Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme’s co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Microbial enzymes e.g. proteases are known as supreme enzymes utilized in different industries 1 . All five classes of proteases including serine, aspartate, threonine, cysteine, and metalloproteases are found and therefore exist in prokaryotes 2,3 . In comparison to other classes, the cysteine proteases seems to be introduced less in bacteria 2 , however, the presence of cysteine proteases in prokaryotes is as common as in eukaryotes 4 .
Although most of the cysteine protease members are endopeptidases, there are few members which have exopeptidase activity exclusively or additionally. Cysteine proteases are defined as proteases that in their catalysis process, the thiol group of a cysteine residue plays the role of a nucleophile 3 . These peptidases catalyze the carboxylic acid derivatives hydrolysis through a two-step pathway in which the formation of general acid-base and an acyl-thiol intermediate hydrolysis occurs 5 . Interest in cysteine peptidases is growing and they are being utilized in different industries such as medicinal applications 6 .
In order to prevent their proteolytic activity on non-substrate proteins, proteases are regulated carefully. Allostery is a reversible swift regulation of enzyme activities without energy consumption. Allosteric enzymes have a site distinct from their catalytic center which controls and regulates enzymatic activity through interaction with small molecules having activatory or inhibitory effects. Although the prominent role of allostery in regulation of different proteases has received much attention, the study of small molecules effects such as inhibitors in regulating them is still in early stages 7 .
Native collagens have a triple-helical structure that common proteases are unable to hydrolyze 8 . Collagenases are proteases which break down the peptide bonds in specific sites of collagen 9 . Bacterial collagenases due to their wide industrial and biological applications are propitious enzymes 10 . Microbial collagenases have been directly utilized in clinical treatments and in laboratory researches as experimental reactants 11 . They can tenderize meat rEla-ligand docking, and molecular dynamics. To comprehend the proteolytic activity of the predicted protease model, the cysteine protease was docked with 4 ligands with supposedly increasing and inhibitory effects on activity. The docking results overall exhibited a second binding site which seemed to be allosteric site of rEla cysteine protease. The docking interaction of enzyme with Triton X-100 was predicted through a hydrogen bond between oxygen atom of Triton X-100 and Ala 32 residue with a distance of 2.63 Å and docking score of − 75.52 kJ/mol (Fig. 5a). Molecular docking of the allosteric cavity and glycerol showed 5 hydrogen bonds with Phe 36, Tyr 45 (2 bonds), Asp 56 and Val 54 in distance of 3.12, 3.01, 2.93, 2.97 and 3.21 Å respectively (Fig. 5b). Figure 5c and d shows the docking results of the rEla predicted model with 2 cysteine protease specific inhibitors E. 64 (c) and Leupeptin (d). E.64 interacts with allosteric cavity (supported by Glu 19, leu 20, Arg 28, Glu 33, Val 34 and Tyr 45) with dock score of − 107.5 kJ/mol but Leupeptin interacts with His 106 residue of active site and Glu 14 with dock score of − 107.01 kJ/mol. The docked complexes were then simulated for a period of 50 ns and the interactions were maintained during 50 ns MD simulations. MD results indicated that after 50 ns of molecular dynamic simulation, the structure was stable and the bonds were preserved (Fig. 6). The RMSD result shows stable behavior (Fig. 6a). E.64 has the highest value indicating the highest conformational changes in protein structure. RMS fluctuation plot (Fig. 6b) overall shows larger fluctuations for the ends of the protein and small fluctuations for the rigid structural elements. The plots indicate that Triton X-100, Leupeptin and E.64 interactions with protein shows considerable fluctuations in different parts of a certain loop consisting of residues from 39 to 61. Figure 6c highlights the aforementioned loop, responsible for the flexibility changes in the enzyme structure.   Supplementary Fig. S4 online) and the SDS-PAGE data indicates that the cysteine protease was for the most part hydrolyzed by proteinase K, but remained approximately intact by trypsin (Fig. 8d).
