Multifect xylanase

For the enzymatic hydrolysis, Accellerase 1500 (78.1 mg BCA protein/ml; lot 1662334068; DuPont, Palo Alto, CA, USA) and Multifect xylanase (45.4 mg BCA protein/ml; lot 301-04296-205; DuPont, Palo Alto, CA, USA) were used. The loadings were 80 mg and 20 mg/g biomass for the Accellerase and Multifect enzymes, respectively. Hydrolysis was conducted at 50 °C for 70 h, with 0.02% sodium azide in 140 mM pH 5.0 citrate buffer. Glucose and xylose release values were then measured using GOPOD and XDH enzymatic assays according to the manufacturer’s instructions (Megazyme, Bray, Ireland).

The deacetylated corn stover solids were washed with 20 mM sodium acetate and 100 mM sodium chloride buffer (pH 4.8) solution. The commercial enzyme product (Multifect^® Xylanase from DuPont™ Genencor^®, Palo Alto, California, United States) was desalted in 10 mL aliquots using two serial HiPrep 26/10 desalting columns (GE Life Sciences, Piscataway NJ) equilibrated in 20 mM sodium acetate buffer (pH 4.8) with 100 mM sodium chloride. Protein containing fractions were pooled and protein concentration determined using the BCA protein assay (Pierce Rockford, IL). Enzyme samples were desalted less than 2 days before use, with fresh material being generated for each experiment as desalted commercial enzymes tend to degrade and precipitate within a few days. The enzyme solution was added to same buffer solution containing deacetylated corn stover solids at 1% with enzyme loading at 3.6–58 mg/g xylan. Enzymatic digestions were performed in 2.5 mL shaker tubes in a shaking incubator at 45 °C and 150 rpm for up to 40 h.