绿色木霉产壳聚糖酶的分离纯化及酶学性质

Purification and Property Investigation of Chitosanase From Trichoderma viride

对绿色木霉所产的壳聚糖酶进行分离纯化,结果表明当壳聚糖酶经过20%和60%的硫酸铵分级沉淀、透析、超滤浓缩、苯乙烯系阴离子交换柱以及Sephdex G-75层析柱的分离纯化,发现该酶的纯化倍数达到29.13倍,回收率达到53.9%,经SDS-PAGE电泳鉴定该酶达到了电泳纯,其分子质量为51.235 ku,最适温度为50℃,最适pH值为5.2,具有较强的底物特异性,K+和Mn2+对酶有一定的激活作用,Mg2+、Fe3+、Ca2+和Pb2+的影响不明显,Zn2+、Cu2+和Hg2+对酶有较强的抑制作用。通过Lineweaver-Burk法求得动力学参数,该酶Km值为0.048 mg/mL,Vmax值为0.6 mg.L-1.min-1。据酶解产物薄层层析实验分析其水解产物,说明此酶主要以内切方式作用于壳聚糖,该酶既能水解GclN-GlcN糖苷键,也能水解GlcNAc-GlcN糖苷键。

Chitosanase from Trichoderma viride was purified through a series of procedures including fractional precipitation of （NH4）2SO4 with 20% and 60% concentration, dialysis, concentration by ultratqhmtion, styrene ion exchange and Sephadex G - 75 gel filtration chromatography, and the enzymology of the purified ehitosanase was investigated. Re- suits show the purification fold of the enzyme is 29.13 and the yield is 53.9%. The molecular weight of the ehitosanase determined by SDS- PAGE is 51.235 ku and the optimum temperature and pH for the chitosanase action is 50 ℃ and 5. 2, respectively. The chitosanase displays ehitosan substracte specificity; K^＋ and Mn^2＋ enhance the enzyme activity and Mn^2＋, Fe^3＋, Ca^2＋ and Pb^2＋ affect the activity indistinctly, whereas Zn^2＋, Cu^2＋ and Hg^2＋ inhibit the activity. By Lineweaver - Burk method, the kinetic parameter Km is 0. 048 mg/mL and Vmax is 0.6 mg·L^-1. min^-1. It is suggested based on the analysis of the hydrolysate by TLC that this enzyme is an endo - type ehitosanase and cleave both GclN - GleN and GlcNAc - GleN linkage.