球孢白僵菌胞外壳聚糖酶的纯化和性质

PURIFICATION AND PROPERTIES OF AN EXTRACELLULAR CHITOSANASE FROM BEAUVERIA BASSIANA

球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀，SephadexG-75凝胶过滤，Chitosan-bead亲和层析，第二次SephadexG-75凝胶过滤，得到电泳纯的一种胞外壳聚糖酶，比活力达到45u/mg。此酶的分子量为36kD；最适酶反应温度为60℃；最适pH为4.0；最适离子强度为0.25mol/LNaCl;37℃以下，pH2.0-5.0之间稳定性好；Cu^2+,Hg^2+,Pb^2+,Ni^2+对该酶有强烈抑制作用；Ag^+,Mn^2+也有较强抑制作用；Fe^2+有轻微激活作用，该壳聚糖酶是一种糖蛋白，含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖；也能轻微水解CMC、DEAE-Cellulose和胶体几丁质；但不能水解片状的壳聚糖和几丁质。

An extracellular chitosanase from the culture supernatant of Beauveria bassiana 1316-V1 was purified to an apparent homogeneity by SDS-PAGE through ammonium sulfate fraction, Sephadex G-75 chromatography, chitosan-bead affinity chromatography, and the second Sephadex G-75 chromatography. The specific activity of purified enzyme reached 45u/mg.The chitosanase had a molecular mass of 36 kD, and showed a maximum activity at 60℃, pH 4.0, and 0.25mol/L NaCl. The enzyme was stable below 37℃ and pH 2.0 ~ 5.0. The chitosanase activity was strongly inhibited by Cu2+, Hg 2+, Pb2+, and Ni2+; also markedly inhibited by Ag+and Mn2+; but slightly activated by Fe2+. The chitosanase is a kind of glycoprotein, and it contains about 12.6% of carbohydrate. The enzyme didn, t degraded flaked chitosan and chitin, but showed a little activity toward CMC, DEAE-Cellulose, and colloid chitin; the most susceptible substrate was 90% deacetylated chitosan.