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  • 03월 10일 13시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능

대한화학회 제105회 학술발표회 및 총회 Protein engineering of Coprinus cinereus peroxidase for the application to polymerization of phenolics

등록일
2010년 2월 19일 12시 19분 35초
접수번호
1289
발표코드
금13C3심 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
발표시간
금 10시 : 20분
발표형식
심포지엄
발표분야
생명화학 - 단백질 과학의 생화학적 접근
저자 및
공동저자
김용환
광운대학교 화학공학과, Korea
The peroxidase has been commonly used in many biotechnology fields, from fine chemical synthesis to environmental clean-up. The peroxidase catalyzes the oxidation of phenolic compounds. Two equivalents of phenol are converted into a highly reactive radical species by each consuming one equivalent of hydrogen peroxide. The CiP was significantly inactivated during the oxidation of phenolic compounds. Conversely, the CiP nearly maintained its initial activity for the oxidation of syringic acid, vanilic acid and ferulic acid. The thermodynamic parameter (ΔΔG_f298K) and turnover capacity (ΔS⁄ΔE) were adapted to explain the CiP inactivation due to covalent bonding between the enzyme and phenolic compounds. In the cases of syringic acid, vanilic acid and ferulic acid, which maintained high residual CiP activities after reaction, the ΔΔG_f298K^0 were more negative and the turnover capacities were higher than the other values. This means that these compounds prefer to form a dimer rather than an enzyme-phenolics complex. Among the inactivation factors, the formation of covalent bonding between the enzyme and phenolic radicals was concluded to be the main mechanism for the inactivation of CiP. The sequence of fragment from inactivated CiP (m/z 838.880) was KGTTQPGPSLGFAEELSPFPGEFRM (residues 219-241, underlined residue F229) and was covalently modified with molecular phenol, whereas a fragment of native CiP (m/z 1211.621) was not. Various mutants of the F229 residue of CiP were constructed to investigate the possibility to block the inactivation caused by radical coupling of phenols with the Phe residue through site directed mutation and expressed in Pichia pastoris by using pPicZA as expression vector. The F229A mutant showed a higher turnover capacity (14 fold higher than that of wild-type) and both the F229I and F229L mutants showed an increase in the turnover capacity of 5 fold. Herein, we proposed that the major inactivation mechanism for CiP is the attack of the F229 residue of CiP by the radical. This attack can form a covalent bond between the phenolic radical and the enzyme. The modified F229 with the phenol polymer at the entrance of active pocket then blocks the active center, which may be called “molecular clothing or shielding”. This work provides a more detailed understanding of the suicide inactivation of peroxidase and suggests the strategy for the development of stable peroxidase against the oxidation of enzyme itself. The radical-resistant robust peroxidase can be efficiently used in diverse fields, from the analytical diagnostic field as a key component of biosensors and immunoassays to the industrial processes as a biocatalyst in the synthesis of fine chemicals including phenolic polymers.

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