Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability.
Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-beta-1,4-xylanase from Bacillus subtilis under acidic conditions. Since the pH dependence of protein stability is governed by the ionisation states of the side chains of its titrable amino acid residues, we explored the strategy of changing pH-stability profiles by altering pK(a) values of key residues through in silico designed mutations. To this end, computational predictions and molecular modelling were carried out using the recently developed pKD software package. Four endoxylanase variants, in which the pK(a) values of either Asp4 and Asp11 or His149 were targeted to shift downwards through incorporation of three to five point mutations, were generated and recombinantly expressed in the cytoplasm of Escherichia coli. All four mutants showed considerably increased functional stability at acid pH levels. They retained approximately 30-70% and approximately 75-95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only approximately 23% and approximately 57%, respectively, for the wild-type enzyme under the experimental conditions. No acidophilic adaptation of the catalytic activity had occurred. In addition, their functional stability and catalytic activity profiles under different temperature and ionic strength conditions were significantly altered. These findings contribute to general understanding of the molecular mechanisms governing the pH-dependent stability of GH11 proteins, and hence they can be applied to enhance the stability and effectiveness of many GH11 endoxylanases used in industry today.
Beliën
Tim
T
Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Center (LFORCE), Katholieke Universiteit Leuven, Leuven, Belgium. tim.belien@pcfruit.be
Joye
Iris J
IJ
Delcour
Jan A
JA
Courtin
Christophe M
CM
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009994:Osmolar Concentration; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases