Mechanically stable porous cellulose media for affinity purification of family 9 cellulose-binding module-tagged fusion proteins.

A mechanically stable cellulose-based chromatography media was synthesized to permit inexpensive affinity purification of recombinant proteins containing the family 9 carbohydrate-binding module (CBM9) fused to either the N- or C-terminus of the target protein. A second-order response surface model was used to identify optimal concentrations of the primary reactants, epichlorohydrin and dimethyl sulfoxide (DMSO), required to cross-link the starting material, Perloza MT100, a compressible but inexpensive cellulose-based chromatography resin. This resulted in a mechanically stable cross-linked affinity chromatography media capable of operating at an order-of-magnitude higher linear velocity than permitted by unmodified MT100. Moments and Van Deemter analyses were used to show that rates of solute mass transfer within the column are largely unaffected by the cross-linking reaction, while the binding capacity decreased by 20% to 7.1 micromol of protein/g resin, a value superior to most commercial affinity chromatography media. In sharp contrast to MT100, the mechanical stability and purification performance of the cross-linked media are not diminished by scale-up or repeated column use.

Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: optimization of process yields.

An organosolv process involving extraction with hot aqueous ethanol has been evaluated for bioconversion of hybrid poplar to ethanol. The process resulted in fractionation of poplar chips into a cellulose-rich solids fraction, an ethanol organosolv lignin (EOL) fraction, and a water-soluble fraction containing hemicellulosic sugars, sugar breakdown products, degraded lignin, and other components. The influence of four independent process variables (temperature, time, catalyst dose, and ethanol concentration) on product yields was analyzed over a broad range using a small composite design and response surface methodology. Center point conditions for the composite design (180 degrees C, 60 min, 1.25% H(2)SO(4), and 60% ethanol), yielded a solids fraction containing approximately 88% of the cellulose present in the untreated poplar. Approximately 82% of the total cellulose in the untreated poplar was recovered as monomeric glucose after hydrolysis of the solids fraction for 24 h using a low enzyme loading (20 filter paper units of cellulase/g cellulose); approximately 85% was recovered after 48 h hydrolysis. Total recovery of xylose (soluble and insoluble) was equivalent to approximately 72% of the xylose present in untreated wood. Approximately 74% of the lignin in untreated wood was recovered as EOL. Other cooking conditions resulted in either similar or inferior product yields although the distribution of components between the various fractions differed markedly. Data analysis generated regression models that describe process responses for any combination of the four variables.