Expression in Escherichia coli and characterization of beta-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 Ni(2+)-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 degrees 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 microg/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 microg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.
Smaali
Issam
I
Institut National de la Recherche Agronomique, UMR 614, 8 rue Gabriel Voisin, B.P. 316, 51688 Reims, Cedex 2, France.
Rémond
Caroline
C
O'Donohue
Michael J
MJ
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004043:Dietary Fiber / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004043:Dietary Fiber; D011994:Recombinant Proteins; D014995:Xylosidases