FIGURE 5.

B. eggerthii ORF1299 and B. intestinalis ORF4213 encode endoxylanases. A, purification of recombinant BeXyn5A and BiXyn5A. B. eggerthii ORF1299 and B. intestinalis ORF4123 were cloned into expression vectors and expressed heterologously as hexahistidine fusion proteins in E. coli. The proteins were purified using cobalt affinity chromatography and gel filtration, and the elution fractions were pooled and analyzed by 12% SDS-PAGE, followed by Coomassie Brilliant Blue G-250 staining. B, thin layer chromatography of products released from WAX by BeXyn5A and BiXyn5A. BeXyn5A or BiXyn5A (0.50 μm each) was incubated with WAX (1% w/v) for 15 h at 37 °C, and the products were resolved by thin layer chromatography followed by staining with methanolic orcinol. Xylo-oligosaccharide standards X₁–X₅ and arabinose (A1) were spotted on the plate in lanes 2 and 1, respectively, to serve as markers for the identification of hydrolysis products. C, reducing sugars released from sWAX by BeXyn5A and BiXyn5A. BeXyn5A or BiXyn5A (0.50 μm each) was incubated with WAX (1% w/v) for 15 h at 37 °C, and the reducing sugars were detected by using the para-hydroxybenzoic acid hydrazide assay. The reducing sugar concentrations were calculated from the absorbance at 410 nm by comparison with a standard curve generated with known concentrations of glucose. C, the values are reported as the means ± S.D. from three independent experiments.