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

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.

Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

“…GalaxyTBM was used for producing the protein structure from a given amino acid sequence. It first generates the structure of reliable core region through a template-based approach, after which the unreliable local regions are recognized and re-modelled through ab initio method 77 , 78 . Among five predicted models, the best one was chosen for refinement.…”

Biodegradation of pretreated chicken feather with keratinolytic bacteria Glutamicibacter arilaitensis

“…Increase in pH alter cysteine residues to lathionine that makes it accessible for keratinase and alkaline pH was suitable for deamination reaction leading to the release of ammonium ion which increases pH and favor keratinolysis [37]. In this study, pH of the medium increased in shake ask method, similarly enzyme activity was higher (92.5%) at pH 7-10 by Bacillus amyloliquefaciens [42]. The pH of medium affects the reaction mixture and aid nutrient transportation across the cell membrane of bacteria.…”

In SilicoInteractions of the Components from theSchinus terebinthifoliusExtract with Human Tyrosinase

“…Figures 2 and 3 show the Verify 3D plot and Ramachandran's plot, respectively, of the model chosen to be used in the next step. The Verify 3D plot shows the percentage of the structure‐amino acid sequence relationship that matches the database, which in this case is 97.7 %, and the literature suggests a cutting‐edge value of 80 % [19] . The Ramachandran's plot shows that the percentage of residues in the most favorable and forbidden regions are 83.2 % and 1.3 %, respectively.…”

“…The Verify 3D plot shows the percentage of the structure-amino acid sequence relationship that matches the database, which in this case is 97.7 %, and the literature suggests a cutting-edge value of 80 %. [19] The Ramachandran's plot shows that the percentage of residues in the most favorable and forbidden regions are 83.2 % and 1.3 %, respectively. These results indicate the stereochemical quality of the built model.…”