TY - JOUR ID - 112897 TI - Enhancement of Pharmaceutical Urate Oxidase Thermostability by Rational Design of De Novo Disulfide Bridge JO - Iranian Journal of Biotechnology JA - IJB LA - en SN - 1728-3043 AU - Rezaeian-Marjani, Leila AU - Imani, Mehdi AU - Zarei Jaliani, Hossein AD - Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran AD - Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran AD - Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Y1 - 2020 PY - 2020 VL - 18 IS - 3 SP - 1 EP - 11 KW - Aspergillus flavus KW - Disulfide bridge KW - gout KW - Site-directed mutagenesis KW - Urate Oxidase DO - 10.30498/ijb.2020.2662 N2 - Background: As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such asgout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structuralinstability most likely due to its subcellular and fungal origin.Objectives: In this research, by using rational design and introduction of de novo disulfide bridge in urate oxidase structure,we designed and created a thermostable urate oxidase for the first time.Materials and Methods: Utilizing site-directed mutagenesis and only with one point mutation we constructed two separate mutants: Ala6Cys and Ser282Cys which covalently linked subunits of enzyme each other. Single mutation to cysteine created three inter-chain disulfide bridges and one hydrogen bond in Ala6Cys and two disulfide bridges in Ser282Cys. Results: Both mutants showed 10 °C increase in optimum activity compared to wild-type enzyme while the Km values for both increased by 50% and their specific activity compromised. The thermal stability of Ser282Cys increased remarkably by comparing Ala6Cys and wild-type enzymes. Estimation of half life for wild-type enzyme demonstrated 38.5 min, while for Ala6Cys and Ser282Cys were 138 and 115 min, respectively. Interestingly, the optimal pH of both mutants was broaden from 7 to 10, which could make them candidates for industrial applications. Conclusion: It seemed that introducing disulfide bridges resulted in local and overall rigidity by bringing two adjacent sites of enzyme together and decreasing the conformational entropy of unfolding state is responsible for the enhancement of thermostability. UR - https://www.ijbiotech.com/article_112897.html L1 - https://www.ijbiotech.com/article_112897_4b8cf8baeb5cc7fb554fc44db6511d85.pdf ER -