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Iranian Journal of Biotechnology، جلد ۱۸، شماره ۳، صفحات ۱-۱۱
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عنوان انگلیسی Enhancement of Pharmaceutical Urate Oxidase Thermostability by Rational Design of De Novo Disulfide Bridge
چکیده انگلیسی مقاله Background: As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such as
gout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structural
instability 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.
کلیدواژه‌های انگلیسی مقاله Aspergillus flavus,Disulfide bridge,gout,Site-directed mutagenesis,Urate Oxidase
نویسندگان مقاله Leila Rezaeian-Marjani |
Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran

Mehdi Imani |
Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Hossein Zarei Jaliani |
Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

نشانی اینترنتی https://www.ijbiotech.com/article_112897_4b8cf8baeb5cc7fb554fc44db6511d85.pdf
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