|
Cellulose resources is the earth's most abundant renewable resources, effective utilization of cellulose resources, can ease the increasingly prominent worldwide food and energy shortages. Cellulase by cellulase three components: endo-glucanase (endoglucanase EG), exo of glucanase (cellubiohydralases CBH), and beta-glucosidase (P-glucosidases, BG) The synergy of the hydrolysis of cellulose to glucose. Cellulase enzyme activity affect the cellulase in industrial production, the use of gene cloning, gene expression, the transformation of cellulase protein molecules and design means to get people expect high enzyme activity of cellulase become hot spots. Trichoderma viride (Trichoderma viride) is one of the highest strain producing cellulase activity, it is widely distributed in nature, often saprophytic on wood, seeds and plant residues. Termites (Coptotermes formoscmus) rotten wood and other cellulose-rich wood substances for food, are rich in cellulose degrading bacteria in the gut microbes. This thesis from years of outdoor placed Kuchiki isolated the the biodegradable cellulose fungal G-1, the the biodegradable cellulose fungal G-2 isolated from the termite gut microorganisms After the colony morphology and the mycelial morphology observation and 18Sr DNA The sequence analysis identified fungal G-1, G-2 of Trichoderma viride Trichoderma viride. Obtained from the G-1 was cloned endoglucanase glycosidase gene (EG Ⅱ, Genbank: HM116999.1) and exo-glucanase glycosidase gene (CBH Ⅱ, GenBank: HM117000.1), obtained from the G-2 was cloned two β-1, 4 - glucosidase gene (BG Ⅰ, Genbank: HM178944.1; BG Ⅱ, Genbank: HM178945.1). Sequence alignment analysis, cloned EG II and CBH Ⅱ gene sequences carried out site-directed mutagenesis, two mutation sequence EG Ⅱ-mut was and CBH Ⅱ-mut was, its amino acid sequence encoded mutate, intended to increase its expression protein enzyme vitality . The EG Ⅱ, EG Ⅱ-mut was CBH Ⅱ remove their own signal peptide sequence, building secretive expression vector pPIC9k. The constructed plasmid was-EG Ⅱ pPIC9k. The constructed plasmid was-EG Ⅱ-mut was, pPIC9K-CBH Ⅱ, onto Pasteur complete red yeast (Pichia pastoris and) GS115 conducted induced expression. By the MD, MM board screening transformants coarse sieve into Congo red staining, enzyme activity was measured and then the DNS method, screened secretion of extracellular enzyme activity of recombinant Pichia pastoris strain GS115-EG II -7 GS115-EG Ⅱ-mut-10, GS115-CBH Ⅱ -2. Pastoris GS115-EG II -7 pastoris GS115-EG II-MUT-10 endo-glucanase activity were 20.915U/ml 24.110U/ml pastoris GS115-CBH II-2 of the exonuclease the glucanase activity reaches 27.925U / ml. The recombinant Pichia yeast strain mutated sequences (EG Ⅱ-mut) than the restructuring of the original sequence (EG Ⅱ) Bi pastoris extracellular secretion endoglucanase enzyme activity increased, the results show that the some site-directed mutagenesis can reveal affect enzyme important functional domains, cellulase gene transformation using molecular biology methods in order to lay the foundation for high cellulase enzyme activity. This thesis is constructed six Saccharomyces cerevisiae expression vector, pYES2/CT/lacZ-EG the II, pYES2/CT/lacZ-EG Ⅱ-mut was pYES2/CT/lacZ-BG Ⅰ pYES2/CT/lacZ-BG II, pYES2/CT-CBH II , pYES2/CT-CBH Ⅱ-mut, for follow-up work to lay the foundation for recombinant strains that transform S. cerevisiae, take restructuring endo glucanase, exo-glucanase and β-glucosidase enzyme The highest activity of the Saccharomyces cerevisiae, to respectively fermentation broth mixed in different proportions in accordance with the highest levels of activity time, screening the ratio of degradation of straw cellulose fastest rate, to achieve high efficiency of cellulose hydrolysis and fermentation carried out simultaneously.
|