This study focused on isolation, characterization, an‎d optimization of protease production from native bacterial strains. Sampling was performed from 17 diverse sources including hot springs, rice fields, northern forests, fish farm effluents, an‎d fermented products like 20-year-old grape vinegar. Samples were cultured on specific media (solid/liquid LB, skim milk agar, casein, an‎d glucose-based media). The methodology comprised three main phases: 1) Primary isolation via serial dilution (10⁻³ to 10⁻⁷) an‎d cultivation on LB agar, 2) Qualitative screening based on clear zone formation on skim milk agar an‎d quantitative activity measurement using Folin-Ciocalteu assay, an‎d 3) Molecular identification through 16S rRNA an‎d rpoB gene sequencing. Among 17 isolated colonies, seven superior strains (C1 an‎d S1-S6) were selec‎ted. Strain C1, isolated from grape vinegar, exhibited exceptional performance in all tests. Quantitative analysis revealed its peak activity (7299 U/ml) at pH=7 an‎d 70°C for 20 minutes. Notably, it maintained significant activity (2499 U) under extreme conditions (pH=11, 90°C, 1 minute). Whole-genome sequencing an‎d phylogenetic analysis (dDDH=98/43%, ANI) identified C1 as Bacillus halotolerans, harboring 4121 protein-coding genes, 8 rRNA, an‎d 97 tRNA genes. With high production an‎d remarkable stability, C1 enzyme presents an ideal can‎didate for detergent, leather, an‎d textile industries. The study provides significant insights into utilizing native microbial resources for industrial enzyme production, offering an efficient alternative to commercial counterparts. The robust enzymatic properties combined with cost-effective production potential position this protease as a promising biocatalyst for harsh industrial processes.

ابوذر سورني

محمد صدقي

بدرالدين ابراهيم سيدطباطبايي , صادق زارعي