Effects of temperature and pH on enzyme activity and stability. Figure 9 shows the effect of temperature and pH on rEla activity and stability. The Enzyme revealed high stability in pH ranges from 5 to 9 and showed highest activity at alkaline pH 8 (Fig. 9a). rEla optimum temperature was 50 °C (Fig. 9b) and enzyme remarkably exhibited over 70% activity at temperature range of 20-60 °C. After 90 min incubation the relative activities of rEla was more than 80% in the pH range of 6-9 ( Fig. 9c) and enzyme preserved more than 70% of its residual activity at10-70 °C (Fig. 9d). In another stability experiment rEla showed 67% and 44% activity after 2 h incubation at pH 5 and 11, respectively (Fig. 9e). Thermostability studies revealed that the enzyme preserved about 60% activity after 3 h incubation at 50 °C (Fig. 9f) Table S1 online. The activation energy was calculated 9.145 kJ/mol by Arrhenius plot (Fig. 9g) which is the amount of energy that must be provided for rEla to form E-S complex. ΔG * , ΔH * and ΔS * at optimum temperature were 79, 6.39 kJ/ mol and − 0.22 kJ/molK, respectively.
To measure the thermodynamic parameters of enzyme irreversible thermo-inactivation, thermal inactivation plot at 50, 70 and 90 °C was drawn (see Supplementary Fig. S5 online). k in was obtained for each of the above temperatures and as shown in Table 1, k in increases gently as temperature goes up. The Arrhenius plot was then designed for mentioned reaction (Fig. 9h). Activation energy E a # value was calculated 22.78 kJ/mol. △G # , enthalpy △H # and entropy (△S # ) were 95.3 kJ/mol, 22.1 kJ/mol and − 0.226 kJ/molK at optimum temperature. Table 1 displays negative amounts of entropy, suggesting the negligible disorder and therefore reasonable thermotolerance of rEla. rEla long-term storage. In order to assess rEla long-term stability, the enzyme was kept in 25, 4, and − 20 °C for 100 days. Along other samples lyophilized enzyme was also incubated at 4 °C. The SDS-PAGE analysis was run after 100 days to observe alterations in enzyme electrophoretic pattern (Fig. 8e) and no noticeable changes were observed. The activity was examined at day 30, 60 and 100 as shown in Fig. 10a. All of the enzyme samples preserved more than 60% activity after 2 months. The rEla activity results after 100 days indicates that www.nature.com/scientificreports/ lyophilized protease exhibited the best stability by retaining 89% activity and the enzyme kept at -20℃ with glycerol, lost its residual activity more than other samples and was only 46% active.
Substrates specificity. The Effects of different substrates on enzyme activity were assessed using conventional protease substrates including casein, azocasein, albumen, gelatin, collagen and l.leucine.p.nitroaniline. The enzyme showed the highest activity toward gelatin and collagen with the relative activity of 130 and 120%, respectively and exhibited the least activity against azocaseine with the relative activity of 32% (Fig. 10b).
Effects of organic solvents, surfactants and metal ions on enzyme activity. The influence of organic solvents, surfactants and metal ions (2 and 5 mM), on the proteolytic activity of purified rEla was investigated at optimum activity conditions (Fig. 11). The effect of several surfactants at the concentration of 1, 4 and 8% (w/v) on enzyme activity was studied (Fig. 11a). The Proteolytic activity was not significantly changed in the presence of 10% ethanol and 90% of activity was preserved in comparison to the control, although adding 20% ethanol leads to reduction of enzyme activity to 57% (Fig. 11b). Exposing the enzyme to isopropanol and acetone ceased the activity. The enzyme retained more than 70% of its activity following exposure to 10% methanol. 10% glycerol decreased the enzyme activity to less than 40%. The observations showed increase in enzyme activity at 1% concentration of Tween 20, Tween 80, Triton X-100 and SDS up to 419,117, 472 and 386% respectively. Triton X-100 at concentration of 4% had the maximum influence and increased proteolytic activity to 490%. Figure 11c shows that enzyme activity was increased in the presence of Ca 2+ ions with final concentration of 2 mM. The enzyme maintained less than 60% of its maximum activity after treatment with 5 mM of KCl, AlCl 3 , BaCl 2 , CaCl 2 and 2 mM concentration of LiCl. The addition of Na + and K + decreased proteolytic activity gradually so the relative activity of purified enzyme was approximately about 80% and 70%, respectively. Moreover, at 5 mM ZnSO 4 , Li + and Mg 2+ enzyme activity was totally lost. Supplementary Table S2 shows effect of different metal ions on some cysteine proteases.   Fig. 11d. IAA and IAM completely ceased the enzyme activity as they are cysteine protease inhibitors, while PMSF (2 mM) failed and the protease maintained more than 60% of its residual activity. EDTA, Urea and β-ME at 5 mM concentration, lowered activity to less than 25%. In order to confirm the type of inhibitor behavior, the enzyme activity was measured in the presence of two specific inhibitors, Leupeptin and E.64 at 2.5, 5, and 10 mM (see Supplementary Fig. S6 online) which belong to the group of small inhibitors 26 . leupeptin competitively inhibited the enzyme activity while E.64 inhibitory impact was uncompetitive (Fig. 12).

