Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine.
Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China.
Xi
X
Cheng
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China.
Xingjian
X
Huang
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China.
Siyu
S
Liu
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China.
Mi
M
Tang
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China. Electronic address: wanfenghu@mail.hzau.edu.cn.
Wanfeng
W
Hu
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China.
Siyi
S
Pan
D004156:Catechol Oxidase / Q000737:chemistry* / Q000302:isolation & purification; D002957:Citrus / Q000201:enzymology*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D013696:Temperature
D006023:Glycoproteins; D010940:Plant Proteins; C053376:pathogenesis-related proteins, plant; C058814:germin; D004156:Catechol Oxidase
Enzyme activity; Germin-like protein; Polyphenol oxidase; Structure characterization