Purification and biochemical studies of chitinase from rice TG9 suspension cells

• Main Authors: Ching-Ying Tseng, 曾競穎
• Other Authors: Hsien-Yi Sung
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/41705879434185148782

碩士 === 國立臺灣大學 === 農業化學研究所 === 91 === Exochitinases and endochitinases were assayed for chitinase activity by SDS-PAGE activity staining in gels containing 4-methylumbelliferyl-β-D-N,N’,N’’-triacetychitotrioside [4-MU-(GlcNAc)3] or ethylene glycol chitin (EGC) embedded as substrate. Several chitinase isoenzymes were presented in a rice (Oryza sativa, cv Taikeng 9) cell suspension culture and detected in the medium during suspension cells growth. Three of them, designated as ch A, ch B and ch C were isolated from the culture filtrate to homogeneity by 40 - 80﹪ ammonium sulfate precipitation, Sephacryl S-100, Mono P and Mono Q column chromatography. By SDS-PAGE activity staining, two endochitinases ch C, ch B and exochitinase ch A had a molecular mass of 29 , 26 and 67 kD, respectively. The optimal temperature of ch A was 60℃ and ch C was 50℃. The optimal pH of ch A, ch B and ch C was 3. The isoelectric point estimated by chromatofocusing was 5-6 (ch A) and 5 (ch B and ch C). For hydrolysis of 4-MU-(GlcNAc)3, Km and Vmax of exochitinase ch A were 2.85 μM and 0.22 μmole/min/mg, respectively. Heavy metal ion of Hg2+ and Woodward’s reagent K significantly inhibited exochitinase ch A activity suggest that ch A belongs to family 18 or family 19 of the glycosyl hydrolases. Using Q-Tof-MS for ch B, amino acid sequencing, the results indicated that endochitinase ch B contains the sequence of FASIAPFGNAEVQR which was found in rice (Oryza sativa, cv Nakdong) class Ⅲ chitinase RCB4.