| Summary: | 碩士 === 靜宜大學 === 食品營養研究所 === 98 === A chitinase was purified from lyophilized powder of mature papaya seeds through successive steps of buffer extraction, ammonium sulfate precipitation, Sephacryl S-100 HR gel filtration, Polybuffer exchanger PBE 94 chromatofocusing and Sephacryl S-100 HR gel filtration. By these steps, the purity of the enzyme was increased 152 fold and the recovery of enzyme activity was 17.5％. The purified chitinase catalyzed the hydrolysis of both chitin and chitosan. The optimum pH for CM-chitin hydrolysis was 4.5, the optimum temperature was 50℃, the Km was 1.88 mg/mL and the Vmax was 1.08 μmol GlcNAc/min/mg, whereas the optimum pH for AE-chitosan hydrolysis was 6, the optimum temperature was 50℃, the Km was 1.20 mg/mL and Vmax was 0.130 μmol GlcN/min/mg. The purified chitinase was thermally stable after holding at 30-50℃ for 60 min. However, at temperatures higher than 50℃, the enzyme activity decreased significantly. The initial heat inactivation of the enzyme followed first-order reaction kinetics. The half-life values of thermal inactivation for the enzyme varied from 51.5 to 3.4 min at 60-70℃. The isoelectric point of the enzyme was pH 4.6, as estimated by isoelectric focusing electrophoresis and zymogram staining. The molecular mass of the enzyme was 31.6 kDa, as estimated by gel filtration. This value was close to that estimated by SDS-PAGE (33.1 kDa), indicating the purified chitinase was a monomeric enzyme. The purified chitinase was deemed to be a class III chitinase because it was recognized by a polyclonal antibody raised against 30 kDa chitinase from jelly fig achene, as examined by Western immunoblotting. The purified enzyme was not fully homogeneous, there was some contamination with trace amounts of a minor isoform of chitinase (28.3 kDa), as analyzed by SDS-PAGE and chitinase activity staining. The minor isoform was not recognized by anti- (jelly fig chitinase) antibody. Chemical modification agents, including diethyl pyrocarbonate (2.5 mM), Woodward’s reagent K (50 mM), N-bromosuccinimide (5 mM) and N-acetylimidazole (2.5 mM) significantly inhibited the activity of the purified chitinase, implying that imidazole groups from histidine, carboxyl groups from aspartic acid and glutamic acid, indole groups from tryptophan and phenolic groups from tyrosine were located at or near the active site of the enzyme. The purified chitinase catalyzed the hydrolysis of chitin, chitosan and their derivatives. However, the enzyme had greater activity toward chitin than toward chitosan. The major product of ethylene glycol chitin hydrolysis catalyzed by the enzyme was chitin oligomer with chain length of 4.
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