Discussion
Bacterial proteases are one of the most important and well-studied parts of hydrolytic enzymes with numerous industrial and medical applications 1 . The utilization of recombinant enzymes has privilege over native proteins, as the amount of purified protein increases significantly and enzymatic properties such as stability are commonly improved 27 .
Here, in this study we heterologously expressed the cysteine protease gene from a novel thermophilic bacterium, Cohnella sp. A01. In silico analysis indicate that rEla is an intracellular protease. It has low sequence similarity to other reported proteases with the highest identity of 46.70%, and is thus a novel alkaline cysteine protease. Phylogenetic analysis of rEla gene sequence indicates that it is a cysteine protease from DJ-1/ThiJ/ PfpI superfamily. Despite growing fast and having representative in most of the organisms, only few members of this superfamily have been characterized biochemically 28 . Sequence alignment of rEla and homologous gene sequences exhibits the conserved catalytic triad His 105 , Cys 106 and Gly 74 . The activity of all cysteine proteases depends on the catalytic dyad consisting of cysteine and histidine. The order of Cys and His (Cys-His or His-Cys) residues differs among the families 29 .
Protease I from Pyrococus horikoshii was considered as the most similar template to build 3D structure. The models constructed with Modeller 9v7, SWISS-model and I_TASSER were superimposed in chimera 1.14 and very low RMSD values verified the close similarity of all generated models. The Ramachandran plot, without any outlier residues, validated the modeled structure. Further verification was carried out using ProSA. The calculated Z-score (− 6.41) displayed a compatible value with native proteins and an acceptable negative balance for the potential energy of the predicted model 30 .
Due to their flexible binding cavity, allosteric sites allow ligands binding to the allosteric pocket in a way that forms the best conformation (low energy conformations as zymogen state) to inhibit the enzyme, therefore in their presence the residual activity of the enzyme would be very low to zero 31 . Docking and MD simulation conclusions indicate that rEla has an allosteric site capable of having interactions with small molecules, resulting in increase or cease of the catalytic activity. E.64 is a cysteine protease inhibitor. The results exhibit the interaction between E.64 and allosteric site of rEla, causing inactivation of the enzyme via stable conformational changes. RMSF result shows fluctuation in residues from 39 to 43 leading to more rigidity of the enzyme. Triton X-100 www.nature.com/scientificreports/ is a nonionic detergent and it was shown that its presence has a great impact on increasing the protease activity.
The results indicate that Triton X-100 interacts with Ala 32 residue of the allosteric site through hydrogen bond and induces the fluctuation changes in residues from 49 to 52 locating in a big loop near active site of the enzyme. Since all RMSF results emphasis on fluctuation changes of residues lying in this area, this particular loop seems to have a significant effect on rEla flexibility and rigidity. Leupeptin docking suggests that it is a competitive inhibitor. It interacts with active site of rEla and prevents the enzyme-substrate complex formation. Therefore, MD simulation results confirm docking outcomes and show maintenance of the interactions after 50 ns.
Docking results indicate that the substrate binding pocket of rEla consists of more than 10 amino acids and is mostly identical for both substrates. Collagen and l.leucine.p.nitroaniline both interact with Pro 126 but have different spatial orientations with respect to the main residues of the active site. This might lead to the higher Proteinase K is a nonspecific endopeptidase which doesn't need specific sites on proteins to digest them 43,44 . On the other hand trypsin as a specific protease, cleaves only the C-terminal of lysine and arginine residues 45 . Thus, proteinase K is able to break down rEla more freely. On the other hand, rEla has 21 specific cleavage sites for trypsin to hydrolyze it. Only 8 of these cleavage sites are on the surface of rEla. Therefore, in comparison with proteinase K, trypsin might be very limited. In addition, due to rEla high rigidity some of these 8 cleavage sites might be inaccessible 46 .
