Study on the antioxidant enzymes 2-Cys and 1-Cys Peroxiredoxin

• Main Authors: Hui-Ming Huang, 黃慧明
• Other Authors: Chi-Tsai Lin
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/33165196534826901828

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 93 === The purpose of this thesis is to study antioxidant enzyme. A full length cDNA of 958 bp encoding a putative 2-Cys Peroxiredoxin (2-Cys Prx) from Antrodia camphorata (A. cam.) fruit body was cloned by polymerase chain reaction (PCR). Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 188 amino acid residues. Computer analysis of its sequence revealing the two cysteines (48 and 165) that form a disulfide bond, was well-conserved among the reported 2-Cys Prx sequences. To further characterize the A. cam. 2-Cys Prx, the coding region was subcloned into an expression vector pAVD 10 and transformed into E.coli BL21 (DE3) pLysS. The expression of the 2-Cys Prx was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme showed two forms by 15 % SDS-PAGE. The enzyme retained 68 % activity after heating at 60 ℃ for 2 min. The enzyme activity was inhibited under acidic pH (pH 2.2). The enzyme activity was only a little decrease under 1 % SDS treatment. The enzyme retained 47 % activity under 0.4 M imidazole treatment. The enzyme showed 42 % activity after 3 h of incubation at 37 ℃ with chymotrypsin. Furthermore, the dimeric enzyme was much resistant to proteolysis after 3 h of incubation at 37 ℃ with trypsin. In addition, the mutants of A. cam. 2-Cys Prx at position 48 、165 or 48 / 165 from Cys to Ser showed lower activity. IV A full length cDNA of 837 bp encoding a putative 1-Cys peroxiredoxin (1-Cys Prx) from A. cam. fruit body was cloned by PCR. Nucleotide sequence analysis of this cDNA revealed that it comprised a complete open reading frame coding for 223 amino acid residues. Computer analysis of its sequence revealing cysteine (46) that hethered with the other subunit to form a disulfide bond was well-conserved among the reported 1-Cys Prx sequences. To further characterize the A. cam. 1-Cys Prx, the coding region was subcloned into an expression vector pET-20b (+) and transformed into E.coli BL21 (DE3) pLysS. The expression of the 1-Cys Prx was purified by Ni2+ -nitrilotriacetic acid Sepharose superflow column. The purified enzyme showed one form by 15 % SDS-PAGE. The enzyme retained 37 % activity after heating at 60 ℃ for 16 min. The enzyme was stable under basic pH (above pH 9.0). The enzyme was inhibited in the present of SDS (above 1 %). The enzyme retained 68 % activity at 0.4 M imidazole. The enzyme retained 15 % activity after 20 min of incubation at 37 ℃ with chymotrypsin. The enzyme retained 9 % activity after 40 min of incubation at 37 ℃ with trypsin.