Isolated Directly From Natural Habitats In The New Cellulase Composition For Discussion

The fossil fuel have been almost exhausted after hundreds years of industrialization. In our country, rapidly developed industrialization make a higher consumption of energy. Therefore, development of new energy resource is not only important for economic stability, but also essential for the sustainable development in the near future. All the energy on earth comes from sunlight, and the energies are mainly converted into the biological energy of biomass. Considering its huge reserves, clean by-products and cheapness, biological resource were naturally been seen as the most suitable replacement of fossil fuel. However, there are still many drawbacks for the high efficiency utilization of this energy.From the basic researches to the optimizations for application, many factors can influence the utilization of biological energy, and each of them may work for the final success. The utilization of the biomass has been researched for decades in our lab. One of the problems we focused on is to develop new cellulases and explain the mechanism of lingo-cellulose degradation in natural environment. But because of a limitation in the cultivability of microbes, the understandings of cellulose degradation were all come from several modal organisms, and cellulose derivatives were often used as the substrates. Furthermore, there is a lack of appropriate method to investigate cellulases in the natural environments.We aimed at the problems above and the natural moldy silage was used in our studies as substrates. We investigated the dominant cellulose-decomposing fungi in the silage, and an aciduric endoglucanase that has industrial value was isolated and investigated. The amino acid residues which were presumed to affect acid tolerance were picked out by homology modeling. The cellulases in natural moldy silage were directly purified without cultivation, and the properties of the cellulases were investigated in details to be compared with those of the isolated fungus. A novel method was finally established to direct isolate cellulases from the natural environment without microbial cultivation, and some novelties were obtained in our research.1 Aspergillus fumigatus is the dominant fungus during the silage decomposition process.In natural moldy silage, a fungus which grows fast at high temperature was isolated and named Y05. The spore-producing ability of the strain was strong and when screened by the traditional cellulose plate, the strain can cover up the existence of other microorganisms. The strain was determined as Aspergillus fumigatus by 18S rDNA and morphology identification. The classification of the strain is: Cellular organisms; Eukaryota; Fungi/Metazoa group; Fungi; Ascomycota; Pezizomycotina; Eurotiomycetes; Eurotiales; Trichocomaceae; mitosporic Trichocomaceae; Aspergillus; Aspergillus fumigatus.1.1 Aspergillus fumigatus Y05 produce aciduric cellulase system, and a 25kDa aciduric endoglucanase was purified from the extracellular proteins.Cornstalk powder could efficiently induce the synthesis of cellulases in strain Y05. The growth and cellulases production of Aspergillus fumigatus Y05 were both investigated in wheat bran and cornstalk powder media. Though wheat bran showed better growth and cellulases production ability at the initial stages, cornstalk powder which has more complex structure showed stronger inductive effect to synthesize cellulases, and higher cellulase activity was obtained. Some modified derivative cellulose, such as microcrystalline cellulose, CF11 and CMC etc, did not show high cellulase inductive ability. Furthermore, Aspergillus fumigatus Y05 grew slowly in these media, indicating that these simple substrates are not favorable to the strain for growth and production of cellulases.For CMC, the optimal temperature for crude celullase from Aspergillus fumigatus Y05 was 65°C, and the optimal pH was 4.2. For the insoluble substrates, such as corncob and filter paper, the optimal temperature and pH were 60°C and 4.4. The enzyme showed stronger acid tolerance, the optimal pH is lower than that of the cellulases from T. reeseiA 2SkD endoglucanase EG25 was purified from the aciduric crude enzymes of Aspergillus fumigatus Y05. The optimum pH of the enzyme was 3.2, which is much lower than those of general fungi cellulases. Even at pH 2.2, EG25 still contained 50% of the activity, and the activity was lost when pH was near 7.0. After incubated in room temperature at different pH (2.2-7.2) for 24 h, the CMCase activities were tested at pH 3.2. The results showed that EG25 was stable at these conditions, and more than 80% activities were preserved, among which the activity at pH 6.2 was highest. Though the optimal pH for EG25 is acidic, the enzyme is stable at high pH. The low activity at high pH was not caused by irreversible inactivation. When the pH was adjusted to the optimal pH, the enzyme still showed high activity.EG25, the enzyme produced by Aspergillus fumigatus Y05, was determined as an endoglucanase of GH12 family by LC/MS/MS. It did not contain CBD, and the molecular weight is 25785.42.1.1 Homology modeling, analysis of mechanisms of aciduric tolerance for GH12 familyHomology modeling was performed to build the structure of EG25 by MODELLER using the crystal structure of A. niger Cel12A (1ks4A) as model structure. Sequnce alignment between the two enzymes showed that Asp95 might be the key site with responsibility for the aciduric tolerance of EG25. This part of work could lead to the relationship between amino acid sequence and optimal pH. 1.4 Separating of extracellular proteins of Aspergillus fumigatus by 2-D PAGE, and celluiase activity staining was firstly used in 2-D PAGE.A method was established to purify the extracellular proteins of Aspergillus fumigatus in solid state fermentation. The proteins were separated by 2-D PAGE.The celllulase activity staining protocols were investigated. The proteins could be separated well and easy to resume activity if iodoacetamide was absent in equilibrium procedure. Celluiase activity staining was first used in 2-D PAGE and achieved good success. This method makes 2-D PAGE provide more information, and it is a good support for identification of proteins. Due to the fact that overfull proteins make against renaturation, this method will get better effect if used in the analysis of slight protein, especially proteins from natural environment.2 The different properties of celluiase between moldy silage and Aspergillus fumigatus indicating the different components composing.Aspergillus fumigatus is the dominant fungi in moldy silage. There are many resemblances between the aqueous extract of moldy silage and those from fermentated broth of Aspergillus fumigatus, such as thermotolerance (optimal reaction temperature is 65°C), and some cellulases with same molecular weight. As the dominant fungus, the proteins produced by Aspergillus fumigatus would certainly be the main components in the aqueous extract of moldy silage. However, there are also many differences between the two samples; for example, the optimal pH of Aspergillus fumigatus crude enzyme is 4.2, while that from moldy silage is 5.8. Furthermore, the fractions from the two samples showed different thermotolerance after molecular sieving. These results indicated the limitation of the traditional screening method by cultivation.2.1 Direct purification of cellulase without cultivation from the moldy silage.There have been no reports about direct purification of cellulases from natural environment. In the previous work in our lab, a cellobiohydrolase had been purified based on the adsorption ability of cellulases. Here, we furthermore purified another cellobiohydrolase and several endo-celllulase by chromatographic separation. This direct purification method will develop the researches of uncultured microbes.2.2 Establishing a strategy to analyze proteins in natural environment by metaproteomics for the first timeUsing moldy silage as the sample, a metaproteomics strategy has been established to analyze the cellulases in complex environment without cultivation. The protocols of this strategy include extracts of proteins from sample, purification, 2D-PAGE, cellulase activity staining and LC/MS identification. Although there are still some shortages, the method could be helpful to the investigation of the degradation of natural ligno-cellulose. The purification without cultivation could show the natural distribution of cellulases. The 2-D-PAGE, cellulose activity staining, and MS identification composed a high efficient screening method for new cellulases.