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Abstract
In order to meet the Renewable Fuels Standard demands for 30 billion gallons of biofuels by the end of 2020, new technologies for generation of cellulosic ethanol must be exploited. Breaking down cellulose by cellulase enzyme is very important for this purpose but this is not thermostable and degrades at higher temperatures in bioreactors. Towards creation of a more ecologically friendly method of rendering bioethanol from cellulosic waste, we attempted to produce recombinant higher temperature resistant cellulases for use in bioreactors. The project involved molecular cloning of genes for cellulose-degrading enzymes based on bacterial source, expressing the recombinant proteins in E. coli and optimizing enzymatic activity. We were able to generate in vitro bacterial expression systems to produce recombinant His-tag purified protein which showed cellulase like activity.
Highlights
Cheap, clean, green energy production is a goal of Department of energy and EPA
Cellulases and other glycoside hydrolases [3,4] are assembled onto multidomain scaffoldin proteins for efficient degradation of cellulosic substrates [4]
The amplicon was cloned into a Gateway System (Invitrogen, using a standard BioRad (USA)) his-tag expression vector and BL-21 E. coli bacteria was transformed with this construct
Summary
Simple, is costly because of the high price of the corn kernels themselves Agricultural waste, such as corn stover (the leaves, stalks, and stripped cobs of corn plants, left over after harvest), is cheap. An additional complication is that while the fermentation reaction that breaks down corn starch needs just one enzyme, the degradation of cellulose requires a whole suite of enzymes, or cellulases, working in concert. Cellulases and other glycoside hydrolases [3,4] are assembled onto multidomain scaffoldin proteins for efficient degradation of cellulosic substrates [4]. The chimeric proteins that are made by recombining natural sequences differ Our objective for this project was to construct chimeric synthetic cellulase genes for production of thermostable cellulases for efficient breakdown of cellulose at high temperature
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