| Summary: | 碩士 === 靜宜大學 === 食品營養研究所 === 92 === Abstract
Chitinase and chitosanase activities were found in pea (Pisum sativum L. Taichung No. 14 ) seeds during germination. Chitosanase was purified from 4 days germinated pea seeds by sequentially applying buffer extraction, 80 % saturation ammonium sulfate fractionation, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography and Superdex 75 HR gel filtration. Using these steps, the purity of the enzyme was increased 52.5 fold with a yield of 7.6 %. The chitosanase had an optimal pH of 4 and an optimal temperature of 60℃ for chitosan hydrolysis. The molecular mass was 29 kDa, as estimated by gel filtration. This value was close to that estimated by SDS-PAGE , indicating the enzyme to be monomer. Metal ion Mn2+（10 mM）and chimical modification agent N-bromosuccinimide (NBS)（5 mM）significantly inhibited the enzyme activity. This enzyme showed activity toward chitosans of varying N-acetyl content (20 to 90 % deacetylation ). Most effectively hydrolyzed were chitosan polymers that were 50 to 60 % deacetylation. With respect to hydrolysis of chitosans with different molecular mass , the enzyme activity decreased as chitosan molecular mass decreased. From the substrate specificity examined, the enzyme also showed activity toward chitin and p-nitrophenyl N-acetyl chito-oligosaccharides, indicating the existence of chitinase in the purified enzyme. End products of chitosan hydrolysis by the enzyme were chito-oligosaccharides. The components of the chito- oligosaccharides were monomer to hexamer and some chito-oligosaccharides had longer chain lengths.
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