Characterization of a chitosanase isolated from a commercial ficin preparation.
A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).
Chiang
Chui-Liang
CL
Department of Food Science, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan, Republic of China.
Chang
Ya-Min
YM
Chang
Chen-Tien
CT
Sung
Hsien-Yi
HY
D001974:Bromosuccinimide / Q000494:pharmacology; D002688:Chitinases / Q000378:metabolism; D048271:Chitosan / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005361:Ficain / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases; D005361:Ficain; D001974:Bromosuccinimide