Volume 15, Issue 60 (12-2025)
NCMBJ 2025, 15(60): 79-92 |
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Yahyaei S, Akhavan Sepahi A, Amani J, Hatef Salmanian A. Cloning and expression of alkaline protease gene from Bacillus strain isolated from hot spring samples of Tehran and Alborz provinces. NCMBJ 2025; 15 (60) :79-92
URL: http://ncmbjpiau.ir/article-1-1786-en.html
URL: http://ncmbjpiau.ir/article-1-1786-en.html
3. Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract: (10 Views)
Aim and Background: Protease enzymes with wide applications in various industries have occupied a major part of the global enzyme market. Different bacterial species produce these enzymes. In the meantime, the proteases that can be active in harsh conditions, such as high temperature and pH, have attracted the attention of researchers. This study aimed to identify protease-producing strains from sources in harsh environmental conditions, such as hot springs.
Materials and Methods: In this study, after screening the bacteria isolated from the soil and water samples of hot springs, a strain of the genus Bacillus was identified based on 16S rDNA molecular analysis. After confirming the presence of the alkaline protease-producing subtilisin gene in this selected strain, this gene was isolated using the PCR technique and cloned into the pET28a expression vector. After confirming the correctness of cloning, the optimization of the production of this recombinant protein was investigated by IPTG induction.
Results: The results of this study showed that the selected strain is most closely related to Bacillus pumilus and Bacillus safensis. After creating the expression vector, the recombinant protease exhibited the highest expression values at 37°C within four hours of induction with 1 mM IPTG. Recombinant protein expression was confirmed by the presence of a 33kDa band in Western blot analysis.
Conclusion: The results of this study determined that the selected strain isolated from the hot springs sample has the potential to be used in various industries with the alkaline protease-producing gene.
Materials and Methods: In this study, after screening the bacteria isolated from the soil and water samples of hot springs, a strain of the genus Bacillus was identified based on 16S rDNA molecular analysis. After confirming the presence of the alkaline protease-producing subtilisin gene in this selected strain, this gene was isolated using the PCR technique and cloned into the pET28a expression vector. After confirming the correctness of cloning, the optimization of the production of this recombinant protein was investigated by IPTG induction.
Results: The results of this study showed that the selected strain is most closely related to Bacillus pumilus and Bacillus safensis. After creating the expression vector, the recombinant protease exhibited the highest expression values at 37°C within four hours of induction with 1 mM IPTG. Recombinant protein expression was confirmed by the presence of a 33kDa band in Western blot analysis.
Conclusion: The results of this study determined that the selected strain isolated from the hot springs sample has the potential to be used in various industries with the alkaline protease-producing gene.
Type of Study: Research Article |
Subject:
Microbiology
Received: 2025/12/20 | Accepted: 2025/12/1 | Published: 2025/12/1
Received: 2025/12/20 | Accepted: 2025/12/1 | Published: 2025/12/1
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