Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover.
Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover.
复制标题
DOI：
10.1186/s13068-017-0757-5
复制
发表时间：
2017
影响因子：
6.3
通讯作者：
Balan V
中科院分区：
文献类型：
--
作者：
Gunawan C;Xue S;Pattathil S;da Costa Sousa L;Dale BE;Balan V
低效率的碳水化合物转化一直是各种木质纤维素生物质预处理技术（包括AFEX、稀酸和离子液体预处理）未解决的问题。先前的工作已经表明，总碳水化合物的22%通常未转化，在用过量的商业酶负载（20 mg酶/g生物质）水解（72 h）后保留为可溶性或不溶性低聚物。这些总未转化碳水化合物中的近三分之一（22%中的7个）存在于未水解固体（UHS）残留物中。这些未转化的碳水化合物的存在导致相当大的糖产率损失，这对生物炼制的整体经济性产生负面影响。目前的商业酶混合物不能有效地消化植物细胞壁聚糖中的特定交联，特别是存在于半纤维素和果胶中的那些。因此，获得关于最易水解的非纤维素聚糖交联的信息成为通过补充水解那些未转化的聚糖所需的酶活性来合理地改进商业酶混合物的关键研究。在这项工作中，不能从AFEX预处理的玉米秸秆（CS）酶促转化为单糖的细胞壁聚糖，其特征在于使用组成分析和糖组分析工具。使用包含纤维素酶和半纤维素酶的商业酶混合物以7%葡聚糖负载（~20%固体负载）水解预处理的CS。在168小时酶水解的时间段内评估UHS和液体水解产物中存在的碳水化合物。细胞壁聚糖特异性单克隆抗体（mAb）被用来表征的类型和丰富的非纤维素多糖存在于UHS在酶水解的过程中。发现4-O-甲基-D-葡萄糖醛酸取代的木聚糖和果胶-阿拉伯半乳聚糖是UHS和液体水解物中mAb识别的最丰富的表位，这表明在这项工作中使用的商业酶混合物不能有效地靶向那些取代的多糖残基。据我们所知，这是第一个报告使用糖组分析作为一种工具，动态监测柠檬酸细胞壁碳水化合物在酶水解过程中。UHS和液体水解产物的糖组分析揭示了一些在酶水解后未被切割和富集的聚糖。主要的多糖包括4-O-甲基-D-葡糖醛酸取代的木聚糖和果胶-阿拉伯半乳聚糖，这表明需要具有葡糖醛酸糖苷酶和阿拉伯呋喃糖苷酶活性的酶来最大化单糖产量。这种方法提供了一种快速的工具，以帮助开发新的酶鸡尾酒，通过补充现有的鸡尾酒与所需的酶活性，实现预处理的生物质在未来的完全解构。本文的在线版本（doi：10.1186/s13068-017-0757-5）包含补充材料，可供授权用户使用。
Inefficient carbohydrate conversion has been an unsolved problem for various lignocellulosic biomass pretreatment technologies, including AFEX, dilute acid, and ionic liquid pretreatments. Previous work has shown 22% of total carbohydrates are typically unconverted, remaining as soluble or insoluble oligomers after hydrolysis (72 h) with excess commercial enzyme loading (20 mg enzymes/g biomass). Nearly one third (7 out of 22%) of these total unconverted carbohydrates are present in unhydrolyzed solid (UHS) residues. The presence of these unconverted carbohydrates leads to a considerable sugar yield loss, which negatively impacts the overall economics of the biorefinery. Current commercial enzyme cocktails are not effective to digest specific cross-linkages in plant cell wall glycans, especially some of those present in hemicelluloses and pectins. Thus, obtaining information about the most recalcitrant non-cellulosic glycan cross-linkages becomes a key study to rationally improve commercial enzyme cocktails, by supplementing the required enzyme activities for hydrolyzing those unconverted glycans. In this work, cell wall glycans that could not be enzymatically converted to monomeric sugars from AFEX-pretreated corn stover (CS) were characterized using compositional analysis and glycome profiling tools. The pretreated CS was hydrolyzed using commercial enzyme mixtures comprising cellulase and hemicellulase at 7% glucan loading (~20% solid loading). The carbohydrates present in UHS and liquid hydrolysate were evaluated over a time period of 168 h enzymatic hydrolysis. Cell wall glycan-specific monoclonal antibodies (mAbs) were used to characterize the type and abundance of non-cellulosic polysaccharides present in UHS over the course of enzymatic hydrolysis. 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan were found to be the most abundant epitopes recognized by mAbs in UHS and liquid hydrolysate, suggesting that the commercial enzyme cocktails used in this work are unable to effectively target those substituted polysaccharide residues. To our knowledge, this is the first report using glycome profiling as a tool to dynamically monitor recalcitrant cell wall carbohydrates during the course of enzymatic hydrolysis. Glycome profiling of UHS and liquid hydrolysates unveiled some of the glycans that are not cleaved and enriched after enzyme hydrolysis. The major polysaccharides include 4-O-methyl-d-glucuronic acid-substituted xylan and pectic-arabinogalactan, suggesting that enzymes with glucuronidase and arabinofuranosidase activities are required to maximize monomeric sugar yields. This methodology provides a rapid tool to assist in developing new enzyme cocktails, by supplementing the existing cocktails with the required enzyme activities for achieving complete deconstruction of pretreated biomass in the future. The online version of this article (doi:10.1186/s13068-017-0757-5) contains supplementary material, which is available to authorized users.