The yield of reducing sugar after enzymatic hydrolysis is shown for different particle size fractions for each pretreatment regime.

<p>The yield of reducing sugar after enzymatic hydrolysis is shown for different particle size fractions for each pretreatment regime.</p

Cellulosic Biomass Pretreatment and Sugar Yields as a Function of Biomass Particle Size

<div><p>Three lignocellulosic pretreatment techniques (ammonia fiber expansion, dilute acid and ionic liquid) are compared with respect to saccharification efficiency, particle size and biomass composition. In particular, the effects of switchgrass particle size (32–200) on each pretreatment regime are examined. Physical properties of untreated and pretreated samples are characterized using crystallinity, surface accessibility measurements and scanning electron microscopy (SEM) imaging. At every particle size tested, ionic liquid (IL) pretreatment results in greater cell wall disruption, reduced crystallinity, increased accessible surface area, and higher saccharification efficiencies compared with dilute acid and AFEX pretreatments. The advantages of using IL pretreatment are greatest at larger particle sizes (>75 µm).</p></div

Representative plot for nitrogen porosimetry experiments.

<p>Nitrogen adsorption isotherms are shown for 32–50 mesh samples of untreated and pretreated switchgrass.</p

Compositional analysis of untreated and pretreated switchgrass.

<p>Compositional analysis was performed on milled biomass prior to fractionation. Data shown are a representation of three independent measurements (see Material and Methods).</p

SEM images of untreated and pretreated switchgrass.

<p>A –untreated, B – AFEX-pretreated, C – dilute acid pretreated, D – ionic liquid pretreated.</p

Crystallinity index derived from XRD pattern.

<p>Data shown are a representation of three independent measurements (see Material and Methods).</p

BET surface areas of the biomass samples (m²/g).

<p>Data shown are a representation of three independent measurements (see Material and Methods).</p

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

<div><p>Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising <i>in silico</i> selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of <i>Arabidopsis thaliana</i> cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from <i>Arabidopsis thaliana</i>, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.</p></div

Alphabetical list of the CWGTs that were screened.

<p>Alphabetical list of the CWGTs that were screened.</p

<i>Arabidopsis thaliana</i> RGP1 scale-up, purification and activity determinations.

<p>(A) Coomassie stained SDS-PAGE of the final chromatographic step yielding almost pure RGP1 (42 kDa). (B) Phosphate-release assay showing autoglycosylating or hydrolytic activity of RGP1 on UDP-glucose. (C) UDP-arabinose mutase activity of RGP1. High-pressure liquid chromatograms of authentic UDP-arabinopyranose (UDP-Ara<i>p</i>) and UDP-arabinofuranose (UDP-Ara<i>f</i>) standards (grey) overlaid with the chromatogram of the reaction mixture of UDP-Ara<i>f</i> with recombinant, purified RGP1 (black).</p