Directed evolution of N-carbamoylase and the characterization of the evolved mutants = N-carbamoylase의 방향적 진화 및 진화된 mutant의 특성 연구

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N-Carbamyl-D-amino acid amidohydrolase (N-carbamoylase), which is currently employed in the industrial production of unnatural D-amino acid in conjunction with D-hydantoinase, has low oxidative and thermal stability, and low expression. First, we attempted a simultaneous improvement of oxidative and thermal stability of N-carbamoylase from Agrobacterium tumefaciens NRRL B11291 by directed evolution using DNA shuffling. In a second generation of evolution, best mutant 2S3 with improved oxidative and thermal stability was selected, purified, and characterized. Temperature, at which 50% of initial activity remains after incubation for 30 min was 73℃ for 2S3, while it was 1℃ for wild-type enzyme. Treatment of wild-type enzyme with 0.2 mM hydrogen peroxide for 30 min at $25℃ resulted in a complete loss of activity, but 2S3 retained about 79 % of initial activity under the same conditions. In addition, through the time course inactivation experiment, oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than wild type at 0.2 mM $H_2O_2, and it also showed an 8- fold increased thermostability at 70℃. The Km value of 2S3 was estimated to be similar to that of wild-type enzyme; however $k_{cat}$ was decreased, leading to a slightly reduced value of $k_{cat}$ /Km compared to wild-type enzyme. DNA sequence analysis revealed that six amino acid residues were changed in 2S3, and substitutions included Q23L, V40A, H58Y, G75S, M184L, and T262A. Stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Q23L, H58Y, M184L, and T262A were found to enhance both oxidative and thermal stability of the enzyme, and of them, T262A showed the most significant effect. Meanwhile, V40A and G75S gave rise to an increase only in oxidative stability. The positions of the mutated amino acid residues were identified in the structure of N-carbamoylase from Agrobacterium sp. KNK 712 and structural analysis of t...
Kim, Hak-Sungresearcher김학성researcher
한국과학기술원 : 생물과학과,
Issue Date
177212/325007 / 000985218

학위논문(박사) - 한국과학기술원 : 생물과학과, 2002.8, [ vii, 90 p. ]


enzyme stability; D-amino acid; N-carbamoylase; Directed evolution; protein exrpession; 단백질 발현; 효소안정성; D-아미노산; N-카바모일라아제; 방향적 진화

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