| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === Abstract
The purpose of this thesis is to study antioxidant enzyme. A full-length cDNA with 764 bp encoding a putative Glutathione peroxidase (GPx) from Antrodia camphorata was cloned. Nucleotide sequence analysis indicated that it comprises a complete open reading fram coding for 159 amino acid residues. The deduced amino acid sequence showed high similarity (31%~61%) with that of GPx from other species. Computer analysis revealing the Cys-36 was well-conserved among the all reported GPx sequence. To further characterize the A. cam GPx, the coding region was subcloned into an expression vector, pYEX-S1, and transformed in Saccharomyces cerevisiae (CK3). The expression of the GPx was purifieded by Ni2+-nitrilotriacetic acid Sepharose superflow. The A. cam GPx enzyme remained about 50% activity after heating 60℃for 8 min. The half-life was 8.5 min. The enzyme was inhibited under acidic pH (2.2) and 0.2 M imidazole. The enzyme remained about 50% activity 1% SDS, and it had much resistant to trypsin attack than chymotrypsin.
As above, a full-length cDNA with 1794 bp encoding a putative catalase from Antrodia camphorata was cloned. Nucleotide sequence analysis indicated that it comprises a complete open reading fram coding for 509 amino acid residues. The deduced amino acid sequence showed high similarity (47%~55%) with that of catalase from other species. Computer analysis revealing the His-63 was well-conserved among the all reported catalase sequence. To further characterize the A. cam catalase, the coding region was subcloned into an expression vector, pET-20b(+), and transformed in E. coli BL21(DE3)pLysS. The expression of the catalase was confirmed by enzyme activity stained on a native PAGE and was purifieded by Ni2+-nitrilotriacetic acid Sepharose superflow. The A. cam catalase enzyme remained about 50% activity after heating 60℃for 15 min. The half-life was 14 min. The enzyme was inhibited under acidic pH (below 5.4) and 0.4 M imidazole. The enzyme remained about 50% activity 0.5% SDS, and it had much resistant to trypsin attack than chymotrypsin.
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