固定化纤维二糖酶的研究

黑曲霉 (AspergillusnigerLORRE 0 12 )的孢子中富含纤维二糖酶 ,将这些孢子用海藻酸钙凝胶包埋后 ,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定 ,半衰期为 38d ,耐热性和适宜的pH范围均比固定化前有所增加 ,其Km 和Vmax值分别为 6 .0 1mmol L和 7.0 6mmol (min·L)。利用固定化纤维二糖酶重复分批酶解10g L的纤维二糖 ,连续 10批的酶解得率均可保持在 97%以上 ;采用连续酶解工艺 ,当稀释率为 0 .4h- 1 ,酶解得率可达 98.5 %。玉米芯经稀酸预处理后 ,其纤维残渣用里氏木霉 (Trichodermareesei)纤维素酶降解 ,酶解得率为6 9.5 % ;通过固定化纤维二糖酶的进一步作用 ,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除 ,酶解得率提高到 84.2 % ,还原糖中葡萄糖的比例由 5 3 .6 %升至 89.5 % ,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。

Studies on Immobilized Cellobiase

Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, which can be used for production of ethanol, organic acids, and other chemicals. Cellulase is a complex enzyme containing endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and cellobiase (EC 3.2.1.21). The hydrolysis of natural cellulose to glucose depends on the synergism of these three components. The mostly used cellulase produced by Trichoderma reesei has high activity of endoglucanase and exoglucanase, but the activity of cellobiase is relatively low. Therefore, improving the activity of cellobiase in cellulase reaction system is the key to enhance the sacchrification yield of cellulosic resources. Aspergillus niger LORRE 012 was a high productivity strain for cellobiase production. It was found that the spores of this strain were rich in cellobiase. In this work, the cellobiase was immobilized efficiently by simply entrapping the spores into calcium alginate gels instead of immobilizing the pure cellobiase proteins. The immobilized cellobiase was quite stable, and its half-life was 38 days under pH 4.8, 50 degrees C. The thermal stability of the immobilized cellobiase was improved, and it was stable below 70 degrees C. The suitable pH range of the immobilized cellobiase was pH 3.0 - 5.0, with the optimal pH value 4.8. The Km and Vmax value of the immobilized cellobiase were 6.01 mmol/L and 7.06 mmol/min x L, respectively. In repeated batch hydrolysis processes, 50 mL of substrate (10 g/L cellobiose) and 10 mL of immobilized beads containing cellobiase were added into a 150 ml flask. After reacting at pH 4.8, 50 degrees C for several hours, the hydrolysate was harvested for assay, and the immobilized beads were used for the next batch hydrolysis with the fresh substrate. This process was repeated, and the yield of enzymatic hydrolysis kept higher than 97% during 10 batches. The continuous hydrolysis process was carried out in a column reactor (inside diameter 2.8 cm, inside height 40 cm) packed with the immobilized beads. Using 10 g/L cellobiose as substrate, the hydrolysis yield reached 98% under 0.4 h (-1) dilution rate and pH 4.8, 50 degrees C. After corncob was treated by 1% dilute acid, the cellulosic residue (100 g/L) was used as substrate, and hydrolyzed by the cellulase (15 IFPU/g substrate) from Trichoderma reesei, at pH 4.8, 50 degrees C for 48 h. The concentration of reducing sugar in the hydrolysate was only 48.50 g/L (hydrolysis yield 69.5%). When the hydrolysate was further treated by the immobilized cellobiase, the cellobiose was hydrolyzed into glucose, and the feedback inhibition caused by the cellobiose accumulation disappeared sharply. By the synergism of immobilized cellobiase and the cellulase from T. reesei left in the hydrolysate, other oligosaccharides were mostly converted to monosaccharides. At 48 h, the reducing sugar concentration was increased to 58.78 g/L, the hydrolysis yield of the corncob residue was improved to 84.2%, and the ratio of the glucose in the total reducing sugar was increased from 53.6% to 89.5%. The reducing sugars converted from corncob could be used further in the fermentation of valuable industrial products. This research results were meaningful in the conversion and utilization of renewable biomass.