Studies on the Purification and Properties of N-acetyl-β-D-glucosaminidase from Cabbage

• Main Authors: Young, Fong-Ping, 楊鳳平
• Other Authors: Chang,Chen-Tien
• Format: Others
• Language: zh-TW
• Published: 1992
• Online Access: http://ndltd.ncl.edu.tw/handle/wuxq39

碩士 === 靜宜大學 === 食品營養學系 === 80 === N-acetyl-β-D-glucosaminidase was purified from cabbage by sequential steps of buffer extraction,ammonium sulfate fractionation. Sephadex G-75 gel filtration, chromatofocusing, and Sepharose CL-6B gel filtration. By these steps, the purity of the enzyme increased by 358 folds, the recovery of activity was 17.4％. The purified enzyme was homogeneous as examined by polyacrylamide gel electrophoresls. The purified N-acetyl-β-D-glucosaminidase had an optium pH of 4.0, optium temperature of 60°C, Km value of 0.94 mM, activation energy of 10.2 Kcal/mole, activation energy of thermal inactivation of 8.9 Kcal/mole for hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminde. The enzyme showed molecular weight of 151,000 as determined by Sepharose CL-6B gel filtration. One subunit with molecular weight of 21,000 was observed as determined by SDSpolyacrylamide gel electrophoresis. Heavy metalions(O.06mM) , Ag+ and Hg+2, significantly inhibited the activity of the enzyme. N-Bromosuccinimide (NBSI; O.06mM), phenylmethylsulfonyl fluoride (PMSF;O.6mM), and iodine (O.6mM) also inhibited the activity of the enzyme. The enzyme showed high activity for hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide but had no action on p-nitrophenyl-N-acetyl-α-D-glucosaminide. The enzyme also hydrolyzed p-nitrophenyl-N-acetyl-β-D-galactosaminide and oligochitin (2-4)with rather low activity. From the chemical modification and Dixon-Webb kinetic studies, the amino acid residues, histidine, tryptophan , tyrosine, and serine are probably located at or near the activite site of the enzyme.