Table 1.
Preliminary screening of isolated bacteria for their proteolytic activity.
Fig 1.
Screening of bacteria for its proteolytic activity- preliminary screening of isolated bacteria for their proteolytic activity on skim-milk agar (A); primary screening of Bacillus sp. HM49 on casein (B), gelatin (C) and skimmed milk (D) agar media at a temperature of 20 °C (pH 8.0) after (i) 24 h, (ii) 48 h, and (iii) 72 h incubation time.
Table 2.
Primary screening of proteolytic bacteria, HM49 on different media at different temperatures.
Table 3.
Protein purification of protease enzyme from isolate HM49.
Fig 2.
(A, B, C). Optimization of protease production by isolate HM49 (2A- fermentation pH; 2B- fermentation Temperature; 2C-fermentation Time). Isolate HM49 depicted optimal enzyme production in pH 8.0 at 20 °C post 72h incubation period. The data in figures represent mean standard deviation (Mean ± SD) of three independent replicates with different letters above the bars indicating significant enzyme activity at p = <0.05 significance level. (D, E). Effects of varying pH and temperature on protease activity. Isolate HM49 recorded its protease enzyme activity from pH 6 (lowest) reaching its optimum at pH 8 followed by a gradual decrease up to pH 12 (2D). Temperature studies revealed its dynamism broadly in 5°C (lowest) to 40 °C range with its optimum activity being recorded at 20 °C (2E). The data in figures represent mean standard deviation (Mean ± SD) of three independent replicates with different letters above the bars indicating significant enzyme activity at p = <0.05 significance level.
Fig 3.
16S rRNA gene-based molecular identification of Bacillus velezensis HM49.
Representative electrophoretic image of amplified PCR product (A); evolutionary neighbor-joining phylogenetic tree (B).
Fig 4.
(A, B). Conserved domain prediction of CAASPR-HM49 protein (A). Evolutionary phylogenetic relationships of CAASPR-HM49 with other peptidases employing the neighbor-joining method (B). (C). Multiple sequence alignment of CAASPR-HM49 with several other typical proteases from the S8 family of extracellular subtilisins.
Fig 5.
(A). Ramachandran plot of CAASPR-HM49 subtilisin like protease refined model. Above 90% residues were spotted in most favoured regions and over 8% residues were localized in additional allowed regions. Presence of residue over 90% in most favoured regions is the recognition for a good model. PDBsum was used for producing this plot. (B). 3D/1D profile of refined CAASPR-HM49 subtilisin like protease model. Above 81% residues displayed a score equal to or greater than 0.2 due to which the good model quality is strongly perceptible. (C). Z-score plot portraying how well the refined model aligns with experimentally generated structures. This plot compares the algorithm-generated models with X-ray diffraction and NMR solved protein structures having amino acid length similar to modelled protein. If the modelled protein lies outside the shaded area, then the model is probably integrated with errors. From the location of our modelled protein on this plot, it is highly evident that it is free from probable errors. In other words, the overall quality score of CAASPR-HM49 subtilisin like protease model is well within (not outside) the range.
Table 4.
Final docking score and binding free energy values of lactose against CAASPR-HM49 subtilisin like protease.
Fig 6.
Three dimensional view of lactose in the binding pocket of CAASPR-HM49 subtilisin like protease (A). Interaction profile of lactose with various residues of this protease (B). Total seven hydrogen bonding interactions and two salt bridges exist between lactose and CAASPR-HM49 subtilisin like protease in docked state (blue solid lines represent hydrogen bonds and golden dotted lines depict salt bridges).
Fig 7.
Commercial detergent compatibility test of protease from isolate HM49 (A). Different letters above the bars indicate significant enzyme activity at 0.05 significance level. Blood stain removal test of protease from isolate HM49 (B). Disintegration of chicken feather (C).