Catalytic properties of β-d-xylosidase XylBH43 from Bacillus halodurans C-125 and mutant XylBH43-W147G

Abstract

Recombinant glycoside hydrolase family 43 member β-d-xylosidase XylBH43 from Bacillus halodurans C-125 was expressed in Escherichia coli with C-terminal His-tag. Compared to structurally homologous β-xylosidase SXA, another GH43 member and the most active xylosidase known to date, the kcat value for xylobiose hydrolysis was 2-fold lower, xylotriose and xylotetraose kcat values were similar, and binding affinities for inhibitors xylose and glucose were 10-fold lower and 1.5-fold higher, respectively. Mutant SXA-W145G had previously been shown to exhibit reduced monosaccharide binding. Characterization of the corresponding mutant XylBH43-W147G shows a similar 2.6-fold Ki(d-xylose) increase. In addition, the Trp mutant displayed lowered substrate inhibition for both natural and artificial substrates, while Km of substrates increased and thermal stability at 50°C decreased ∼100-fold. The superior xylooligosaccharide kcat values for XylBH43 make this enzyme valuable both as a saccharification enzyme, and as a source of genetic diversity in on-going protein engineering efforts targeted at optimizing GH43 enzymes for biomass saccharification.