pH 9.0, followed by serratiopeptidase assay, whereas, the optimum temperature for serratiopeptidase activity was measured by performing the assay at diverse temperatures ranging from 20 °C, 28 °C, 37 °C, 50 °C, and 60 °C for 30 min in sodium borate buffer of pH 9.0.
Effect of inhibitors on enzyme activity
The influence of chelating agents such as ethylene diamine tetra acetic acid (EDTA) and phenyl methyl sulfonyl fluoride (PMSF) on the activity of serratiopeptidase was determined by incubating the purified enzyme with 1 mM, 5 mM, and 10 mM concentrations of EDTA and PMSF solutions for 30 min at 37 °C, and the comparative activities were obtained by performing serratiopeptidase assay. The activity of the control (purified enzyme without any inhibitor) was determined and considered as 100%. The influence of chelating agents on the enzyme activity was also determined by casein clearing zone technique [22]. The purified enzyme S.AMP13 was incubated with inhibitors at concentrations of 1 mM, 5 mM, and 10 mM for 30 min at 37 °C and introduced into wells of casein agar plate after 24 h incubation; the activities were assessed by measuring the clearing zone.
Effect of metal ions
The serratiopeptidase activity was assessed before and after the inactivation of metal ions from purified protease using 10 mM EDTA. In order to study the influence of metal ions on enzyme activity before chelation, the purified enzyme (1 mg/mL) was incubated for a time interval of 1 h at 37 °C with different metal ions at a concentration of 5 mM of Mg+2, Zn+2, Ca+2, Cu+2, Na+, and K+ in 50 mM of sodium borate buffer pH 9.0, and serratiopeptidase activity was determined as described earlier. For analyzing the metal ion effect after chelation, the purified enzyme was incubated with 10 mM EDTA, followed by incubation with different metal ions in sodium borate buffer 50 mM (pH 9.0) at 37 °C for 1 h, and the samples were evaluated by performing the serratiopeptidase assay. The activity of purified serratiopeptidase in a buffer without a chelating agent and metal ions (control) was assessed.
Determination of kinetic parameters
The kinetic constants of the purified enzyme were determined by measuring the serratiopeptidase activity at different casein (substrate) concentrations (1.2 to 9.8 mg/mL). The Km and Vmax values were determined using the Michaelis-Menten graph, and the Lineweaver-Burk double-reciprocal graph was plotted with calculated values.
Analysis of amino acid sequence
The purified enzyme was excised from the SDS-PAGE gel as a single protein band and named as S.AMP13 and dehydrated. After drying, the gel pieces were reduced with 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate, incubated at 56 °C for an hour and alkylated by incubating with iodoacetamide for 45 min at room temperature, then digested by incubating with trypsin solution overnight at 37 °C. The resulting tryptic digested peptides were extracted, and the supernatant was dispensed into an autosampler vial for peptide analysis by LC-MS. The tryptic digested peptides of 10-μL sample were injected in C18 UPLC column (75 μm × 150 mm), 1.7-μm particle size for the separation of peptides using a Water ACQUITY UPLC system, followed by analysis on the Q-TOF (model—Synapt G2) instrument for MS and MSMS. The column was eluted with a flow rate of 0.3 mL/min using buffers A (0.1% by volume formic acid (FA) in water) and B (0.1% by volume formic acid in acetonitrile (ACN)). The raw data was processed by Mass Lynx 4.1 Waters, peptide editor software, to get the complete integrated sequence of the sample. The individual peptide MSMS spectra were matched to the database sequence for amino acid sequence, and protein identification was assigned by searching a Swiss-Prot database containing all known alkaline metalloprotease from Streptomyces and other bacterial sources based on ProteinLynx Global SERVER (PLGS) score software, Waters. The obtained protein sequence was aligned with the similar proteins using “Clustal X2” [23] for the construction of pairwise sequence alignment from the sequence set. A dendrogram (guide tree) was constructed to analyze the phylogenetic relation of the newly identified protein by neighbor-joining (NJ) statistical method using MEGA 6.06 (Molecular Evolutionary Genetics Analysis) software. Conserved domain and motif in the sequence were identified using the conserved domain database of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and motif scan (http://myhits.isb-sib.ch/cgibin/motif_scan).
Prediction of structure
SWISS-MODEL server (http://swissmodel.expasy.org/interactive) was used to predict the 3D structure of the designed protein based on homology or comparative modeling, and the templates with the highest quality have been selected for model building. The best model was selected based on the QMEAN score. The computed model was structurally validated and analyzed by using PROCHECK of the PDBsum server, and the secondary structure and functions of protease were predicted using ProFunc server.