Department of Life Sciences

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Browsing Department of Life Sciences by Author "Rabbia Muzaffar"

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    CLONING, EXPRESSION AND CHARACTERIZATION OF TREHALOSE SYNTHASE FROM PYROBACULUM CALIDIFONTIS
    (University of Management and Technology Lahore, 2017) Rabbia Muzaffar
    Trehalose is a multifunctional disaccharide sugar which is produced in different organisms like plants, animals, fungi and other microbes. It is known for its ability to preserve the important proteins and biomolecules of organisms inside the body under stress conditions i.e. temperature fluctuations and desiccation. Due to this potent nature, it can be used in pharmaceutical, food and cosmetic industries as stabilizer. It is difficult to attain this commercially valuable product from those organisms in high amount. Therefore, enzymes can be used for in vitro synthesis of trehalose at large scale. The most efficient enzyme for the production of trehalose is the Trehalose synthase, a glycosyltransferase. It involves a one-step pathway that convert maltose into trehalose. The enzyme of trehalose synthase was successfully cloned and expressed from Pyrobaculum calidifontis. The ORF of the gene was Pcal_1359. Thegene was expressed in E. coli expression system through pET-21a (+) vector. The protein was produced as insoluble inclusion bodies. That was refolded by various strategies. The considerable activity was observed by refolding with Urea. The 3D structure was not available, therefore it was made by homology modelling technique. Furthermore, the structure was refined by Molecular dynamics (MD) simulation studies that have indicated that the enzyme can survive at high temperature range from 300K to 400K. The docking studies was also performed by using maltose as substrate and acrabose, cathomycin and ValidamycinA as inhibitors. The active site residues of Pcal_1359 was Asp119, His139, Arg222, and Glu308. The binding affinity was highest with cathomycin that indicated it as a potential inhibitor for the enzyme.