链霉菌壳聚糖酶的纯化及其酶学性质

摘要

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 × 10-3μ mol/(mL· min),the maximum rate was 0.318 μ mol/(mL· min),and the Michaelis constant Km was 1.0 × l0-2mg/mL.Furthermore,ChA showed relative substrate specificity.K +,Na +,Li +,Mg2 +,Ca2 +,Ba2 +,Zn2 +,Cu2 +,and Co2 + had labile effects on the enzyme,while Mn + 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.%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1h后酶活为初始酶活的33.3％,40℃温育1h后酶活为初始酶活的22.2％.ChA的酶促反应初速率为6.2×10-3 μmol/(mL· min),Vmax为0.318 μmol/(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.