Directly Fermentation Of Microcrystalline Cellulose To Ethanol By Use Of Saccharomyces Cerevisiae Codisplaying Of Cellulolytic Enzyme

Screening of cellulose-degrading microorganisms and constructing of cellulase gene surface display Saccharomyces cerevisiae has been viewed as the most effective method to face a series of emergency problems, such as energy consuming, higher fuel price and environment pollution.In this article, a new strain of high efficiency cellulose-degrading is isolated by using Congo red method and detecting the activities of cellulosic enzymes. The strain was identified Trichoderma longibrachiatum CD-6(CGMCC No.5395), depending on the analyzed results which conclude morphology observation, taxonomic status detection and internal transcribed spacer rDNA(ITS) gene sequence. The result of cellulose-degrading activity demonstrated that this strain can produce abundant cellulases with high activity, the enzyme activity towards CMCase、FPA、 xylose which is50.65IU/ml,105.09IU/ml,172.62IU/ml, respectively.Three kind of cellulase genes were cloned, which were gene cbhⅡ from Trichoderma longibrachiatum, egⅢ from Trichoderma reesei, and bgl from Aspergillus niger, respectively, and analyzed the sequence homology. Two surface display vectors have been constructed, and transformed into Saccharomyces cerevisiae competent cells, and then recombinant Saccharomy cescerevisiae which display cellulase on the surface were constructed.The activities and properties of recombinant cellulase were analyzed.The CMC enzyme activity of the strains CEN-pYD-EG-BGL and CEN-pYED-CBH-EG-BGL reached to304.68IU/ml and426.79IU/ml, respectively. The filter paper activity analysis showed, the maximum activity of recombinant strains CEN-pYE-CBH,CEN-pYD-EG-BGL and CEN-pYED-CBH-EG-BGL is322.27IU/ml,293.86IU/ml,294.69IU/ml. The threshold of pH and temperature stability of strain CEN-pYED-CBH-EG-BGL was ranging from4.0-10.0and20℃-50℃, respectively.Direct fermentation microcrystalline cellulose to ethanol by using recombinant Saccharomyces cerevisiae strains CEN-pYE+pYD(control), CEN-pYE-CBH, CEN-pYD-EG-BGL, CEN-pYED-CBH-EG-BGL. Analyze the degadation ability of recombinant strains,The results illustrated that CEN-pYED-CBH-EG-BGL has the strongest hydrolysis ability. The highest concentration of ethanol was2.63g/L.The yield was0.3g/g(in grams of ethanol produced per gram of microcrystalline cellulose consumed). This indicated that simultaneous and synergistic saccharification and fermentation of microcrystalline cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.