Purification and characterization of a robust thermostable protease isolated from Bacillus subtilis strain HR02 as an extremozyme

Since the hot water of Genow, a village in Isin rural district in the central district of Bandar Abbas, Hormozgan Province, Iran, has a rich source of thermophilic bacteria, the current study aimed to find a new thermophilic protease enzyme with suitable properties to be used in different industries.Water and sediment samples were collected from the hot water of Genow, and finally, 20 colonies were isolated. Among these isolated colonies, two bacterial strains grew on the skim milk agar medium, and a clear halo was formed around the colony, which was accurately identified by 16S rRNA gene sequence analysis. The comparison of 16S rRNA gene sequence analyses of isolated strains HR01 and HR02 with registered sequences of 16S rRNA genes in NCBI showed that the two isolates had the most similarity to Bacillus sonorensis and Bacillus subtilis, respectively. Among the two bacterial strains, the highest enzymatic activity was observed in B. subtilis strain HR02, from which the protease purification process was performed. A putative native B. subtilis strain HR02 protease (BSHR02PR) was purified by the UNO Q-6 ionic exchange chromatography method. Biochemical analyses revealed a monomeric enzyme, BsHR02Pro, with a molecular weight of 25 kDa, showing the maximum activity at 70°C and pH 8.0. Moreover, the purified enzyme was stable up to 80 °C and in a pH range of 6.0-12.0. The steady-state kinetic analysis for colloidal casein showed that the Km , Vmax and kcat values of the purified enzyme were 25.7 μM, 93.2 μM min-1 and 2.18 s-1 , respectively.The hot water of Genow is a rich source of protease-producing bacteria. Sediments are a better source for the isolation of these types of bacteria than spring water. Overall, our results demonstrated a potential bacterial enzyme BsHR02Pro as a suitable catalyst to be used in the various industries.