人工反转型葡萄糖苷酶的分子进化及抗产物抑制研究

Currently, lowering the cost of converting cellulosic biomass into liquid fuels can be achieved by improved cellulose-hydrolyzing organisms or enzymes, improved processes for biomass pretreatment, but the most economic strategy is to improve conversion of cellulosic biomass to sugars. The main handicap of improve conversion of cellulosic biomass to sugars is the low conversion rate using the commercial cellulase. Complete degradation of hemicelluloses requires endo-β-1, 4-glucanase, cellobiohydrolase, and β-glucosidase play a critical role in releasing fermentable sugars from intermediate oligosaccharides in saccharification of lignocellulose that inhibits the endoglucanases and limits the enzymatic hydrolysis. All β-glucosidases described so far act through a retaining mechanism and the two-step mechanism employed by retaining enzymes such as β-glucosidases allows these enzymes to transglycosylate, especially in high concentration of products, which will lower the product accumulations. However, the reaction of inverting glycosyl hydrolases is irreversible once the linkage is cleaved with the addition of a water molecule, and the inverted mechanism benefits to glycosyl hydrolases hydrolysis. Previously, we have developed a new DNA shuffling method named nonhomologous DNA shuffling. In this program, we will construct an artificial inverting β-glucosidase from an inverting GH43 β-xylosidase by using this technology, combining with a high-throughput screening method and 1H-NMR characterizing of catalytic mechanism. Subsequently, we will analyze the enzymatic properties of the artificial inverting β-glucosidase and investigate the capability for improving the conversion rate in saccharification of cellulose. After the execution of this program, our result will provide a new clue to the lowering the cost of converting cellulosic biomass into liquid fuels.

产物的抑制效应是限制木质纤维素酶酶法降解的瓶颈，至今仍未取得突破性进展。β -葡萄糖苷酶是控制木质纤维素多酶降解的关键节点，已报道所有β -葡萄糖苷酶都是保留型酶，保留型催化是可逆的，决定了β -葡萄糖苷酶不能将底物纤维二糖、纤维三糖等寡糖完全水解，导致中间产物积累，抑制了纤维素酶解效率。反转型糖基水解酶的催化过程是不可逆的，理论上可以实现底物的完全转化。因此，本研究将建立不同家族非同源基因改组技术(nonhomologous DNA shuffling）,应用于GH43反转型β-木糖苷酶和保留型GH3β-葡萄糖苷酶之间的基因改组，定向筛选反转型β-葡萄糖苷酶，进一步研究人工反转型β-葡萄糖苷酶的酶学特性及其抗产物抑制的分子机制，研究成果将为蛋白质体外定向进化提供新的方法，也为提高木质纤维素的糖化率提供新思路。

产物的抑制效应是限制木质纤维素酶酶法降解的瓶颈，至今仍未取得突破性进展。β -葡萄糖苷酶是控制木质纤维素多酶降解的关键节点，已报道所有β -葡萄糖苷酶都是保留型酶，保留型催化是可逆的，决定了β -葡萄糖苷酶不能将底物纤维二糖、纤维三糖等寡糖完全水解，导致中间产物积累，抑制了纤维素酶解效率。糖基水解酶(Glycosyl Hydrolase)存在有两种不同的催化机制，一种是保留型，其酶催化的水解反应是可逆的，酶解反应容易达到平衡状态；另一种是反转型，其催化过程是不可逆的，催化反应一直向水解方向进行，理论上反转型催化可以实现100%转化率。反转型催化是异头位碳原子的构型转化催化机制，即经典的一步法酸/碱催化。水分子被酶活性中心的催化碱激活后，在催化酸的帮助下攻击异头碳中心，随后异头碳上羟基由β构型转变构α构型。本研究首先采用非理性的蛋白质分子进化方法，以GH43 反转型β-木糖苷酶为母本进行基因改组和突变，另一方面我们通过母本蛋白的结晶与结构解析，发现反转型β-木糖苷酶与底物空间距离在5 Å内的氨基酸要远多于保留型β-葡萄糖苷酶，结合保留型与反转型两种催化机制的研究，我们发现反转型酶的两个催化氨基酸之间的距离比保留型酶的距离大，主要原因是反转型酶的催化过程中有水分子的直接参与，需要较大的空间同时容纳底物与水分子，这种机制有助于我们深入系统地分析氨基酸在蛋白结构与功能中的作用，为后续的理性改造与设计提供理论基础。在上述基础上，我们尝试通过Rosetta_design的方法进行反转型β-葡萄糖苷酶的分子构建，设计了约5000个候选蛋白，其中一些具有β-葡萄糖苷酶，但活性较低，需进一步设计与优化，从而提高其催化纤维寡糖的效率。