枇杷果肉酶促褐变的研究——I.枇杷果肉中多酚氧化酶的分离、纯化和动力学特征

Studies on the Enzymic Browning of Loquat Fruit I.Purification of polyphenol oxidase ＆ its property

枇杷果肉制成丙酮粉,经硫酸铵分级沉淀、透析,DEAE-纤维素柱层析、ACA_(34)柱层析和FPLC分离等方法,分离和纯化得到多酚氧化酶的两个同工酶(酶A和酶B).酶B由一个或几个分子量为42×10~3D的亚基组成.酶的动力学研究表明:酶A、酶B的K_m(多巴胺为底物)均为2.40×10^(-3)mol/L,以邻苯二酚为底物时,酶A和酶B的K_m分别为0.163×10^(-3)mol/L和1.54×10^(-3)mol/L.酶底物特异性表明,酶B是邻位二酚酶,不能作用于单酚和间位二酚.

Two isozymes of polyphenol oxidases in the acetone powder of loquat (Eriobotrya japonica. Lind. )wereextracted and purified through (NH_4)_2SO_4 fraction, dialysis and chromatography (FPLC) separation. Thesetwo enzymes (enzyme A & enzyme B) were purified to one band on polyaerylamide dis-gel electrophoresis.The subunit weight of enzyme B was 42 000 D as determined by SDS-gel electrophoesis. Enzyme B has cate-cholase activity but has no activity towards monophenols. The K_m values were 0. 613 mmol/L (catechol),2. 4Cmmol/L(dopamine)for enzyme A and 1.539 mmol/L(cateebol),2. 375 mmol/L(dopamine) for enzyme B.