Biophysical studies on mechanisms of cellulose biodegradation

点击翻译按钮获取中文摘要

Depolymerization of cellulose to saccharides is an important step in the production of ethanol as a bio-fuel. Native cellulose undergoes biodegradation by the concerted action of a variety of enzymes having a multimodular organization of carbohydrate binding modules (CBM) and catalytic domains responsible for binding the enzyme to cellulose and for the catalytic degradation of the biopolymer, respectively. The rate-determining step of the enzymatic attack is the initial adsorption of the enzyme on a cellulose microfiber after which digestion occurs rapidly. We propose to employ the spectrum of spectroscopic, surface imaging and surface analytical techniques to understand the mechanism of cellulase adsorption onto cellulose fibres using isolated CBMs, engineered enzymes that possess their CBM(s) but have inactive catalytic sites, and others that lack one or more of their CBMs.  Mutant enzymes, varying in the length of the peptide tether between the CBM and catalytic domain, will be engineered. With the isolated CBMs and the catalytically inactive enzymes, we will measure the rates of enzyme adsorption to cellulose fibres. The rate of cellulose degradation using the same type of enzymes but with active catalytic groups will be compared and the rate-determining step of the enzymatic digestion of cellulose will be identified. Finally, infrared reflection absorption spectroscopy (IRRAS) will be used to identify the nature of the bond between the enzyme and the cellulose fibres. High-resolution scanning atomic force microscopy (AFM) will be used to identify the nature of the binding sites responsible for enzyme adsorption onto cellulose.  Using this approach we will develop new strategies for engineering of more effective enzymes for depolymerization of cellulose leading to more efficient biofuel production.