Purification and Characterization of a Novel Extracellular Haloprotease Vpr from Bacillus licheniformis Strain KB111

Abstract

Research background. Halo-alkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments are investigated for industrial applications. Experimental approach. The whole-genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS. Results and conclusions. The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease was 7 and 50 °C, respectively while the enzyme exhibited maximal activity in the broad salinity range of 2–4 M NaCl. It was fully stable at an alkaline pH range of 7–11 at 50 °C with a half-life of 90 min. Metal ions such as K+, Ca2+ and Mg2+ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acids profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain, and a C-terminal domain. Novelty and scientific contribution. A novel extracellular haloprotease from B. licheniformis was purified, characterized, and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.

Hydrolase Activities of Sourdough Microorganisms

“…A thermostable serine protease from B. licheniformis (LMG7561) reduced staling, promoted softness, and the effect was additive to known anti-staling agents (such as amylases) [74]. In another study, a serine protease with properties to be used in the food industry was purified and characterized from B. licheniformis KB111 [75]. The Bacillus genus is involved in bread spoilage [76].…”

Microbial-derived salt-tolerant proteases and their applications in high-salt traditional soybean fermented foods: a review

“…Table 1 collects information on some microbial-derived STPs reported since 2015. Notably, the strains have been isolated from various high-salt environments such as saline soils, seawater, lakes, and marine sediments, and used in various industries (Daoud et al 2018 ; Flores-Santos et al 2020 ; Foophow et al 2022 ; Yang et al 2020 ). STPs produced by different microorganisms differ not only in salt tolerance, but also in structure and enzymatic properties such as the optimum temperature, pH, and metal ion inhibition.…”

“…This strain has shown potential to produce an extracellular serine protease, which further expands the range of STP species available for study and application. A recently discovered extracellular haloprotease has been successfully isolated and purified from Bacillus licheniformis KB111 (Foophow et al 2022 ). With their distinctive enzymatic properties, these proteases have found a wide range of applications in diverse industries including detergents, leather production, food processing, medicine, textiles, and environmental protection.…”

“…Fungi represent a significant source of STPs, with notable species including A. niger , A. oryzae , Aspergillus reticularis , Penicillium citrus , and Penicillium nalgiovense (Foophow et al 2022 ; Lee et al 2010 ). Many of these fungus-derived STPs have demonstrated potential applications in traditional high-salt fermented foods, such as soy sauce, shrimp paste, and fish paste.…”