Bioenergy/Biofuels/Biochemicals

Journal of Industrial Microbiology & Biotechnology

, Volume 41, Issue 3, pp 489-498

Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis

  • Sanjay K. SinghAffiliated withDepartment of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky
  • , Chamroeun HengAffiliated withUSDA Agricultural Research Service, Western Regional Research Center
  • , Jay D. BrakerAffiliated withUSDA Agricultural Research Service, National Center for Agricultural Utilization Research
  • , Victor J. ChanAffiliated withUSDA Agricultural Research Service, Western Regional Research Center
  • , Charles C. LeeAffiliated withUSDA Agricultural Research Service, Western Regional Research Center
  • , Douglas B. JordanAffiliated withUSDA Agricultural Research Service, National Center for Agricultural Utilization Research
  • , Ling YuanAffiliated withDepartment of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky Email author 
  • , Kurt WagschalAffiliated withUSDA Agricultural Research Service, Western Regional Research Center Email author 

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Abstract

Directed evolution of β-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K t 0.5 by −1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K t 0.5 by ~3 °C; none of the individual amino acid changes measurably affect K t 0.5 . Saturation mutagenesis of L186 identified the variant L186K as having the most improved K t 0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K t 0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k cat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K m, K ss (substrate inhibition), and K i (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Keywords

Glycosyl hydrolase Directed evolution Gene shuffling Thermal stability Protein engineering