Abstract
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.
Highlights
Dachigam National Park (DNP) situated in the Zabarwan range of Himalayas lies to the north-east part of Srinagar, the state capital of Jammu and Kashmir, India
The great Himalayan mountain range is expected to contain a wide variety of proteolytic bacteria that could remain active over a wide range of temperatures, limited studies mainly on the cold-active proteases from psychrophilic bacteria [6,7]
Out of 92 isolated morphologically different strains of bacteria, 39 bacteria showed the proteolytic activity upon preliminary screening, of which isolate HM48 formed a remarkable hydrolytic zone of clearance around its colonies and was chosen for further study (Figure 2)
Summary
Dachigam National Park (DNP) situated in the Zabarwan range of Himalayas lies to the north-east part of Srinagar, the state capital of Jammu and Kashmir, India. The park is ecologically significant as it is known to harbor a unique variety of Biomolecules 2021, 11, 117. Biomolecules 2021, 11, 117 plants and animals, including the critically endangered Kashmir red stag, locally known as “Hangul”, endangered Himalayan black bear, Himalayan brown bear, Himalayan gray langur, musk deer and snow leopard. The national park has been extensively studied for its phytodiversity and soil physicochemical characteristics [1], as well as its endangered animals [2,3,4], but is poorly understood as far as its soil bacterial studies are concerned. The great Himalayan mountain range is expected to contain a wide variety of proteolytic bacteria that could remain active over a wide range of temperatures, limited studies mainly on the cold-active proteases from psychrophilic bacteria [6,7]
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