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<p>Proteases constitute a major class of industrial enzymes, with alkaline proteases garnering significant interest due to their catalytic efficiency and stability under alkaline conditions, which are paramount for applications in detergents, waste treatment, and bioremediation. The escalating demand for biocatalysts that maintain functionality under polyextremophilic conditions, such as concurrent high temperature, alkaline pH, and organic solvents, drives the exploration of microbial diversity in underexplored ecological niches. In this study, we report the isolation and multi-faceted characterization of <i>Bacillus halotolerans</i> strain C1, a novel isolate from Iranian extreme environments, exhibiting exceptional alkaline protease production. Through a rigorous screening of 70 bacterial isolates, strain C1 was selected as the superior protease producer based on both qualitative and quantitative analyses. The protease was partially purified to homogeneity via ammonium sulfate precipitation and gel filtration chromatography, yielding a single band on SDS-PAGE corresponding to a molecular mass of approximately 27&#xa0;kDa. The enzyme demonstrated a remarkably high specific activity of 5300 U/g. Biochemical profiling revealed unparalleled stability, retaining significant activity across a broad pH spectrum (5.0–11.0) and a wide thermal range (40–90&#xa0;°C), with optimum activity observed at pH 7.0 and 70&#xa0;°C (7299.67 U/g). Whole-genome sequencing unveiled a 4.121&#xa0;Mb circular chromosome encoding 4121 predicted coding sequences. Comparative genomic analysis confirmed species-level identity as <i>B. halotolerans</i> (dDDH value &gt; 98%), and identified a subtilisin-like serine protease gene (aprE) exhibiting 100% amino acid identity to its <i>B. subtilis</i> homolog, yet residing within a unique genomic locus. Notably, genome mining elucidated 12 biosynthetic gene clusters (BGCs) for secondary metabolites, including nonribosomal peptide synthetases (NRPS) for fengycin, bacillaene, and the siderophore bacillibactin, alongside two distinct bacteriocin clusters encoding the sactipeptide subtilosin A and a novel class IV lanthipeptide. Concurrently, antimicrobial resistance (AMR) profiling identified genes conferring resistance to multiple drug classes, including macrolides (mphK), rifamycins (rphB), and cationic antimicrobial peptides (mprF), mediated through efflux pumps (ykkCD, bmr) and ribosomal protection proteins (vmlR). Critically, the co-localization of these stress-responsive elements with the protease-coding region suggests a genetically encoded, coordinated adaptive strategy to environmental extremism. Our findings posit <i>B. halotolerans</i> C1 as a formidable source of a robust, multi-tolerant alkaline protease and provide a comprehensive genomic blueprint that underscores the imperative of integrating phenotyping with genomic mining for the discovery and rational engineering of next-generation industrial biocatalysts.</p>

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Isolation, characterization, and genomic analysis of Bacillus halotolerans C1 as a robust alkaline protease source

  • 期刊论文

  • 发表日期：

    2025年12月8日

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  • Maryam Rezvani,
  • Aboozar Soorni,
  • Mohammad Sedghi

摘要

Proteases constitute a major class of industrial enzymes, with alkaline proteases garnering significant interest due to their catalytic efficiency and stability under alkaline conditions, which are paramount for applications in detergents, waste treatment, and bioremediation. The escalating demand for biocatalysts that maintain functionality under polyextremophilic conditions, such as concurrent high temperature, alkaline pH, and organic solvents, drives the exploration of microbial diversity in underexplored ecological niches. In this study, we report the isolation and multi-faceted characterization of Bacillus halotolerans strain C1, a novel isolate from Iranian extreme environments, exhibiting exceptional alkaline protease production. Through a rigorous screening of 70 bacterial isolates, strain C1 was selected as the superior protease producer based on both qualitative and quantitative analyses. The protease was partially purified to homogeneity via ammonium sulfate precipitation and gel filtration chromatography, yielding a single band on SDS-PAGE corresponding to a molecular mass of approximately 27 kDa. The enzyme demonstrated a remarkably high specific activity of 5300 U/g. Biochemical profiling revealed unparalleled stability, retaining significant activity across a broad pH spectrum (5.0–11.0) and a wide thermal range (40–90 °C), with optimum activity observed at pH 7.0 and 70 °C (7299.67 U/g). Whole-genome sequencing unveiled a 4.121 Mb circular chromosome encoding 4121 predicted coding sequences. Comparative genomic analysis confirmed species-level identity as B. halotolerans (dDDH value > 98%), and identified a subtilisin-like serine protease gene (aprE) exhibiting 100% amino acid identity to its B. subtilis homolog, yet residing within a unique genomic locus. Notably, genome mining elucidated 12 biosynthetic gene clusters (BGCs) for secondary metabolites, including nonribosomal peptide synthetases (NRPS) for fengycin, bacillaene, and the siderophore bacillibactin, alongside two distinct bacteriocin clusters encoding the sactipeptide subtilosin A and a novel class IV lanthipeptide. Concurrently, antimicrobial resistance (AMR) profiling identified genes conferring resistance to multiple drug classes, including macrolides (mphK), rifamycins (rphB), and cationic antimicrobial peptides (mprF), mediated through efflux pumps (ykkCD, bmr) and ribosomal protection proteins (vmlR). Critically, the co-localization of these stress-responsive elements with the protease-coding region suggests a genetically encoded, coordinated adaptive strategy to environmental extremism. Our findings posit B. halotolerans C1 as a formidable source of a robust, multi-tolerant alkaline protease and provide a comprehensive genomic blueprint that underscores the imperative of integrating phenotyping with genomic mining for the discovery and rational engineering of next-generation industrial biocatalysts.