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。中试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。广泛认为木聚糖可以排列在纤维素微纤维的表面上。表面木聚糖的酶促降解是重要的，因为木聚糖限制酶促接近纤维素微纤维，并且木聚糖转化成木糖为生物燃料生产提供了额外的底物。木聚糖本身被其他组分如木质素的存在所掩盖而不受酶的攻击。我们最近的发现表明，两个离散的木聚糖结构域在纤维素的亲水表面和疏水表面上以不同的构象与纤维素缔合（Bromley等人2013，Busse Wicher等人2014）。因此，这些木聚糖结构域对生物质预处理的敏感性不同，对酶攻击的可及性也不同。为了了解酶对木聚糖的可及性如何受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可及性。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。测定将使用广泛范围的可从Novozymes获得的特定重组水解酶和多糖氧化酶（例如纤维素酶或木聚糖酶）以及广泛可获得的其他酶。测定将测量通过化学或酶促方法去除不同细胞壁组分的影响。在Novozymes放置期间，已以工业相关方式预处理的植物材料将被包括在这项工作中。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，其中木质素缺乏，纤维素结晶度，木聚糖缺乏和葡甘露聚糖缺乏的成分经过修改。我们将使用实验室中现有的许多拟南芥突变体。学生将通过基因杂交产生更多有缺陷的细胞壁组合突变体的细胞壁组成将通过糖分析（HPLC）、酶谱分析（HPLC）和酶谱分析来表征。（PACE、DASH、质谱）和木质素定量（第六个月;新的突变体月18）将使用来自Novozymes的不同酶比较野生型植物中木聚糖的消化率以建立基线可及性。将选择细胞壁的处理以使对木聚糖和纤维素排列的破坏最小化。木聚糖的消化率将通过测量天然细胞壁的酶消化释放的寡糖来评估（第12个月）。木聚糖对酶的可及性将在诺维信的木质纤维素突变体和预处理的作物残留物中进行研究（第24个月）。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，将研究最有趣的可及性变化（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)