Purification and properties of beta-N-Acetylhexosaminidase from cabbage.
beta-N-Acetylhexosaminidase was purified from the extract of cabbage by sequential steps of ammonium sulfate fractionation, chromatofocusing, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 256 fold with a recovery of 8%. The purified enzyme was homogeneous as examined by native PAGE. It showed an optimal pH of 4, an optimal temperature of 60 degrees C and a Km of 0.94 mM for hydrolysis of pNp-beta-GlcNAc. The molecular mass of the enzyme determined from filtration through Sephacryl S-200 was 150 kDa. Three subunits with molecular mass of 64, 57 and 51 kDa were observed as determined by SDS-PAGE. NBS (0.025 mM), DEPC (3 mM) and WRK (30 mM) significantly inhibited the activity of the enzyme. The enzyme also showed activity toward pNp-beta-GalNAc, N,N'-diacetylchitobiose, N,N',N"-triacetylchitotriose and N,N',N",N"'-tetraacetyl chitotetraose but showed no activity toward pNp-alpha-GlcNAc, chitin and ethylene glycol chitin.
Department of Food and Nutrition, Providence University, Shalu, Taiwan, R.O.C.
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Chang
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Young
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Chang
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Sung
D001937:Brassica / Q000201:enzymology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D001619:beta-N-Acetylhexosaminidases; D004047:Diethyl Pyrocarbonate