假单胞菌ⅧT39壳聚糖酶的纯化和性质研究

PURIFICATION AND THE PROPERTIES OF CHITOSANASE FROM PSEUDOMONAS SP Ⅷ T 39

粗酶液经硫酸铵盐析、透析、脱盐、SephadexG 1 0 0柱层析 ,假单胞菌ⅧT 39壳聚糖酶被纯化了 1 9 72倍 ,SDS -PAGE鉴定为一条带 .试验表明 ,该酶为内切型水解酶 ,无CMCase活性 ,最适pH5 .6～ 6.0 ,最适作用温度 60～ 70℃ ,纯酶作用于可溶性壳聚糖的米氏常数为 1 67mg/mL ,最大反应速度为 2 5 6μmol/mL min .SDS PAGE测定的相对分子质量为 5 1 3× 1 0 3.Mn2 +、AL2 +、Co2 +对该酶有激活作用 ,Ag+、Cu2 +、Fe2 +、Fe3+、Zn2

The chitosanase from pseudomonas sp. Ⅷ T 39 is purified about 20-fold by ammonium sulfate fractionation,dialysis and Sephadex G-100 gel filtration.The purified enzyme is demonstrated by SDS-PAGE to be a homogeneous protein.It showed that the enzyme is an endo splititing type and non CMCase activity.The optimal conditions are pH5.6～6.0 and 60～70℃ respectively.The Km and Vmax of the enzyme for soluble chitosan are 1.67 mg/mL and 2.56mol/mL·min respectively.Its MV is about 51.3×10 3 by SDS-PAGE.Mn 2+ ,Al 3+ ,Co 2+ enhance the enzyme activity,whereas Ag +,Cu 2+ ,Fe 2+ ,Fe 3+ ,Zn 2+ inhibit the enzyme activity significantly.