Molecular study and cloning of Methioninase gene from halophilic bacilli in cells susceptible to industrial application
Subject Areas :
Forough Sahirad
1
,
Mansour Bayat
2
*
,
محدثه لاری پور
3
1 - - Master's degree in Industrial Microbiology, Department of Biology, Faculty of Basic Sciences and New Technologies, Tehran Branch, Islamic Azad University, Tehran, Iran
2 - Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
3 - Associate Professor of Medical Mycology, Department of Microbiology, Faculty of Biological Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran
Keywords: Methioninase, Bacilli, cloning, Molecular, Halophile, PCR,
Abstract :
Methioninase is an enzyme that has been widely studied in the field of pharmacy, especially cancer therapy. Halophilic bacillus are a group of gram-positive bacteria that live in saline environments. These organisms are potential reservoirs of enzymes that are widely used in industries. Therefore, the aim is molecular investigation and coloning of the methioninase gene from halophilic bacilli is in the susceptible cell for use in industry. Bacillus strains were isolated and identified from a total of 20 saline water samples in persian gulf-bandarabbas, and then the methioninase gene was isolated from the bacilli by PCR method. The amplified fragment was inserted into the the pTG19 transfer vector by TA cloning method. In the next step, the recombinant vector was transformed into E.coli uragami. The expression level of pectinase enzyme was evaluated using real-time PCR method. Out of 12 Bacillus strains isolated, only 2 strains had methioninase gene. In order to determine the molecular identity of the Bacillus genus carrying the methioninase gene, housekeeping primers were used. Finally, the expression of methioninase gene in Escherichia coli Oragami bacteria was confirmed by PCR product sequence. As a result of this research, it was possible to find local halophilic bacilli producing methioninase enzyme and its gene was successfully transferred from Bacillus bacterium to E.coli bacterium for high production efficiency. According to the results, the optimal temperature of enzyme activity is at It was 37 degrees.
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