拮抗链霉菌R_(15)β-葡萄糖苷酶的纯化及特性研究

Purification and properties of β-glucosidase from antifungal Streptomyces spp. R_(15)

从山东泰安郊区大白菜植株根际土壤中分离筛选获得一株拮抗链霉菌（Streptomyces spp. ）R15菌株。采用固体发酵、粗酶液 （NH4）2SO4分级沉淀、DEAE-52阴离子交换柱层析和Sephadex G75凝胶柱层析等步骤从链霉菌（Streptomyce spp.）R15中分离纯化获得β-葡萄糖苷酶。结果表明, 纯化倍数16.28, 回收率为14.59％, SDS-PAGE电泳测得相对分子量为63.89 kDa。经TLC分析酶解产物, β-葡萄糖苷酶与水杨苷反应生成D-葡萄糖和水杨醇。以水杨苷为底物时, 该酶的Km值为10.644 mmol/L, Vmax为0.525 μmol·L-1·min-1。该酶的最适温度为65℃, 70℃以下相对稳定;最适pH为5, pH 3~7之间相对酶活在75％以上。Cu2＋、Hg2＋和Fe2＋对酶有抑制作用, 其中Hg2＋和Cu2＋的抑制作用最强, 酶活力完全丧失;而Ca2＋, Mn2＋对酶均有激活作用, 相对酶活分别高达137.18%和119.52%。

The β-glucosidase is an important component of the cellulase complex. It not only hydrolyzes cellobiose and cello-oligosaccharide, but also prevents accumulation of cellobiose and reduces the inhibition of cellulase. By using of solid fermentation, fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-52 and Sephadex G75 chromatography, β-glucosidase from antifungal Streptomyces spp.R15 obained from the chinese cabbage rhizosphere in the suburb of Taian, Shandong Province was purified. The results showed that the purification fold and yield were16.28 and 14.59%, respectively. The molecular mass of the enzyme was estimated to be 63.89 kDa by 12% SDS-PAGE. To make an analysis of the enzyme reaction by TLC, β-glucosidase can react with salicin to form D-glucose and salicyl alcohol, which proved that the protein was β-glucosidase. By using of salicin as substrate, the Km and Vmax values for β-glucosidase were 10.644 mmol/L and 0.525 μmol·L-1·min-1, respectively. The optimum temperature was 65℃, the β-glucosidase was more stable when temperature was lower than 70℃. The optimum pH was 5, the relatively activity was above 75% when pH was between 3 and 7.Cu2＋, Hg2＋ and Fe2＋ could inhibit the activity of β-glucosidase, the relatively activity were 0, 3.60%, 62.98% respectively. The inhibitory effect of Hg2＋, Cu2＋ were the strongest of them, the activity was completely lost. Ca2＋, Mn2＋ could stimulate the activity, the relatively activity were 137.18% and 119.52% respectively. Fe3＋, Ba2＋, Mg2＋, K＋, Al3＋ and Zn2＋ had no significant effect on the activity at the level P〈0.05.