Cloning, expression and characterization of novel fungal endoglucanases

• Main Author: Ren, Hua Nan
• Format: Others
• Published: 2009
• Online Access: http://spectrum.library.concordia.ca/976771/1/MR80149.pdf; Ren, Hua Nan <http://spectrum.library.concordia.ca/view/creators/Ren=3AHua_Nan=3A=3A.html> (2009) Cloning, expression and characterization of novel fungal endoglucanases. Masters thesis, Concordia University.

Global energy consumption is projected to double between 1980 and 2020. Furthermore, about 65% of present energy consumption is derived by burning nonrenewable fossil fuels that increase atmospheric levels of CO2 , the major greenhouse gas implicated in global warming. It is therefore important that carbon neutral alternative energy sources be developed. For transportation fuels bioethanol derived from renewable energy sources is considered among the most attractive alternatives. Presently, most bioethanol is produced from corn kernels and sugar cane sucrose, with the United State and Brazil being the world's major producers. Cellulose is another possible feedstock that can be used to produce bioethanol. Compared with the currently used feedstocks, cellulose is very abundant in nature and its use for biofuels production would not compete with the food or animal feed industries. In order to use cellulose for bioethanol production, the cellulose must be converted into the fermentable sugar glucose. Currently, the high cost of converting the cellulose into glucose is a major impediment to using cellulose as a feedstock for the production of bioethanol. The goal of my research was to address this issue by discovering new fungal enzymes that could improve the cellulose hydrolyzing efficiency of existing commercial cellulase systems. In this study I identified, cloned, functionally expressed and characterized six endoglucanases, AfumEgl2010, AnidEgl2020, FgraEgl2010, FgraEgl1020, FgraEgl2020 and NcraEgl2010. Three of these endoglucanases, AfumEgl2010, AnidEgl2020, FgraEgl2010, were characterized in detail in this study. Only AfumEgl2010 harbours a carbohydrate-binding module (CBM). All three of the endoglucanases that were subject to detailed characterization show highest activity at pH 5.0. The temperature optima of these three endoglucanases were determined to be 40°C, 60°C and 70°C. All of them were stable during 30 minutes pre-incubation at 60°C. The kinetic parameters of these three endoglucanases and four other endoglucanases, the Trichoderma reesei (T. reesei ) Eg2/Cel5A and ApulSEQ15654, StheSEQ13822, GtraSEQ630 (previously identified by undergraduate student Christopher St-Francois) were determined at 37°C and the pH optima of each endoglucanase. The Km values ranged from 2.0 to 29 mg/ml. The Vmax values ranged from 5.7 to 41 omole/mg/min. The degree of synergism when these endoglucanases were combined with the T. reesei Cbh1/Ce17A was also determined.