Stabilization of D-hydantoinase by intersubunit-crosslinking and characterization = Subunit 간의 crosslinking에 의한 D-hydantoinase의 구조 안정화 및 특성연구

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D-Hydantoinase is a multimeric enzyme that is active and stable in dimer form but easily deactivated when it is dissociated into monomers. Many multimeric enzymes and proteins undergo unstable monomer stage in the deactivation. D-Hydantoinase was crosslinked into dimer by EDC (1-Ethyl-3-(3-Dimenthylaminopropyl) carbodiimide Hydrochloride) to investigate if these multimeric enzymes could be stabilized by solidification of their multimeric structure with crosslinker. The cross-linked D-hydantoinase was decreased a little activity but showed remarkable improvement in stability to Heat, low pH and organic solvent. Cross-linked D-hydantoinase was not neary inactivated at 70℃ for 30 minutes and exhibited 60% of initial activity at 55℃ in 25 hours but native enzyme lost 40% of initial activity at 55℃ for 30 minutes. Moreover, 50% of native enzyme was deactivated at 50 mM phosphate buffer, pH 6.0 in 1 hour but 80% of crosslinked enzyme was active in same condition. In order to increase solubility of substrate including HPH (Hydroxyphenyl Hydantoin), organic solvent was added to reaction mixture. HPH was solved in reaction solution containing 20% methanol about three folds larger amount than in the solution without methanol. Furthermore, the cross-linked D-hydantoinase was very stable in the present of 20% methanol. Only 20% of the enzyme was deactivated at 55℃ for 4 hours in reaction mixture with 20% methanol but only 26% of native enzyme was active at same condition. WhenHPH and Hydantoin among substrates were converted for about 15 hours, N-carbamoyl D-amino acid of the product was generated by more 56% with cross-linked enzyme than with native enzyme.
Park, Tae-Gwanresearcher박태관researcher
한국과학기술원 : 생물과학과,
Issue Date
151585/325007 / 000973675

학위논문(석사) - 한국과학기술원 : 생물과학과, 1999, [ iv, 43 p. ]


protein engineering; crosslink; EDC; enzyme; 안정성; 효소; 단백질 공학; stability

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