Enzyme accessibility of xylan polysaccharides in plant cell wall biomass

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)