| Summary: | 博士 === 靜宜大學 === 食品營養學系 === 100 === In this study, fresh-cut bamboo shoots were coated with 0.1% chitosan and stored at room temperature for 12, 24, and 48 h. Storage at room temperature for 12-48 h significantly increased chitinase and chitosanase activities in the outer sheaths and edible portion of the shoots, but most notably in the outer sheath portion. Two chitosanase isoforms, A and B, were purified from the sheaths of 48-h-treated bamboo shoots by following sequential steps of buffer extraction, ammonium sulfate fractionation (40%-80% saturation), Sephacryl S-100 HR gel filtration and preparative Rotofor cell isoelectric focusing electrophoresis. Isoforms A and B had molecular masses of 24.5 and 16.4 kDa (as estimated using SDS-PAGE) and the isoelectric points of 4.30 and 9.22 (as estimated using isoelectric focusing electrophoresis). Using chitosan as the substrate, the optimal pH for the activities of isoforms A and B were 3 and 3-4, and the optimal temperatures were 70 ℃and 60 ℃. The kinetic parameters Km and Vmax for isoforms A were 0.539 mg/mL and 0.262 μmole min-1 mg-1, respectively; for isoforms B, the kinetic parameters were 0.183 mg/mL and 0.092 μmole min-1 mg-1, respectively. The chitosans were susceptible to degradation by both enzymes and could be converted to low molecular weight chitosans between 28.8 and 11.7 kDa. Furthermore, the most susceptible chitosan substrates were 50%-70% and 40%-80% deacetylated for isoform A and B, respectively. Both enzymes could also degrade chitin substrates with lower efficacy. Chemical modification reagents N-bromosuccinimide and Woodward’s Reagent K strongly inhibited both enzymes. Aspartic acid (or glutamic acid) and tryptophan residues were probably located at or near the active sites of both enzymes.
Commercial crab chitosan was hydrolyzed using partially purified bamboo shoot chitosanase or papaya latex preparation at pH 4.0 or 6.5 and 50 ℃for 24 or 18 h. Two major low molecular weight chitosan (LMWC) fractions, Fraction A and Fraction B, were obtained by precipitating the hydrolysate with 50% and then 90% ethanol, respectively. The molecular mass of Fraction A was 344.6-23.0 kDa, whereas that of Fraction B was 56.6-11.7 kDa. Both fractions were soluble in water. However, LMWC fractions obtained using chitosan hydrolysis at pH 4.0 were acidic, whereas LMWC fractions obtained using chitosan hydrolysis at pH 6.5 were basic.
The purified chitosanase isoform A and B could be immobilized on glutaraldehyde-activated magnetic Fe3O4 nanoparticles. After Immobilization, the optimal pH of both isoforms and the optimal temperature of isoform A for chitosan hydrolysis were unchanged; but the optimal temperature of isoform B increased from 60 ℃to 70 ℃. Immobilization significantly increased the thermal stability of both isoforms, but most notably that of isoform B. After re-using for 10 times at 30 ℃ to 80 ℃for chitosan hydrolysis both immobilized isoforms retained 89%-35% of their initial activities. Both immobilized isoforms would be useful in producing low molecular weight chitosan.
|
|---|
