Enzymological Charateristics Of Lignocellulose-Degrading Dominant Microbial Strains And Analysis Of The Products Derived From Enzymatic Hydrolysis Of Lignocellusic Biomass

Cellulase, hemicellulase and ligninase secreted by microorganisms have a synergistic effect on the bioconversion and utilization of lignocellulosic biomass. The conversion of lignocellulose into value-added products still faces technical bottlenecks. Energy crises, environmental pollution and food crises force humans to develop technologies for sustainably utilizing available resources, such as biofuels and other value-added products.In this study, cellulose-degrading bacterial strains isolated from municipal wastes and peat lands were used to hydrolyze cellulose of American agave plant. By employing the agave-agar plates method to conduct legal analyses of endo-cellulase activity of 18 bacteria strains, screening out agave species with the degradation advantages:Duganella 55S2, Bacillus 65S3 and Pseudomonas CDS3. Using ethanol analysis assay kit and HPLC to determine its transformation products, we determined the abilities of Bacillus 65S3 and Pseudomonas CDS3 degraded agave were superior to the other 16 bacteria in producing bio-ethanol and xylitol. The highest yield of 65S3 Bacillus degrading agave in producing bio-ethanol was 0.92g/g. The highest yield of CDS3 Pseudomonas degrading agave in producing xylitol was 0.98g/g. The three bacterial strains had a different degrees of morphological change in degradation of agave, as determined by scanning electron microscopy. Therefore agave was an ideal raw material for bio-ethanol and xylitol, Bacillus 65S3 and Pseudomonas CDS3 were able to produce high value-added products, showing great potential for the bio-refining industry.Trichoderma reesei is a widely used cellulase producer. To further improve the degradation ability of T. reesei, we used QM9414 as parental strain, successfully transformed the transcription factor gene Ace2 and obtained engineered fungal strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Of the five strains, T/Ace2-2 increased total filter paper maximum enzyme activity by 2 times that of QM9414, the host strain maximum enzyme activity, and on the first sixth day, the xylanase activity was 1.12 times greater than their host fungi enzyme activity. Research through HPLC on yield of degradation bark generating xylitol by genetically modified fungi T/Ace2-2, xylose -MA medium seeded with T/Ace2-2, on the 6th day, the yield of bark converting to xylitol was up to 0.12 g/g. This shows the engineering fungi T/Ace2-2 had a significant impact on the morphological change in degradation of bark by scanning electron microscopy. Therefore, T. reesei genetically modified fungi T/Ace2-2 could efficiently degrade lignocellulose like bark without pretreatment, achieving cleaner production and a lower cost for the xylitol industry.To improve the degradation ability of T. reesei, we successfully expressed lignin peroxidase gene lipH8 in QM9414 and obtained three genetically modified strains:T/Lip4-3, T/Lip8-5 and T/Lip10-7. By lignin peroxidase activity study of engineering fungi T/Lip4-3, T/Lip8-5 and T/Lip10-7, confirming the peroxidase enzyme activity on the 15th day, engineering fungi T/Lip10-7 reached a maximum of 64.47IU/ml. We then analyzed degradation product of wood by engineered fungi T/Lip10-7 by LC-MS, examining its products such as levulinic acid and benzene. Engineered bacteria T/Lip10-7 also showed obvious internal structure change after the degradation of wood through scanning electron microscopy. Therefore, genetically modified T. reesei strain T/Lip10-7 can be used to degrade cellulose like wood, and to facilitate the production of high value-added products.To study the structure, function and exegetical modification of T. reesei cellulase and xylanase enzymes, we conducted feature analysis using amino acid sequences of T. reesei endoglucanase EGⅣ and xylanase xynl, xyn2. By using Discovery studio for homology modeling of EGⅣ, and by assessing the prediction of structure through the Line Plot graphs and Profiles-3D method, we obtained some generally positive results. Performing docking experiments of EGIV and xynl, xyn2 by Molegro Virtual Docker (MVD) and software, and conservatively analyzing the binding site of ligand and enzyme, we showed that six highly conserved sites exist in xynland xylobiose ligand:Glu 75, Asp 33, Met 79, Phe 121, Glu 164 and Trp 166. The result of xyn2 was similar, but the overall level of conservation was higher than xynl, with the highly conserved sites being:Phe 45, Val 46, Trp 79, Arg 122, Asn 44, Glu 86, Tyr 88 and Glu 177. Performing molecular docking of T.reesei cellulase EGIV, we obtained 22 cellobiose ligand binding sites, of which 17 sites were highly conserved:His 1, Gly 2, His 3, Pro 20, Trp 34, Ala 36, Gly 42, Val 44, Ser 45, Ala 48, His 57, Lys 58, Asn 169, Ala 171, Tyr 174, Pro 175 and Cys 177. Based on the above critical amino acid sites, we rationally designed modification of T. reesei cellulase and hemicellulase, hoping to get industrial-valued enzymes.In summary, a Bacillus strains (65S3), a Pseudomonas strains (CDS3) and genetically modified T. reesei strains T/Ace2-2 and T/Lip10-7 can be used in hydrolysis and fermentation of lignocellulosiC resources, generating environment friendly bio-ethanol, xylitol, and other high value-added products.