Enzyme accessibility of xylan polysaccharides in plant cell wall biomass

纤维素生物能源的一个主要技术挑战是有效和廉价地从纤维素和其他多糖中释放糖。试点生物燃料生产过程涉及使用能源密集型苛刻的生物质预处理，并且还需要添加大量的酶来分解生物质。人们普遍认为木聚糖可以排列在纤维素微原纤维的表面。表面木聚糖的酶降解是重要的，因为木聚糖限制了酶促进入纤维素微原纤维，木聚糖转化为木糖为生物燃料生产提供了额外的底物。木聚糖本身被其他成分（如木质素）的存在所掩盖，不受酶的攻击。我们最近的发现表明，在纤维素的亲水性和疏水性表面上，两个离散的木聚糖结构域以不同的构象与纤维素相关联（Bromley et al. 2013, Busse Wicher et al. 2014）。因此，这些木聚糖结构域将不同地易受生物质预处理的影响，并且将不同地容易受到酶的攻击。为了了解酶对木聚糖的接近是如何受到细胞壁结构和组成的影响，学生将测量木聚糖对探针和酶的接近程度。可及性可能受到物理限制（木质纤维素的“开放性”）、一种聚合物被另一种聚合物屏蔽以及木聚糖的构象和结构的影响。检测将使用诺维信提供的广泛范围的特异性重组水解酶和多糖氧化酶（如纤维素酶或木聚糖酶），以及其他广泛可用的酶。测定将测量通过化学或酶的手段去除不同细胞壁成分的影响。在诺维信安置期间，以工业相关方式进行预处理的植物材料将包括在这项工作中。第二，一组转基因植物将提供不同成分的木质纤维素样品。方法、途径、里程碑：学生将准备一组生物质样品，其中包括木质素缺乏、纤维素结晶度缺乏、木聚糖缺乏和葡甘露聚糖缺乏的改性成分。我们将在实验室中使用许多现有的拟南芥突变体。学生将通过遗传杂交产生更多有缺陷的细胞壁组合（0-12个月）。突变体的细胞壁组成将通过糖分析（HPLC）、酶谱分析（PACE、DASH、质谱）和木质素定量（6个月；新突变体18个月）进行表征。将比较使用诺维信不同酶的野生型植物对木聚糖的消化率，以建立基线可及性。将选择对细胞壁的处理，以尽量减少对木聚糖和纤维素排列的破坏。木聚糖的消化率将通过测定天然细胞壁酶解释放的低聚糖来评估（第12个月）。将在诺维信研究木质纤维素突变体和预处理作物残留物中木聚糖对酶的可及性（第24个月）。将通过产生生物量突变体的新组合和使用额外的降解酶进行探测来研究可及性方面最有趣的变化（第36个月）。其他探针（如抗体或CBMs）的可及性研究（第36个月）

A major technological challenge for cellulosic bioenergy is to release the sugars from cellulose and other polysaccharides effectively and cheaply. Pilot biofuel production processes involve the use of energy-intensive harsh biomass pretreatments, and also require the addition of high quantities of enzymes to break down the biomass.It is widely accepted that xylan can be arrayed on the surface of cellulose microfibrils. The enzymatic degradation of surface xylan is important as xylan restricts enzymatic access to the cellulose microfibrils, and the conversion of xylan into xylose provides an additional substrate for biofuel production. The xylan is itself masked from enzymatic attack by the presence of other components, such as lignin. Our recent discoveries suggest that two discrete xylan domains associate with cellulose in different conformations on the hydrophilic vs. the hydrophobic surfaces of cellulose (Bromley et al. 2013, Busse Wicher et al. 2014). Consequently, these xylan domains will be differently susceptible to biomass pretreatments, and will be differently accessible to enzymatic attack.To understand how access of enzymes to xylan is affected by cell wall structure and composition, the student will measure xylan accessibility to probes and enzymes. The accessibility is likely influenced by physical constraints ('openness' of the lignocellulose), the shielding of one polymer by another, as well as the conformation and structure of the xylan. Assays will use the extensive range of specific recombinant hydrolases and polysaccharide oxidases (e.g. cellulases or xylanases) available from Novozymes, and other enzymes that are widely available. Assays will measure the affect of removal through chemical or enzymatic means, of different cell wall components. Plant material which has been pretreated in industrially relevant ways will be included in this work during the placement at Novozymes. Second, a panel of genetically modified plants will provide lignocellulose samples with different compositions.Methods, approaches, milestones:The student will prepare a panel of biomass samples with modified composition of lignin deficiency, cellulose crystallinity, xylan deficiency and glucomannan deficiency. We will use many of the existing Arabidopsis mutants available in the laboratory. The student will generate more combinations of defective cell walls through genetic crosses (0-12 months).The cell wall composition of the mutants will be characterised by sugar analysis (HPLC), enzymatic profiling (PACE, DASH, Mass spectrometry) and lignin quantification (month 6; new mutants month 18).The digestibility of xylan in wild type plants using different enzymes from Novozymes will be compared to establish baseline accessibility. Treatments of cell walls will be selected that minimise disruption to the arrangement of xylan and cellulose. The digestibility of xylan will be assessed by measuring oligosaccharides released by enzymatic digestion of native cell walls (month 12).Xylan accessibility to enzymes will be studied in the lignocellulose mutants and in pretreated crop residues at Novozymes (month 24). The most interesting alterations in accessibility will be investigated by generating new combinations of biomass mutants and probing with additional degradative enzymes (month 36).Studies of accessibility to other probes, such as antibodies or CBMs (month 36)