Gunawan Christa, Xue Saisi, Pattathil Sivakumar, da Costa Sousa Leonardo, Dale Bruce E, Balan Venkatesh
Biomass Conversion Research Lab (BCRL), Chemical Engineering and Materials Science, Michigan State University, 3815 Technology Boulevard, Lansing, MI 48910 USA.
DOE Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, East Lansing, MI USA.
Biotechnol Biofuels. 2017 Mar 29;10:82. doi: 10.1186/s13068-017-0757-5. eCollection 2017.
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--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--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.
对于包括氨纤维膨胀（AFEX）、稀酸和离子液体预处理在内的各种木质纤维素生物质预处理技术而言，碳水化合物转化效率低下一直是个未解决的问题。此前的研究表明，通常有22%的总碳水化合物未被转化，在用过量商业酶负载量（20毫克酶/克生物质）水解（72小时）后，这些碳水化合物仍以可溶性或不溶性低聚物的形式存在。在这些未转化的总碳水化合物中，近三分之一（22%中的7%）存在于未水解固体（UHS）残渣中。这些未转化碳水化合物的存在导致了相当大的糖产率损失，对生物炼制的整体经济性产生了负面影响。目前的商业酶混合物无法有效消化植物细胞壁聚糖中的特定交联键，尤其是半纤维素和果胶中存在的一些交联键。因此，获取最难降解的非纤维素聚糖交联键的信息成为一项关键研究，以便通过补充水解那些未转化聚糖所需的酶活性来合理改进商业酶混合物。
在这项工作中，使用成分分析和糖组分析工具对无法通过酶促转化为单糖的AFEX预处理玉米秸秆（CS）中的细胞壁聚糖进行了表征。使用包含纤维素酶和半纤维素酶的商业酶混合物在7%葡聚糖负载量（约20%固体负载量）下对预处理的CS进行水解。在168小时的酶水解过程中评估了UHS和液体水解产物中存在的碳水化合物。使用细胞壁聚糖特异性单克隆抗体（mAb）来表征酶水解过程中UHS中存在的非纤维素多糖的类型和丰度。发现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是UHS和液体水解产物中mAb识别的最丰富表位，这表明本工作中使用的商业酶混合物无法有效靶向那些取代的多糖残基。
据我们所知，这是第一份使用糖组分析作为工具在酶水解过程中动态监测难降解细胞壁碳水化合物的报告。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酶和阿拉伯呋喃糖苷酶活性的酶来最大化单糖产率。这种方法提供了一种快速工具，通过在现有混合物中补充所需的酶活性，以协助开发新的酶混合物，从而在未来实现对预处理生物质的完全解构。
