Purification and properties of chitinase from cabbage stems and roots

• Main Authors: Hsueh Yi-Ling, 薛伊玲
• Other Authors: Chang Chen-Tien
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/20219019073885186177

碩士 === 靜宜大學 === 食品營養學系 === 82 === Chitinase has been purified from the extract of cabbage stems and roots through successive steps of ammonium sulfate fractionation, Sephadex G-75 gel filtration, chromatofocusing, and Sephacryl S-200 gel filtration. By these steps, the purity of the enzyme increased by 63 fold and the recovery of the enzyme was 18%. The purified enzyme was homogeneous as examined by SDS-polyacrylamide gel electrophoresis. The purified chitinase had an optimal pH of 6, an optimal temperature of 60 degrees centigrade. The enzyme had a Km value of 86 .mu.M for hydrolysis of ethylene glycol chitin and showed a substrate inhibition at high concentration of ethylene glycol chitin. The molecular weight of the enzyme determined by SDS-Polyacrylamide gel electrophoresis was 41,000 daltons. Heavy metal ions (1.5 mM), Ag+, Hg2+ and Fe2+, significantly inhibited the activity of the enzyme. N-.beta.romosuccinimide (NBSI; 0.5 mM) and 1, 2-cyclohexanedione (CHD; 0.5 mM) also inhibited the activity of the enzyme. The enzyme showed activity for hydrolysis of ethylene glycol chitin, colloidal chitin and native chitin. Howerever, it had no action on p-nitrophenyl- .beta.-D-N, N''- diacetyl chitobiose and p-nitrophenyl-.beta.-D- N, N'', N"- triacetyl chitotriose. It also showed no muramidase activity for hydrolysis of Micrococcus lysodeiktius. Bovine serum albumin (0.05 %), Triton X-100 (0.5 %) and glycerol (50 %) provided significant protection of the enzyme from freezing inactivation.