Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain.
A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.
Hung
Tung-Hsin
TH
Department of Food and Nutrition, Providence University, Shalu, Taiwan 433-01, Republic of China.
Chang
Ya-Min
YM
Sung
Hsien-Yi
HY
Chang
Chen-Tien
CT
D001963:Bromelains / Q000737:chemistry*; D002688:Chitinases / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D013696:Temperature
D001963:Bromelains; D006867:Hydrolases; D006026:Glycoside Hydrolases; C019868:chitosanase; D002688:Chitinases; C091760:stem bromelain