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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOREFINING PROCESSES

Location: Bioproduct Chemistry and Engineering Research

Title: Catalytic properties of ß-D-xylosidase XylBH43 from Bacillus halodurans C-125 and mutant XylBH43-W147G

Authors
item Wagschal, Kurt
item Jordan, Douglas
item Braker, Jay

Submitted to: Process Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 9, 2011
Publication Date: July 19, 2011
Citation: Wagschal, K.C., Jordan, D.B., Braker, J.D. 2011. Expression, characterization, and site-directed mutagenesis of ß-D-xylosidase XylBH43 from Bacillus halodurans C-125. Process Biochemistry. doi:10.1016/j.procbio.2011.07.009.

Interpretive Summary: The gene encoding a glycoside hydrolase family 43 xylosidase (XylBH43) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a tag useful for purification and the recombinant gene product was expressed in E. coli. A series of artificial substrates was used to determine the substrate specificity of the enzyme. This is important since it serves to illuminate the potential usefulness of the enzyme for breaking down biomass for use as a feedstock for chemicals and fuel. Of the artificial substrates tested, XylBH43 hydrolyzed derivatives of the sugars xylose and arabinose, which are chemically similar but differ in structure. The usefulness of the enzyme under potential biomass utilization process conditions was explored by determining that the pH maximum was 6.5, and thermal instability occurred above 50 °C. XylBH43 was also tested on natural substrates, which gives a clearer indication of the reactions the enzyme can be expected to perform in an industrial setting, and it was found that XylBH43 is the second fastest enzyme known for hydrolysis of xylobiose. A site-directed mutant was prepared, and it was shown that this mutation lowered the affinity of the enzyme for inhibitory xylose product, and also decreased substrate inhibition, at the expense of decreased thermal stability. 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.

Technical Abstract: Recombinant glycoside hydrolase family 43 â-D-xylosidase XylBH43 from Bacillus halodurans C-125 was expressed in E. coli with C-terminal His-tag. Compared to structurally homologous beta-xylosidase SXA, the most active xylosidase known to date, kcat 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, and characterization of the corresponding mutant XylBH43-W147G showed 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 increased and thermal stability at 50C 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.

Last Modified: 4/19/2014
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