Studies on the thermodynamic stability of proteases brings us closer to the factors that ascertain the enzyme stability 47 , therefore in order to comprehend rEla's behavior under various physiological conditions, the thermodynamic parameters of the protease were calculated 48 . As we have shown in this study, rEla has structural stability and remains active at high temperatures.
The low k in at 50 °C (0.0026) and notable high t 1/2 (256 min) confirmed the thermostable structure of the enzyme. The Arrhenius plot for irreversible thermos-inactivation was linear. Positive amount of △G # value indicates that the inactivation of enzyme is not spontaneous. The high Ea for inactivation means high amount of energy is needed for denaturation of rEla and therefore the enzyme is stable with temperature.
The rEla cysteine protease exhibited maximum activity at 50 °C similar to the cysteine protease extracted from Cissus quadrangularis 39 . Cysteine proteases isolated from Zingiber montanum rhizome, Ficus johannis, Calotropis procera, Z. officinale, Ficus johannis, exhibited optimum temperature at 60 °C which are all in the optimum temperature range of rEla 4,32,39,41 . The enzyme activity was stable with temperature at the range of 10 to 70 °C and it displayed thermostable features and maintained up to 60% of its proteolytic activity after incubation at 50 °C for 3 h. In a study held by K. Jamir and K. Seshagirirao cysteine protease isolated from Zingiber montanum rhizome 50% inactivation was observed by incubating the protein for 45 min at 40 °C and the enzyme was completely inactivated at 70 °C 32 while, rEla retained about 40% of its maximum activity after 3 h incubation at 90 °C. High thermal stability is an important aspect of proteases required for using them in different applications. Increasing temperatures might cause destruction of non-covalent bonds within a folded protein, which leads to unfolding the protein 49 . Despite the great universal demand for enzymes that can tolerate extreme environmental conditions, the total number of them are very limited 50 . On the other hand, these proteases are capable of keeping the contamination problems to a minimum which would be very helpful in producing and applying them in large quantities 51 .
As we showed rEla was stable in pH range 5-9. Although enzyme highest activity was observed at alkaline range at pH 8, it showed better stability in acidic range. To the best of our knowledge aside from the Zingiber montanum extracted cysteine protease with optimum pH 9, the enzyme had the highest optimum pH among previously studied cysteine proteases. The enzyme's remaining activity after 3 h treatment was 60% at pH 5, while it was less than 40% at pH 11. In the study held by Kizukala Jamir, Kottapalli Seshagirirao, ZCPG cysteine protease from Zingiber montanum rhizome was active in the pH range from 6.0 to 11.0 and preserved more than 61% and 81% of its activity at pH 7.0 and 10.0 respectively 32 . Thus rEla protease is considered alkaline protease as  www.nature.com/scientificreports/ it can perform high activity at alkaline pH, e.g., pH 9. Alkaline proteases optimum temperature is usually around 60 °C and they have an extensive substrate specificity. These properties are essential for proteases applications in detergent industry 5 . Molecular weight, optimum temperature and pH and pI of some cysteine proteases from different sources are summarized in Table 2.
In order to figure out the best method to store rEla for a longer period of time, we incubated the dissolved enzyme at 4, 25 and − 20 °C and also lyophilized enzyme at 4 °C for 100 days. Glycerol at final concentration   One of the important features of alkaline proteases is their substrate specificity 32 . Therefore, we investigated substrate specificity of the purified protease with different substrates, and rEla activity was higher when gelatin and collagen were used. Based on our research, no cysteine protease with specificity for collagen substrates has been reported so far. This specific feature along with unique thermostability increases the chance of industrial and medical applications for the enzyme.
It has been noted that alkaline proteases are in need of divalent metal ions such as Ca 2+ and Mg 2+ to show their maximum activities. These cation ions role in keeping active conformation of enzyme in high temperatures, is of a great importance, owning to the fact that they protect the protease from thermal denaturation 53 . Supplementary Table S2 represents the effect of several metal ions on rEla and other cysteine proteases in comparison. Among the examined ions, Ca 2+ increased enzyme activity to 135% at 2 mM concentration, suggesting it as possible rEla cofactor.
