Porter Stephanie E, Donohoe Bryon S, Beery Kyle E, Xu Qi, Ding Shi-You, Vinzant Todd B, Abbas Charles A, Himmel Michael E
Chemical and Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, USA.
Biotechnol Bioeng. 2007 Sep 1;98(1):123-31. doi: 10.1002/bit.21409.
Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.
乙醇是当今美国从可再生原料生产的主要液体运输燃料。玉米谷物是乙醇生产的主要原料，历史上大部分玉米谷物都在湿磨厂进行加工，生产出谷朊粉饲料、谷朊粉、淀粉和胚芽等产品。从谷物中提取的淀粉用于在糖化和发酵步骤中生产乙醇；然而，淀粉的提取效率并非100%。为了更好地了解湿磨过程中的淀粉提取，我们开发了荧光探针，可用于通过共聚焦显微镜在样品中直观地定位淀粉和纤维素。这些探针基于两种碳水化合物结合模块（CBM）的结合特异性，CBM是源自碳水化合物降解酶的小的底物特异性蛋白质结构域。使用分子克隆技术将CBM与绿色荧光蛋白（GFP）或红色荧光蛋白DsRed（RFP）融合。使用这些工程探针，我们发现淀粉特异性探针的结合与玉米纤维样品中的淀粉含量相关。我们还证明胚乳内部存在淀粉，这可能是玉米纤维中淀粉含量高的原因。我们还意外地发现，纤维素特异性探针并不与大多数玉米纤维样品结合，而只与经过热化学过程水解以去除残留淀粉和大部分半纤维素的玉米纤维结合。我们的研究结果应该会引起那些致力于提高玉米谷物转化为乙醇效率的人的兴趣。
