Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 13 4 2021 12 22 Isolation, Molecular Cloning, and Expression of yyxA Serine Protease Gene Extracted from Bacillus licheniformis in Escherichia coli Isolation, Molecular Cloning, and Expression of yyxA Serine Protease Gene Extracted from Bacillus licheniformis in Escherichia coli 35 54 3136 10.22103/jab.2021.17638.1320 FA Zahra Aghaei Jeshvaghani Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Ramin Hoseini Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Gazvin, Iran Journal Article 2021 09 18 Objective Proteases are among the most important industrial enzymes. Microbial proteases, especially from Bacillus sp., are most widely exploited industrially. The aim of this study was isolation, cloning, sequencing, expression, and bioinformatics study of yyxA serine protease gene extracted from Bacillus licheniformis. Materials and methods In this study, after extraction of bacterial DNA, the yyxA serine protease gene was isolated from Bacillus licheniformis using the polymerase chain reaction technique and cloned into the pTG19-T vector. The molecular structure, its biochemical and phylogenetic properties were investigated. The three-dimensional structure of the cloned enzyme was predicted using the I-TASSER, PHYRE2, RAPTORX, and Modeller tools. Confirmation of yyxA gene expression was performed by SDS-PAGE and dot blot analysis. Results Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed high similarity to the sequences of other Bacillus species, such as B. subtilis, B. gobiensis, and B. pumilus. After evaluating the drawn models, it was found that the models provided by PHYRE2 and I-TASSER software were desirable ones for predicting the three-dimensional structure of this protease. Recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-yyxA. Optimization of recombinant protein production was investigated. The highest expression values were obtained at 37 ° C for 4 hours with one mM IPTG. Conclusions The nucleotide sequence of the yyxA gene is 1212 nucleotides long, encoding a protein with 403 amino acids. Studies have shown that the enzyme encoded by this gene is in the category of stable enzyme and will be expressed in solution in Escherichia coli. These advantages make the enzyme as a suitable candidate for use in industry. Objective Proteases are among the most important industrial enzymes. Microbial proteases, especially from Bacillus sp., are most widely exploited industrially. The aim of this study was isolation, cloning, sequencing, expression, and bioinformatics study of yyxA serine protease gene extracted from Bacillus licheniformis. Materials and methods In this study, after extraction of bacterial DNA, the yyxA serine protease gene was isolated from Bacillus licheniformis using the polymerase chain reaction technique and cloned into the pTG19-T vector. The molecular structure, its biochemical and phylogenetic properties were investigated. The three-dimensional structure of the cloned enzyme was predicted using the I-TASSER, PHYRE2, RAPTORX, and Modeller tools. Confirmation of yyxA gene expression was performed by SDS-PAGE and dot blot analysis. Results Cloning was confirmed by sequencing. Based on the results of phylogenetic studies, the obtained protein sequence showed high similarity to the sequences of other Bacillus species, such as B. subtilis, B. gobiensis, and B. pumilus. After evaluating the drawn models, it was found that the models provided by PHYRE2 and I-TASSER software were desirable ones for predicting the three-dimensional structure of this protease. Recombinant protein production was successfully induced by IPTG induction in the host containing the plasmid pET28a-yyxA. Optimization of recombinant protein production was investigated. The highest expression values were obtained at 37 ° C for 4 hours with one mM IPTG. Conclusions The nucleotide sequence of the yyxA gene is 1212 nucleotides long, encoding a protein with 403 amino acids. Studies have shown that the enzyme encoded by this gene is in the category of stable enzyme and will be expressed in solution in Escherichia coli. These advantages make the enzyme as a suitable candidate for use in industry.

https://jab.uk.ac.ir/article_3136_96958c21313002ad2a99effb4704924d.pdf