Glycerol is an efficient chaperone which is broadly used for in vitro studies. It enhances the stability of proteins through increase in hydration and prevention of protein aggregation 54 . Therefore rEla activity was expected to increase or remain uninfluenced. But as discussed before the remarkable decrease might probably be due to the fact that glycerol has three hydroxyl groups, but doesn't have a hydrophobic chain and it makes the microenvironment around the enzyme less suitable for catalytic activity 55 . On the other hand as docking result suggests, glycerol binds with allosteric site of the enzyme and might effect on rEla active conformation.
Since non-ionic surfactants such as SDS are very routine additives in detergent industries, the employed enzyme stability in their presence is very important 51 . SDS at 2 and 4% concentration, increased rEla activity up to 386 and 362% respectively. SDS at low concentration attracts sulfhydryl group of hydrogen atom in cysteine residue and increases proteolytic activity. This cysteine probably facilitates the binding of substrate-enzyme reaction 32 . The substantial increase in protease activity at the presence of surfactants is due to their synergetic interaction with the cationic surfactants and therefore, decreasing the positive charge density and reducing the possibility of cationic inhibition of the enzyme active site 56 . On the other hand, due to the existence of hydrophobic environment surrounding the SH groups within the cysteine proteases, nonionic surfactants have better chance to increase interactions with the probable substrates 57 . See the effect of organic solvents and surfactants on catalytic activity of several cysteine proteases on Supplementary Table S3 online. The enzyme had not any activity in the presence of IAA and IAM, indicating that it belongs to the class of cysteine proteases, whereas IAA and IAM bind covalently with the thiol group of cysteine residue in enzyme catalytic site, the substrate is thereby prevented from binding to the active site. On the other hand, the serineprotease inhibitor PMSF did not have significant inhibitory effect and enzyme retained about 67% of its relative activity after incubating with 2 mM PMSF. As shown in Table 3. β-ME had the opposite effect on rEla in comparison with other cysteine proteases and inhibited the protease slightly at the concentration of 2 mM, however reduced the enzyme activity to 20% at higher concentration (5 mm) suggesting the importance of disulfide bonds in preserving rEla active conformation 58 . In addition, the enzyme was inhibited by EDTA to the extent of 56% and 40% at 2 mm and 5 mm concentration, respectively. Unlike other studied cysteine proteases that EDTA failed to inhibit the enzymes, the observed partial inhibitory effect on rEla cysteine protease might indicate the requirement of Ca 2+ ions for optimum activity of the enzyme. As EDTA is a chelating agent that binds to calcium ions which are essential for the optimum activity of rEla. rEla cysteine protease seemed to be less resistance to urea than Ficus johannis cysteine protease which maintained completely active against 6 mM urea, and lost up to 70% of proteolytic activity at 5 mM concentration of urea. www.nature.com/scientificreports/ Competitive inhibitors enhance the Michaelis constant (K m ), but doesn't change the maximum velocity (V max ). Thus, they are assumed to be structural analogues of the substrate competing to take the same active site on the enzyme 59,60 . Therefore, accordingly Leupeptin is a competitive inhibitor of rEla (Fig. 12a). E.64 is an uncompetitive type of inhibitor, which changes both Km and V max (Fig. 12b). Although, uncompetitive inhibitors do not interact with free enzyme, they are capable of merging with the ES complex to decelerate or prevent the product formation 59 . These data support the outcome of docking and MD simulations indicating that Leupeptin binds to active center while E.64 interacts with allosteric site and the enzyme's conformational changes lead to inactivation.
