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.
Dachigam National Park (DNP), in Zabarwan mountains of north-western Himalaya constitutes a region of high biodiversity with greater endemism. DNP is known for its unique micro-climate together with distinct vegetational zones providing home to variety of threatened and endemic plant, animal, and bird species. However, studies on soil microbial diversity in fragile ecosystems of north-western Himalaya in general and DNP in particular are lacking. This was thus a maiden attempt to study variations in soil bacterial diversity of DNP with respect to changing soil physico-chemical properties, vegetation, and altitude. Soil parameters depicted significant variations among different sites with highest values for temperature, OC, OM and TN being 22.2 ± 0.75 °C, 6.53 ± 0.32%, 11.25 ± 0.54%, 0.545 ± 0.04% from site-2 (low altitudinal grassland site) in summer and lowest of 5.1 ± 0.65 °C, 1.24 ± 0.26%, 2.14 ± 0.45% and 0.132 ± 0.04% at site-9 (high altitudinal mixed pine site) in winter. Bacterial CFU showed significant correlations with soil physico-chemical attributes. This study led to the isolation and identification of 92 morphologically varied bacteria with the highest (15) from site-2 and lowest (04) from site-9 which post BLAST analysis (via 16S rRNA analysis) depicted presence of only 57 distinct bacterial species under taxonomic phylum, Firmicutes and Proteobacteria. Nine species were widely spread (i.e., isolated from > 3 sites), however, most bacteria (37) were restricted to a particular site. Diversity indices ranged between 1.380 to 2.631 (Shannon–Weiner’s index); 0.747 to 0.923 (Simpson’s index) with highest values for site-2 and lowest for site-9. Index of similarity was highest (47.1%) between riverine sites (site-3 and site-4) whereas two mixed pine sites (site-9 and site-10) showed no similarity.
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