假单胞菌H3壳聚糖酶的纯化及部分酶学性质

对 Pseudmona sp.H3产生的壳聚糖酶粗酶液采用(NH_4)_2SO_4盐析、Sephadex G-25脱盐、Sepharose Q-XL阴离子交换层析和Superdex G-75分子筛层析进行纯化,经SDS-PAGE鉴定为单蛋白带,分子质量约为33.8ku,酶反应最适温度为40℃,最适pH为5.0,降解壳聚精(D.A.90.14％)Km值为3.59g/L,V_(max)值为 3.80mmol/(L·min);Ba~(2+)、K~+、Co~(2+)对该酶有激活作用,而Zn~(2+)、Mn~(2+)、Al~(3+)、Cu~(2+)则对酶有抑制作用;此外,该酶除了能降解壳聚糖以外,还具有CMCase活性。

Characteristics and Purification of Chitonsanase from Pseudomona sp.H3

The crude enzyme of chitosanase from Pseudomona sp. H3 was purified using 90% sat- uration of ammonium sulfate, Sephadex G - 25 column, ion-exchange chromatography of Sepharose Q -XL column, and gel filtration of Superdex G - 75 column. The purified chitosanase was homoge- neous on SDS-PAGE and its molecular mass was 33.8 ku. The enzyme is at its optimal activity under 40℃ temperature, and pH 5.0. Its kinetic parameter K_m is 3.59g/L and V_(max) is 3.80mmol/ (L·min) for chitosan(D. A. 90.14%) hydrolysis. Metal ions of Ba~(2+), K~+ and Co~(2+) can activate the enzymatic, but Zn~(2+), Mn~(2+), Al~(3+) and Cu~(2+) can inhibit the activity of the enzyme.