Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two beta-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)(6)-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni2+-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 C for XylBH39 and pH 8 and 60 degrees C for XylBH43) and were relatively stable over the pH range of 3.5-8.5. In contrast, XylBH39 was more thermostable. At 60 degrees C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k(cat)/K-M revealed that XylBH43 hydrolyzed p-nitrophenyl-beta-D-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-alpha-L-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-beta-D-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 mu g/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an alpha-L-arabinofuranosidase. Together, these enzymes were able to release 521 mu g/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.