Document Type : Research Paper
Authors
1
Ph.D. student of Agricultural Biotechnology, Faculty of Agriculture, University of Hamedan, Hamedan, Iran.
2
Associate Professor, Department of Biotechnology, Faculty of Agriculture, University of Hamedan, Hamedan, Iran
3
Department of Biology, Faculty of Science, University of Guilan, Rasht, IR Iran
4
Scientific Board Member of DNA and Genomic Data Bank, Iranian Biological Resource Center (IBRC), ACECR, Tehran, Iran.
10.22124/japb.2024.28131.1550
Abstract
Proteases, as one of the most widely used industrial enzymes, play a role in breaking protein molecules and converting them into peptides and amino acids. Serine proteases, which are considered as endopeptidases, are divided into four subgroups, among which alkaline serine proteases are considered the most important group from an industrial point of view. In this study, serine alkaline metalloprotease gene from Salinivibrio proteolyticus from Bakhtegan hyper saline lake was cloned and recombinantly expressed. The obtained results confirmed the production of a monomeric protein with a molecular weight of about 66 kDa. The results of this research indicate that chaperone plasmids do not have a significant effect on the soluble expression of the studied protease, and even if used, it disrupts the protein purification process due to the presence of high amounts of chaperone proteins in the soluble phase. Enzyme activity measurement by zymography method and also with Folin-Ciocalteu reagent showed its protease activity on casein substrate. By observing the inhibitory effect of EDTA with a concentration of 1 mM as an inhibitor of metalloproteases and the effect of reducing the enzyme activity in the presence of PMSF with a concentration of 1 mM as an inhibitor of serine proteases, it is concluded that the investigated protease belongs to the category of serine metalloproteases. Also, the proteolytic activity of the enzyme on the casein substrate with a concentration of 0.65% was calculated as 1.65 ± 0.08 U/mg based on the spectrophotometer results and using the L-tyrosine standard curve. Finally, in order to achieve the optimal specific activity of the enzyme, the evaluation of different reaction conditions such as optimal temperature and pH and its desired metal ions should be considered.
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