一株绿色木霉产外切β-葡聚糖苷酶条件及其纯化与性质的研究

Isolation, purification and characterization of exo-1,4-β-D-glucanase produced by a strain of Tridchoderma viride sp.

从树林中腐烂木块上采集并筛选出一株分泌外切β葡聚糖苷酶(exo1,4βD glucanaseorcellobiohydrolase,CBH)的绿色木霉菌株.在100mL的锥形瓶中装入40mL培养基的产酶状况最好,其通气量最佳,培养液初始pH为8时绿色木霉产生的CBH活力最高.随绿色木霉生长时间的延长,CBH的活力与培养基中的葡萄糖含量呈交替的一高一低的波浪状变化.通过硫酸铵分部分离、葡聚糖G100凝胶过滤、DEAE纤维素52阴离子交换柱层析等方法使绿色木霉所产的CBH达到了电泳纯,纯化倍数为16.3.CBH的反应最适温度为60℃,最适pH为6.0,Km(底物为羧甲基纤维素钠)为17.34mg/mL.

A strain of Tridchoderma viride sp. producing exo-1,4-β-D-glucanase (cellobiohydrolase,CBH) was isolated from a piece of rotten wood. Culturing in 100 mL conical flasks with 40 mL liquid medium provided optimal aeration and, with the liquid medium at an initial pH of 8, produced the maximal amount of CBH activity. Along with the growth of the mold, the activity of the CBH produced as well as the amount of glucose in the medium changed, fluctuating between high and low then low and high in turns. Through a series of purification procedures, including ammonium sulfate precipitation, sephadex G-100 gel filtration, and DEAE-cellulose 52 ion-exchange column chromatography, pure electrophoresis grade CBH was obtained at 16.3 times the concentration of the primary product. The optimal pH for CBH activity was about 6.0, the optimal temperature about 60 ℃,and the K_m (CMC-Na as substrate) was 17.34 mg/mL.