title: Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis.
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.
authors.affiliation: Department of Plant and Soil Sciences, Kentucky Tobacco Research and Development Center, University of Kentucky, Lexington, KY, 40546, USA.
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Singh
authors.forename: Chamroeun
authors.initials: C
authors.lastname: Heng
authors.forename: Jay D
authors.initials: JD
authors.lastname: Braker
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Chan
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Jordan
authors.forename: Ling
authors.initials: L
authors.lastname: Yuan
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D019020:Directed Molecular Evolution; D004187:Disaccharides / Q000378:metabolism; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009596:Nitrophenols / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014994:Xylose / Q000235:genetics / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004187:Disaccharides; D009596:Nitrophenols; C024719:4-nitrophenyl; D014994:Xylose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C004173:xylobiose