(599c) Purification and Characterization of a GH11 Xylanase from Biobutanol-producing Clostridium beijerinckii G117
Most biobutanol-producing Clostridium species do not express the necessary enzymes to hydrolyze polysaccharides (cellulose, xylan etc.) and thus rendering them unable to ferment lignocellulose directly for biofuel production. In this study, we show that a biobutanol-producing C. beijerinckii strain G117 can express natively endo-1,4-beta-xylanase, which hydrolyze xylan, into culture medium in the presence of 1% beechwood xylan. Xylanase activity can reach up to 2.66 U/ml within 14 h of fermentation. By using salting-out and size-exclusion chromatography, crude xylanase from the supernatant can be purified 8.7-fold with an overall yield of 32.2%. This purified xylanase has a molecular weight of 22.6 kDa, making it one of the smallest clostridial xylanases reported in literature. Conserved domain analysis reveals that the xylanase belongs to glycoside hydrolase family 11 (GH11) without any carbohydrate-binding domain (CBD). When beechwood xylan is used as substrate for the purified enzyme, only xylo-oligosaccharides are produced, suggesting that this is an endo-xylanase. Km and Vmax of this hydrolytic reaction is 19.1 mg/ml and 2766 U/mg, respectively, but the enzyme activity can be further enhanced by adding 10 mM Co2+, 1 mM Mn2+ or 50 mM cysteine.