链霉菌壳聚糖酶的纯化及其酶学性质

分离纯化从烟台近海土壤筛选的链霉菌来源壳聚糖酶,并对其酶学性质进行研究。通过(NH4)2SO4分级沉淀分离得粗酶,透析后经Sephadex G-100柱纯化,得到2种壳聚糖酶(ChA和ChB)。SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤确定ChA的相对分子质量,研究ChA的最适底物水解条件、热稳定性、水解动力学及金属离子对酶活性影响。结果表明:ChA为单亚基蛋白,相对分子质量为4.16×104,在220和280 nm处呈现两个紫外吸收峰,催化水解壳聚糖的最适pH为5.0～5.5,最适温度为55℃。热稳定性实验表明:30℃温育1 h后酶活为初始酶活的33.3%,40℃温育1 h后酶活为初始酶活的22.2%。ChA的酶促反应初速率为6.2×10-3μmol/(mL.min),Vmax为0.318μmol/(mL.min),Km为1×10-2mg/mL,且对底物表现相对专一性。K+、Na+、Li+、Mg2+、Ca2+、Ba2+Zn2+、Cu2+和Co2+对ChA活力均表现为抑制作用,过渡金属离子Mn2+对酶有激活作用,重金属离子Hg2+、Ag+、Cd2+和Pb2+对酶均有较强的抑制作用。Mn2+和Zn2+的动力学研究表明,Mn2+对酶为混合型激活作用,Zn2+对酶为竞争性抑制作用。

Purification chitosanase from Streptomyces hygroscopious and its catalytic properties

The chitosanase purified from Streptomyces hygroscopious isolated from the offshore in Yantai, and then properties of chitosanase were investigated to provide a theoretical basis for its application. The crude enzyme of chitosanase was extracted by ammonium sulfate fraction precipitation, and two types of enzyme, （ChA and ChB）, were obtained by gel filtraction chromatography with Sephadex G-100. The catalytic properties of ChA were further studied. The molecular weight of ChA is 41.6 kDa, which was mono-unit protein, and the maximum UV absorbances were at 220 nm and 280 nm. The optimum pH of ChA was 5.0-5.5 when chitosan was used as substrate and the optimum temperature was 55 ℃. Thermal stability experiment showed that 33.3% of activity was retained after heating at 30℃ for 1 h ,22. 2% of activity was retained after heating at 40 ℃ for 1 h. The initial reaction rate of ChA was 6. 2 × 10^-3 μmoL/ （ mL- min）, the maximum rate was 0. 318 μ moL/（ mL. min）, and the Michaelis constant Km was 1.0 × 10^ -2mg/mL. Furthermore, ChA showed relative substrate specificity. K ＋ , Na ＋, Li＋ , Mg2 ＋ , Ca2 ＋ , Ba2 ＋ ,Zn2 ＋ , Cu2 ＋ , and Co2＋ had labile effects on the enzyme, while Mn2 ＋ could activate the enzyme. Theheavy metal ions Hg2 ＋ , Pb2 ＋ , Ag ＋ , and Cd2 ＋ inhibited the enzyme activity. Mn2 ＋ could make a mixed activation effeet on the enzyme,but Zn2＋ displayed a competitive inhibition on the enzyme.