Purification and Characterization of Catalase-Peroxidase from Flavobacterium meningosepticum

• Main Authors: Man-Chun Yu, 余曼君
• Other Authors: Yaw-Kuen Li
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/41790810980867021919

碩士 === 國立交通大學 === 應用化學系 === 87 === A catalase-peroxidase was purified to at least 90% homogeneity from Flavobacterium meningosepticum. The purification involved a series of chromatographic separations on DEAE, HiTrap Q, and phenyl-sepharose columns. The purified enzyme is a dimeric protein with a Mr value of 63 and 128 kDa estimated by SDS-PAGE and gel filtration, respectively. The UV spectrum of the enzyme demonstrates a distinct absorption band withλmax = 406 nm indicating a protoheme present. This enzyme is stable in pH 7 - 10 and posses a pH optimum in this range. Interestingly, it exhibits an unusual thermostability in 50 - 60 ℃. When enzyme was assayed as peroxidase activity, pyrogallol is the only effective substrate with Km = 2.92 mM. The Km value of H2O2 is 41.5 mM when the enzyme functions as catalase. Divalent metal ions, with the exception of Cu2+, and EDTA have no significant effect on catalase activity. 3-amino-1,2,4-triazole, a specific catalase inhibitor, displayed negligible inhibition on the purified enzyme while NaN3 remained effective.