Preparation Of Endoglucanase From Marine Aspergillus Niger And Combination Of Cellulases

Cellulases are key resources in biorefineries, where they are used to produce alternative fuels and chemicals from lignocellulosic biomass. They consist of endoglucanases (endo-1,4-β-glucanase), exoglucanases (exo-1,4-β-glucanase) and cellobiases (β-glucosidase). To study the properties and compositions of cellulases from different microorganisms is very important for cellulose hydrolysis. A marine Aspergillus niger which has the ability to produce abundant cellulase under solid state fermentation was isolated from the East China Sea seabed mud. In our previous work, we had studied the properties of a cellobiase purified from the crude enzymes at high salinity in detail. In this paper, the medium conditions of solid-state fermentation by the strain were optimized. One kind of endoglucanase isolated from the crude cellulase produced by marine A. niger was studied in detail. Cellulases from marine A. niger and Trichoderma reesei Rut-C30 were recomposed and the characters of different components in the cellulases were investigated. Degradation of rice straws by cellulases produced by T. reesei, marine A. niger and the compound cellulase was also studied.The marine A. niger could take good use of agricultural residues to produce cellulases. The medium conditions for cellulase production under solid state fermentation by marine A. niger were optimized. Using rice straw and 40% wheat bran as substrate containing 70% nutrient solution could lead maximal filter paper activity,endoglucanase activity to 9.64 ±0.37 U·gds-1,205.24±6.94 U·gds-1 on the sixth day, cellobiase activity to 76.27±.13 U·gds-1 on the seventh day.An efficient purification procedure was established to separate and purify endoglucanase from the crude cellulases of marine A. niger. One kind of endoglucanase was purified by ammonium sulfate precipitation, ultra-filtration, anion exchange chromatograph and hydrophobic interaction chromatography. The specific activity of the purified endoglucanase increased to 557.27 U·mg-1 from the crude endoglucanase (37.72 U·mg-1). The purified endoglucanase was characterized in detail. The results showed that the molecular weight of the endoglucanase was about 34 kDa by SDS-PAGE. The optimal temperature and pH for the activity of the endoglucanase were 60 ℃ and 4.5, respectively. The endoglucanase activity retained 87% at 50 ℃,57% at 60℃ after 9h, respectively, and the endoglucanase was stable over a broad pH range. The metal ions Cu2+ and Fe2+ were found as inducers while Mn2+, Ni2+ and Zn2+ have significantly inhibited the enzyme activity. In the solution with NaCl concentration in the range of 20～60 g·L-1, the endoglucanase activity was higher than that in the NaCl-free solution, and the residual activity of about 89% at salt concentration (NaCl 20%) revealed for its halotolerance nature. The kinetic constants Km and Vmax determined for endoglucanase, with carboxylmethyl cellulose as a substrate, were 11.7mg·mL-1 and 943.4U·mg-1, respectively.The combination of two different cellulases from marine A. niger and T. reesei was studied. The results were found that the cellulase blends had maximized filter paper activity with 43.9% synergistic enhancement compared with that predicted when the ratio of FPase and Cellobiase in the enzyme blends was 1:2. The compound cellulase was prepared according to this fixed enzyme composition was also investigated. The results showed that it was between character of cellulases from marine A. niger and that of T. reesei. The optimum pH of filter paper activity, endoglucanase and cellobiase were all 5.0 and the optimum temperatures of the three kinds of cellulases were 50℃,50℃ and 60℃. The compound cellulase could retain higher activity than the cellulase from T. reesei when incubated at 50℃ for 48 hours. Its halotolerance was also better than that from T. reesei. It could still retain more than 50% of its initial enzyme activity when NaCl concentration was 20%.Moreover, the compound cellulase evaluated for its hydrolytic effectiveness on diluted acid pretreated rice straws was superior to the use of unblended T. reesei or marine A. niger cellulases, under corresponding conditions (20 U·(g substrate)-1,20 (g substrate)·L-1 and 50℃ for 48h). Hydrolysis experiments using the compound cellulase resulted in the formation of 8.4 g·L-1 reducing sugar within 48 h corresponding to 6.5 g·L-1 by T. reesei enzymes and 4.1 g·L-1 by marine A. niger enzymes. The compound cellulase hydrolyzed 54.72% of the cellulose releasing 7.6 g·L-1 glucose from the pretreated rice straw within 48 h corresponding to 24.08%, 3.4 g·L-1 glucose and 41.04%,5.7 g·L-1 glucose cellulose hydrolysis by marine A. niger and T. reesei cellulases, respectively. Glucose hydrolysis yields by the compound cellulase increased 33.3% and 123.5% compared with unblended marine A. niger and T. reesei cellulases. In conclusion, the compound cellulase of the marine A. niger and T. reesei cellulases was more effective for the rice straw cellulose hydrolysis.