Volume 14, Issue 3 (12-2019)                   MGj 2019, 14(3): 231-241 | Back to browse issues page

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rezakhani Z, hesampoor A. Engineering of Escherichia coli phytase enzyme thermostability. MGj. 2019; 14 (3) :231-241
URL: http://mg.genetics.ir/article-1-1623-en.html
Abstract:   (877 Views)
Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Natural and recombinant production of this enzyme is one of the important issues in protein engineering field. In this study, the synthetic Phytase gene Bac phy-wild was cloned into PUC57 vector and transformed to susceptible Escherichia coli-DH5α. In order to replace the polar amino acid with non-polar amino acid within the target mutagenic protein; we used a primer designed to tryptophan (S392W) for targeted mutagenicity in the amino acid serine 392 position. After confirming the mutation at the target site, the mutated gene was registered at the NCBI gene bank. To investigate the expression and temperature stability of the mutant enzyme and compare it to WT phytase, the WT and mutated Bac Phy-Mut genes were transferred to pET26b (+) expression vector. Recombinant vector construct was transferred to a to Escherichia coli-BL21. After screening of recombinant clones, SDS-PAGE analysis and protein concentration were used to evaluate the expression of recombinant protein. The results showed the presence of protein with a molecular weight of 42 kDa. Also the study of physicochemical properties of WT and mutated phytase showed that the optimum temperature was unchanged at 55 ° C and the pH was optimized to be 5. Comparison of thermal stability of the mutated phytase in compare with WT at 40, 50, 60, 70 and 80 ° C was improved by 18, 24, 19, 9 and 6%, respectively. Hence, the results of the current research showed that by using targeted mutagenesis method we succeed to improve the mutated phytase with higher thermal stability which could be obtained and mutant phytase could be used in the agricultural and environmental industries as food and feed additive of livestock, poultry and Fish as well as in medical application.
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Type of Study: Applicable | Subject: Subject 01
Received: 2020/02/2 | Accepted: 2020/02/2 | Published: 2020/02/2

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