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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Publications at this Location » Publication #284255

Title: Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

item Jordan, Douglas
item Wagschal, Kurt
item GRIGORESCU, ARABELA - Northwestern University
item Braker, Jay

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2012
Publication Date: 10/26/2012
Citation: Jordan, D.B., Wagschal, K.C., Grigorescu, A.A., Braker, J.D. 2013. Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates. Applied Microbiology and Biotechnology. 97:4415-4428.

Interpretive Summary: Enzymatic saccharification of plant biomass is deemed the most economical route for providing fermentable sugars that can be converted to transportation fuels and other bioproducts. Given the scale of the endeavor, it behooves us to obtain the most economically efficient enzymes available. This work focuses on discovering highly active ß-xylosidases, which act in the breakdown of xylan, the second most abundant polysaccharide after cellulose in plant biomass. Among the four ß-xylosidases examined, one of them is the most active enzyme known, by a factor of two, that acts on natural substrates, xylobiose, xylotriose, and xylotetraose. The enzyme has good stability and protein production characteristics that should translate its activity in the bioreactor to a factor of two improvement.

Technical Abstract: The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. ß-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 has been identified as a source of highly active ß-xylosidases. Biochemical details of four GH43 ß-xylosidases are examined here. Sedimentation equilibrium experiments indicate the quaternary state of three of the enzymes are monomer, homodimer, or homotetramer. kcat and kcat/Km of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of some GH43 ß-xylosidases. Ki values for D-glucose (83.3 to 357 mM) and D-xylose (15.6 to 70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (Kt^0.5^ ~ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from Lactobacillus brevis ATCC 367 displays the highest reported kcat and kcat/Km on natural substrates xylobiose (407 s^-1^, 138 s^-1^mM^-1^), xylotriose (235 s^-1^, 80.8 s^-1^mM^-1^) and xylotetraose (146 s^-1^, 32.6 s^-1^mM^-1^).