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Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. One property that could use improvement is its relatively high affinities for d-glucose and d-xylose (K i ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K d-glucosei and K d-xylosei twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k d-xylobiosecat and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K i values that are 1.5–2.7-fold (d-glucose) and 1.9–4.6-fold (d-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k d-xylobiosecat than that of the wild type. At 0.1 M d-xylobiose and 0.1 M d-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.

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Abbreviations

SXA:

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium

4NPX:

4-nitrophenyl β-d-xylopyranoside

4NPA:

4-nitrophenyl α-l-arabinofuranoside

MUX:

4-methylumbelliferyl β-d-xylopyranoside

X2:

D-xylobiose

SXA-C3:

SXA containing these mutations W145G, T265A, P328L, and N516D

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Authors and Affiliations

  1. National Center for Agricultural Utilization Research, USDA Agricultural Research Service, 1815 N. University Street, Peoria, IL, 61604, USA

    Douglas B. Jordan & Jay D. Braker

  2. Western Regional Research Center, USDA Agricultural Research Service, Albany, CA, 94710, USA

    Kurt Wagschal & Chamroeun Heng

  3. Kentucky Tobacco Research and Development Center, Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA

    Zhanmin Fan & Ling Yuan

Authors
  1. Douglas B. Jordan
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  2. Kurt Wagschal
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  3. Zhanmin Fan
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  4. Ling Yuan
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  5. Jay D. Braker
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  6. Chamroeun Heng
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Correspondence to Douglas B. Jordan.

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The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.

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Jordan, D.B., Wagschal, K., Fan, Z. et al. Engineering lower inhibitor affinities in β-d-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145. J Ind Microbiol Biotechnol 38, 1821–1835 (2011). https://doi.org/10.1007/s10295-011-0971-2

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  • DOI: https://doi.org/10.1007/s10295-011-0971-2

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