重组酿酒酵母表达纤维素酶和膨胀素的活性分析-activity analysis of recombinant saccharomyces cerevisiae expressing cellulase and expansin.docxVIP

暨南大学硕士学位论文 暨南大学硕士学位论文 PAGE PAGE VI Abstract Cellulose is the cheapest and most abundant recycled resource on the earth. Converting cellulose to ethanol will be an effective way to solve the worlds energy shortage. But so far there is no good way to make full use of cellulose. The Saccharomyces cerevisiae has a high rate of converting sugar to alcohol, and is an ideal microorganism for ethanol production. However, it lacks the ablity to produce cellulases and can not degrade cellulose directly. In this study, Trichoderma reesei endoglucanaseⅠ (EGⅠ) gene and Aspergillus niger β-glucosidase Ⅰ (BGL Ⅰ ) gene were integrated into the genome of S. cerevisiae. The recombinant strain obtained the ability to degrade amorphous cellulose and produce ethanol directly. Furthermore, preliminary analysis was conducted to detect the activity of T. reesei swollenin, as well as the effect of swollenin in pormoting the degradation of cellulose by cellulases.The encoding region of T. reesei eg1 was obtained by RT-PCR. The eg1 and A. niger bgl1 were then cloned into vector pδRCMB, resulting the recombinant plasmid pδRCMB-bgl1-eg1. The two cellulase genes were then integrated into the genome of S. cerevisiae W303-1A through δ DNA integration by electroporation. The recombinant strain S. cerevisiae-eb was screened by the resistance to G418, PCR and Congo-red staining. The EGⅠ and BGLⅠ enzyme activities were analyzed in the culture supernatants. The secreting expression of recombinant EGⅠ and BGLⅠ was further confirmed by SDSand zymogram. The direct fermentation of PASC as the sole carbon source was performed by recombinant strain S. cerevisiae-eb. The expression of EGⅠ by S. cerevisiae-eb reached the highest endoglucanase activity of 0.10 IU/mL after 120 h of cultivation. The optimal temperature was 60 ℃ and optimal pH was 6.0. The recombinant BGLⅠ reached the highest β-glucosidase activity of 4.83 IU/mL at 144 h. The optimal temperature and pH for BGLⅠ were at 60 ℃ and pH 4.5,