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 K_M and V_max 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.

中文翻译：

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克什米尔喜马拉雅山达奇甘国家公园土壤中解淀粉芽孢杆菌HM48菌株热稳定高电位蛋白酶的生化特性和功能分析

从达奇加姆国家公园的土壤中分离出一种新型的温度稳定的碱性蛋白酶产生细菌，已知该细菌栖息于喜马拉雅山西部的多种特有动植物物种中。通过形态学，革兰氏染色和生化技术，然后使用16S rRNA方法进行分子鉴定，将该高产蛋白酶的分离株鉴定为淀粉芽孢杆菌HM48菌株。解淀粉芽孢杆菌的细胞外蛋白酶HM48通过用硫酸铵（80％）沉淀进行纯化，然后进行渗析和凝胶过滤色谱，从而将其纯度提高5.8倍。纯化的酶的SDS-PAGE分析证实分子量约为≈25kDa。该酶在pH 8.0的较宽温度范围（10–90°C）中显示出异常的活性，在70°C时仍保持最大值，是该蛋白水解芽孢杆菌的最高报道，K _M和V _max为11.71 mg / mL和357.14 µmol / mL / min。该酶对各种金属离子，表面活性剂，氧化剂（H ₂ O ₂），有机溶剂，并与广泛使用的洗涤剂表现出出色的相容性。这种蛋白酶通过举例说明在70°C时可以去除血液和蛋黄污渍，并有效分解鸡毛，从而显示出有效的洗涤性能，使其对于洗衣目的和废物处理至关重要。为了进行功能分析，菌株HM48的蛋白酶基因扩增产生了约700 bp的核苷酸序列，当对照NCBI中的可用序列进行检查时，显示出与解淀粉芽孢杆菌类似枯草杆菌蛋白酶的丝氨酸蛋白酶相似。。该蛋白酶及其优先级最高的底物β-酪蛋白的结构是通过蛋白质建模生成的。通过未来的算法验证了这些蛋白质模型，然后进行了蛋白质-蛋白质（来自HM48和β-酪蛋白的蛋白酶）对接。这些蛋白质在对接状态下的相互作用情况也产生了，从而阐明了它们更精细的细节。这些属性使得这种热稳定蛋白酶新颖并且适用于高温工业和环境应用。