Production And Properties Of Salt Tolerant Cellulase And β-Glucosidase From A Marine Aspergillus Niger

Cellulase, consisted of endoglucanase, exoglucanase and β-glucosidase, can hydrolyze cellulose into glucose. P-glucosidase can transform cellobiose into glucose. Hydrolyzing cellobiose is a critical step of enzymatic cellulose hydrolysis.Under high salinity condition, salt tolerant cellulase can efficiently hydrolyze cellulose into glucose. Salt tolerant cellulase has important significance for treatment of papermaking and cellulosic materials pretreatment sewage, efficient utilization of cellulose in marine litter, algae and seaweed and reclamation of saline and alkaline lands.A marine fungus with salt tolerant cellulase was selected. The production process, properties and universal salt tolerant mechanism of β-glucosidase was studied. The results provided the foundation for practical application of salt tolerant cellulase. This article was consisted of the following:Firstly, a marine fungus was obtained to produce salt tolerant cellulase. According the morphology, ITS sequence and optimum growth salinity of5%NaCl (w/v) solution salinity, the marine fungus was identified as a halophile Aspergillus niger. Optimum pH, temperature and NaCl concentration of cellulase activity was4.5,58℃and12%(w/v). Cellulase activity in12%NaCl (w/v) solution was1.33times higher than that in NaCl free solution. The glucose production of hydrolyzing cellulose of cellulase in black liquor for12h increased by11.5percent compared with that in tap water. Cellulase from the marine Aspergillus niger was salt tolerant and thermostable.Secondly, a natural medium was designed to environment friendly produce cellulase under solid state fermentation conditions. The optimum natural medium was consisted of76.9%Eichhornia crassipes (w/w),8.9%raw corn cob (w/w),3.5%raw rice straw (w/w),10.7%raw wheat bran (w/w) and natural seawater (2.33times weight of the dry substrates). Incubation for144h, cellulase production was17.80U/g dry substrates.Thirdly, responses of cellulase activity and total extracellular proteins to different carbon sources under submerge fermentation were analyzed. The results showed that responses of cellulase activity and total extracellular proteins to different carbon sources were remarkably different. When corn cob or rice straw was used as carbon sources, endoglucanase, P-glucosidase and cellulase production was2.00and1.70U/mL,3.25and2.62U/mL,0.13and0.08U/mL. Endoglucanase, P-glucosidase and cellulase activity was1.39and1.31U/mg protein,3.25and2.62U/mg protein,0.181and0.165U/mg protein. Two-dimension electrolphoresis showed15different kinds of proteins in total extracellular proteins when corn cob or rice straw was used as carbon sources.Forthly, P-glucosidase was produced by immobilizing the marine Aspergillus niger on loofa sponges. Biomass of mycelia immobilized on loofa sponge carrier could be3.1g/L and the immobilization percentage could be over95%. The immobilization technology reduced the production circle, time to the maximum production by free or immobilized mycelia was7or4.5d. P-glucosidase production of the fourth and fifth bacthes was more than that by free mycelia β-glucosidase activities of five repeat batches were all over110U/mL.Fifthly, properties and thermodynamics of β-glucosidase was analyzed at different salinities. Crude P-glucosidase activity in6%NaCl (w/v) solution was the maximum. In free or6%NaCl (w/v) solution, optimum temperature and pH of crude β-glucosidase activity was same,66℃and5.0. Crude β-glucosidase activity in6%NaCl (w/v) solution was1.46folds higher than that in NaCl free solution. At62℃,65℃,67℃and70℃, half life of crude in6%NaCl (w/v) solution were more than twice of these in NaCl free solution. Gibb’s free energy for denaturation and melt temperature of crude β-glucosidase was about2kJ/mol higher and slightly warmer than that in NaCl free solution. After purification, molecular weight of β-glucosidase was measured to be about110kDa. The increasing of activity and half life of pure P-glucosidase at high salinity was similar with that of crude β-glucosidase. P-glucosidase from the marine haloversatile Aspergillus niger was salt tolerant and thermostable.Sixthly, molecular model of β-glucosidase was constructed and the probable mechanism for salt tolerance was explained. Homology molecular model for the kind of β-glucosidase was constructed. Many glutamic acid and aspartic acid residues, acidic amino acid residues, on the surface of β-glucosidase molecular model, endow β-glucosidase molecular with a high electronegativivty surface. The interaction between high electronegativivty surface and molecular core probably enhances the kind of β-glucosidase thermostability at high salinity. The high concentration of Na+probably isomerizes β-glucosidase and catalytic sites of the isomer were probably more efficient to intreract with the substrate of cellobiose.Efficient producing of cellulase and β-glucosidase, studying properties of cellulase and P-glucosidase at high salinity, the explanation of mechanism for β-glucosidase salt tolerance was valuable for hydrolyzing cellulose at high salinity.