Biochemical Characterization and Functional Analysis of Heat Stable High Potential Protease of Bacillus amyloliquefaciens Strain HM48 from Soils of Dachigam National Park in Kashmir Himalaya.
A novel temperature stable alkaline protease yielding bacteria was isolated from the soils of Dachigam National Park, which is known to be inhabited by a wide variety of endemic plant and animal species of Western Himalaya. This high-potential protease producing isolate was characterized and identified as Bacillus amyloliquefaciens strain HM48 by morphological, Gram's staining and biochemical techniques followed by molecular characterization using 16S rRNA approach. The extracellular protease of B. amyloliquefaciens HM48 was purified by precipitating with ammonium sulfate (80%), followed by dialysis and Gel filtration chromatography increasing its purity by 5.8-fold. The SDS-PAGE analysis of the purified enzyme confirmed a molecular weight of about ≈25 kDa. The enzyme displayed exceptional activity in a broad temperature range (10-90 °C) at pH 8.0, retaining its maximum at 70 °C, being the highest reported for this proteolytic Bacillus sp., with KM and Vmax of 11.71 mg/mL and 357.14 µmol/mL/min, respectively. The enzyme exhibited remarkable activity and stability against various metal ions, surfactants, oxidizing agent (H2O2), organic solvents and displayed outstanding compatibility with widely used detergents. This protease showed effective wash performance by exemplifying complete blood and egg-yolk stains removal at 70 °C and efficiently disintegrated chicken feathers making it of vital importance for laundry purpose and waste management. For functional analysis, protease gene amplification of strain HM48 yielded a nucleotide sequence of about 700 bp, which, when checked against the available sequences in NCBI, displayed similarity with subtilisin-like serine protease of B. amyloliquefaciens. The structure of this protease and its highest-priority substrate β-casein was generated through protein modeling. These protein models were validated through futuristic algorithms following which protein-protein (protease from HM48 and β-casein) docking was performed. The interaction profile of these proteins in the docked state with each other was also generated, shedding light on their finer details. Such attributes make this thermally stable protease novel and suitable for high-temperature industrial and environmental applications.
['Mushtaq|Hina|H|0000-0003-3549-2546', 'Jehangir|Arshid|A|', 'Ganai|Shabir Ahmad|SA|', 'Farooq|Saleem|S|0000-0001-5226-6212', 'Ganai|Bashir Ahmad|BA|', 'Nazir|Ruqeya|R|']
D000818:Animals D000069977:Bacillus amyloliquefaciens D002364:Caseins D002645:Chickens D004492:Edetic Acid D004795:Enzyme Stability D005241:Feathers D005843:Geography D006358:Hot Temperature D006863:Hydrogen-Ion Concentration D007194:India D007477:Ions D007700:Kinetics D008670:Metals D008826:Microbial Sensitivity Tests D062105:Molecular Docking Simulation D008970:Molecular Weight D016877:Oxidants D010447:Peptide Hydrolases D059748:Proteolysis D012336:RNA, Ribosomal, 16S D015203:Reproducibility of Results D012988:Soil Microbiology D012997:Solvents D013379:Substrate Specificity D013501:Surface-Active Agents
Kashmir Himalaya alkaline protease 16S rRNA KM Vmax protein modeling protein–protein docking soil bacteria