【摘要】 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。更多还原

【Abstract】 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 μmol/(mL·min),and the Michaelis constant Km was 1.0×10-2 mg/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.The heavy metal ions Hg2+,Pb2+,Ag+,and Cd2+ inhibited the enzyme activity.Mn2+ could make a mixed activation effect on the enzyme,but Zn2+ displayed a competitive inhibition on the enzyme.更多还原