P20: Development of a novel thermoacidophilic endo-1,4-β-xylanase

Improved acid hydrolysis methods for the conversion of lignocellulose to fermentable sugars are essential to bringing biofuels to a competitive market. We used a systems biology approach to develop a suite of thermoacidophilic lignocellulosic degrading enzymes from microorganisms that could completely depolymerize hemicellulose under high temperature and low pH conditions. Genomic sequencing of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was used to identify numerous glycosyl hydrolases that were expressed, purified and characterized for substrate utilization as well as optimum pH and temperature. Here we report on the improved expression and purification in Escherichia coli for one of the most important of these enzymes, an endo-1,4-β-xylanase (Xyl)  which has activity on the xylose-β-1,4-xylose linkage of the xylan backbone from hemicellulose. The purified recombinant enzyme (rXyl) had optimum xylanase activity at 70°C and pH 6.0 with specific activity ranging from 102 U/mg (Somogyi-method) to 791U/mg (DNS-method) using wheat arabinoxylan as substrate.  The temperature range of activity was 50 to 80°C and the pH range was 5 to 9. Following optimization for protein expression in E. coli under batch fermentation conditions >170 mg/g cell mass rXyl was produced and ~65% purified to homogeneity by affinity chromatography. The activity range of this enzyme makes it potentially useful in industrial applications including biofuel production, animal feed processing, and paper brightening and fluffing.