Production, Optimization, and Partial Purification of Alkali-Thermotolerant Proteases from Newly Isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12

Abstract

Proteases that can remain active under extreme conditions such as high temperature, pH, and salt concentration are widely applicable in the commercial sector. The majority of the proteases are rendered useless under harsh conditions in industries. Therefore, there is a need to search for new proteases that can tolerate and function in harsh conditions, thus improving their commercial value. In this study, 142 bacterial isolates were isolated from diverse alkaline soil habitats. The two highest protease-producing bacterial isolates were identified as Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12, respectively, based on 16S rRNA sequencing. Optimal protease production was detected at pH 8, 37 °C, 48 h, 5% (w/v) NaCl for Bacillus subtilis S1 (99.8 U/mL) and pH 9, 37 °C, 72 h, 10% (w/v) NaCl for Bacillus amyloliquefaciens KSM12 (94.6 U/mL). The molecular weight of these partially purified proteases was then assessed on SDS-PAGE (17 kDa for Bacillus subtilis S1 and 65 kDa for Bacillus amyloliquefaciens KSM12), respectively. The maximum protease activity for Bacillus subtilis S1 was detected at pH 8, 40 °C, and for Bacillus amyloliquefaciens KSM12 at pH 9, 60 °C. These results suggest that the proteases secreted by Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12 are suitable for industries working in a highly alkaline environment.

Production of Alkaline protease from Bacillus amyloliquefaciens TAS-2 and Optimizing fermentation conditions

“…Among these strains, B. amyloliquefaciens TSA-2 showed maximum protease activity at pH 9, which indicated that it could be producing alkaline proteases. Similar observations were made in previous studies on Bacillus species, which also showed peak protease production at pH 8 and 9 [14]. Furthermore, B. amyloliquefaciens TSA-2 produced the most protease at a temperature of 40-45°C, suggesting that these bacteria were mesophilic.…”

“…However, an increase in temperature beyond this range resulted in a reduction in protease synthesis, indicating that high temperatures disrupted the molecular structure of the bacteria, causing them to stop producing proteases. Other studies have suggested that the ideal temperature for protease generation in Bacillus subtilis is 37°C [14]. In the case of B. amyloliquefaciens TSA-2, a 24-hour incubation period was found to be ideal for maximum protease synthesis.…”

“…It was also observed that the maximum synthesis of extracellular proteases occurs during the late logarithmic and early stationary phase, when bacteria begin secreting proteases to break down complex nutrients in the absence of carbon and nitrogen sources [19]. According to previous reports, the ideal pH range for Bacillus species proteases is 6-10, and their stability values span a wide pH and temperature range (37-60°C) [14]. After optimization, the protease production for B. amyloliquefaciens increased from 38 U/mL to 60.8 U/mL, suggesting that these bacteria could potentially generate proteases that are highly halotolerant [20].…”

Growth Media Optimization for Production of Alkaline Protease from Industrial Wastewater using Bacillus subtilis PTCC 1254

“…The most important group of enzymes so far used are the alkaline serine proteases [6]. Alkaline proteases as additives are *Corresponding Author Institutional Email: Najafpour@nit.ac.ir (G. Najafpour Darzi) extensively used in pharmaceuticals, dehairing of leather, textile, food and detergent industries [7,8]. The major sources of alkaline proteases consist of plants, animal organs and microorganisms.…”

Effects of Oxygen Transference on Protease Production by Rhodotorula mucilaginosa CBMAI 1528 in a Stirred Tank Bioreactor

“…Microbial proteases represent two-thirds of commercial proteases worldwide [ 5 ]. Microorganisms are preferred over the others for the large-scale production of many enzymes for the following reasons: they are present everywhere in nature; they can be produced in relatively higher quantities than in plants and in animals; they grow quickly and simply; they have unique physiological and biochemical properties, as well as simple culture conditions; they are produced by cells of easy manipulation [ 6 , 7 ]; and they are secreted from microorganisms, facilitating the down streaming stages. Moreover, the proteolytic enzymes found in microbes are small, dense, and structurally spherical and have many applications in various industrial sectors [ 5 ].…”