Biophysical studies on mechanisms of cellulose biodegradation

将纤维素解聚为糖类是生产乙醇作为生物燃料的重要步骤。天然纤维素通过多种酶的协同作用进行生物降解，这些酶具有碳水化合物结合模块(CBM)和催化结构域的多模块组织，分别负责将酶结合到纤维素和催化降解生物聚合物。酶攻击的速度决定步骤是酶在纤维素超细纤维上的初始吸附，之后迅速发生消化。我们建议使用光谱、表面成像和表面分析技术来了解纤维素酶在纤维素纤维上的吸附机制，使用分离的CBM，拥有CBM但具有非活性催化中心的工程酶(S)，以及其他缺乏一个或多个CBM的工程酶。我们将设计出突变酶，在CBM和催化结构域之间的肽系绳长度不同。利用分离的CBM和催化失活的酶，我们将测量酶对纤维素纤维的吸附速度。将比较使用相同类型的酶但具有活性催化基团的纤维素的降解速度，并确定酶消化纤维素的速度决定步骤。最后，红外反射吸收光谱(IRRAS)将被用来鉴定酶和纤维素纤维之间的键的性质。高分辨率扫描原子力显微镜(AFM)将被用来识别负责酶在纤维素上吸附的结合部位的性质。通过使用这种方法，我们将开发新的策略来设计更有效的酶来解聚纤维素，从而更高效地生产生物燃料。

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.