Purification and Characterization of Peptide:N-Glycanase from Pig Sperm

• Main Authors: Shiou-Ming Fann, 范修明
• Other Authors: Su-Fang Chien
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/48714446499176028078

碩士 === 淡江大學 === 化學學系 === 88 === The purpose of this experiment is to isolate and purify the oligosaccharide chain—cleaving enzyme peptide:N-glycanase from pig sperms.methods：extract the peptide:N-glycanase from pig sperms and the clear solution was freeze-dry. Then followed by chromatographies: DEAE-Sephadex A-50, Sephadex G-100 column, hydrophobic interaction chromatography: Phenyl-Sepharose. PNGase can be purified to at least 800-fold. The physical properties of this enzyme are: it's molecular weight is about 67,000 was determined by SDS polyacrylamide gel electrophoresis; pH optimum is at pH5.8; enzyme is very stable at pH between 4.6－9.2; Km by synthetic substrate is 0.36 mM; The hydrolysis of the substrate is proportional to the increasing of incubation time when p-nitroanilinylaspartic acid substrate was used. The purified enzyme doesn't contain other glycosidases such as β-N-acetylglucosamindase.α-L-fucosidase.α-mannosidase and protease. We also used nature dansylated substrates such as asialofetuin glycopeptide. ovalbumin glycopeptide and other glycopeptide substrates. The hydrolysis was observed by thin layer chromatography.Fetuin was quickly hydrolyzed in 30 min while ovalbumin glycopeptide needs overnight to hydrolyzed with the same amount of substrates and enzyme. Side chain free α1- acid glycopeptide & ovalbumin CNBr derived glycopeptide is completely hydrolyzed after overnight incubation in the mixtures contain suitable amount of enzyme and substrates.