Bioinformatics, homology, template search and sequence alignment. To find homology proteins, BLAST (http://blast .ncbi.nlm.nih.gov/Blast .cgi) was carried out against non-redundant protein database 62,63 . The same process was used for searching homologous sequences of protease in the protein data bank (PDB) to recognize the most similar structures 64 .The alignment was performed with 6 similar sequences from pfp1 family, to find conserved domains. PRALINE at http://www.ibi.vu.nl/progr ams/prali newww / with the BLOSUM62 substitution matrix and gap penalty of 12 was used for this alignment 65 . The presence of signal peptide and the location of its cleavage site was predicted using SignalP 5.0 server (http://www.cbs.dtu.dk/servi ces/Signa lP/). The phylogenetic tree of cysteine protease sequences were analyzed using7 MEGA X program (https ://www. megas oftwa re.net/) and iTOLv 5 visualizing tool at https ://itol.embl.de/ 66,67 . Secondary and tertiary structure prediction. Software's developed to predict secondary and 3D protein structures simulate the best and the closest structure to the submitted sequence by applying some algorithms which work based on previously discovered protein structures. To minimize the uncertainty, we compared the results of predictions from at least two commonly used programs for each purpose. Therefore, amino acid sequence of cysteine protease with related PDB file were submitted to the software. Protein secondary structure was predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre 2/) 68 . For 3D structure prediction, SWISS-MODEL (https ://swiss model .expas y.org/), MODELLER 9v7 and I-TASSER server (https ://zhang lab.ccmb.med. umich .edu/I-TASSE R/) were used [69][70][71] . The prediction outputs were imported into Chimera 1.14 (http://www. cgl.ucsf.edu/chime ra) to evaluate the structures and obtain the optimal energy of best predicted cysteine protease structure 72 . To validate the accuracy of predicted structures, Z-Score point (https ://prosa .servi ces.came. sbg.ac.at/prosa .php) was calculated and Ramachandran plot (http://mordr ed.bioc.cam.ac.uk/~rappe r/rampa ge.php/) was constructed 73 . The active site of the proposed model was predicted using 3DLigandSite (http:// www.sbg.bio.ic.ac.uk/3dlig andsi te/) and evaluated using the Chimera 1.14 program 72,74 . Protein-ligand docking studies. The predicted structure was docked with two surfactants (Triton X-100 and glycerol), two inhibitors (leupeptin and E.64) and one substrate (l.leucine.p.nitroaniline) using Molegro Virtual Docker V.6.0 (MVD) 75 . The chemical structures of ligands were obtained from the PubChem. The predicted structure sidechains and obtained ligands were minimized, and the potential cavities (active site and allosteric site) were identified using the built-in cavity algorithm of MVD. For each of the protease-ligand complexes, 20 test runs were performed. The modeled Enzyme was docked with collagen as specific substrate using HAD-DOCK 2.4 76 . The substrate structure was obtained from RCSB protein data bank (https ://www.rcsb.org/). rEla docking results in complexed with collagen and l.leucine.p.nitroaniline were further visualized using Discovery Studio Visualizer v20.1.0.19295 (https ://disco ver.3ds.com/disco very-studi o-visua lizer -downl oad/).

Molecular dynamics simulation. MD simulation of the protein-ligand complexes (protease-Triton
X-100, protease-E.64 and protease-leupeptin complexes) were performed using GROMACS v 4.6.5 with CHARMM36 all-atom force field 77 . The topologies and parameters of ligands were provided by CGenFF server (https ://cgenff .umary land.edu/) which are compatible with the CHARMM36 all-atoms force field. Proteinligand complexes were soaked in a cubic box of water molecules. Neutralization of the enzyme charges was carried out by adding Na + and Clions. The system energy was minimized by the steepest descent algorithm to eliminate bad contact and clashes. At last, after releasing all restraints 50 ns MD runs were performed. All bonds were limited by the LINCS algorithm 78 .
Cloning, expression and purification of the cysteine protease 1759. The gene of cysteine protease 1759 from Cohnella sp.A01 was cloned in expression vector pET-26b(+). The gene was amplified using PCR specific forward and reverse primers (forward: 5′-GGA ATT CCATATG ACC CGG ACG AAA AAC-3′ T m = 60 °C and reverse: 5′-GGG TTCGA ATCA GTG GTG GTG GTG GTG GTG TCG GCG CCC GTA CAG -3′ T m = 66 °C) with Nde I and Hind III recognition sites (shown in italics) respectively. The expression vector and PCR product were then purified using the PCR purification kit and digested with restriction enzymes mentioned above. Ligation of  Protease assay. The protease activity was determined using casein 1%. 50 µl of dialyzed enzyme was added to 70 µl of casein 1% in the same buffer. The enzyme co-factor, CaCl 2 with final concentration of 2 mM was added to the solution. The mixture was then incubated at 50℃ for 30minutes. To terminate the reaction, 125 µl of trichloroacetic acid 15% was used. To precipitate the non-hydrolyzed casein, the tube content was centrifuged at 10,000×g for 5 min at 4 °C. Blank contained reaction cocktail without enzyme. The supernatant absorbance was measured at 280 nm and the amount of hydrolyzed proteins was assessed using tyrosine as a standard. One unit of enzymes activity was defined as the amount of enzyme that can produce 1 µmol of tyrosine per minutes at pH 8 and 50 °C.
Evaluation of temperature and pH on the cysteine protease activity and stability. To determine the cysteine protease temperature profile, enzyme's activity was measured in temperature ranges from 10 to 90 °C. For estimating thermostability in different temperatures, enzyme was incubated in 10, 20, 30, 40, 50, 60, 70 and 80 °C for 90 min and the residual activity was observed. In another experiment to analyze thermal stability of cysteine protease, it was incubated in 50, 70, 90℃ for 3 h and the enzyme activity was measured every hour at optimum temperature. In order to assess the effect of pH on cysteine protease activity, reaction buffer (50 mM) was prepared in various pH 2-14 (composed of acetate, phosphate and glycine) to find the optimum enzyme activity. For pH stability, enzyme was incubated in 5 mM mixed buffer with different pH (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) for 90 min and afterward the protease activity was calculated in pH 8 and in addition pH stability was evaluated by incubating the enzyme in 5 mM mixed buffer at pH values of 5 and 11 for 3 h and the measuring its activity in optimal conditions every hour.
Kinetic and thermodynamic parameters study. Casein concentration of 1.25 to 80 mg/ml were used to determine the kinetic parameters of cysteine protease and the Michaelis-Menten plot was drawn in GraphPad Prism 6 to calculate Maximum velocity (Vmax) and Michaelis-Menten constant (Km) values. The thermodynamic parameters of rEla including ΔH * , ΔG * and ΔS * values were calculated as follow: where, K B is the Boltzmann constant (i.e. R/N, which is 1.38 × 10 −23 J/K), T is the temperature in Kelvin, ℏ is the Planck constant (6.63 × 10 −34 J/mol K), R is the gas constant (8.314 J/K mol).
To determine the irreversible thermo-inactivation parameters, according to the temperature stability plot the following equation was used, where A 0 is the initial enzyme activity, A t is the enzyme activity at intended time, k is the inactivation rate constant: www.nature.com/scientificreports/ To assess thermodynamic properties (ΔE # , ΔH # , ΔG # , ΔS # ), the Arrhenius plot was designed for irreversible thermal denaturation of enzyme at 50, 70, and 90℃. The thermodynamic parameters were calculated by Eqs. (6)(7)(8)(9) where k B is Boltzmann constant, h is Planck constant and k in is the inactivation rate constant: The Gibbs free energy of inactivation (ΔG # ) of the protease was calculated from: Moreover, enzyme half-life at 50, 70 and 90 °C was estimated using the Eq. (10).
Long-term storage of rEla. In order to study the enzyme long-term stability at 25 °C, 4 °C and − 20 °C enzyme was incubated at mentioned temperatures for 100 days and its activity was measured after 30, 60 and 100 days and it was compared to lyophilized sample to figure out the best method of storing the enzyme. To keep enzyme at − 20 °C, we considered two samples one with glycerol added to the buffer (final concentration 20%) to avoid protein damage and denaturation and one without it 52 .
Protease specificity. In order to determine protease specificity, enzyme activity was measured using azocasein, albumin, gelatin, collagen and l.leucine.p.nitroaniline as substrates. In the case of azocasein, 90 µl the substrate 1% and 10 µl of enzyme solution was incubated for 30 min in optimum temperature and pH. The reaction was then terminated by adding cold TCA 15%. The solution was centrifuged 15 min in 4000×g. Absorbance of the supernatant was determined in 440 nm. In assay with l.leucine.p.nitroaniline, 30 µl substrate (10 mM), 100 µl protease, 120 µl distilled water and 250 µl Tris-HCL (100 mM) was incubated for 30 min in optimum temperature and pH. To cease the reaction, 100 µl of acetic acid 30% (w/v) was used. Samples were centrifuged 10 min in 4000×g. Absorbance of the supernatant was determined in 410 nm. Assays with albumin, collagen and gelatin were also carried out the same way as explained for azocasein.