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United States Department of Agriculture

Agricultural Research Service

Research Project: Biorefining Processes

Location: Bioproduct Chemistry and Engineering Research

Title: Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation

Authors
item Singh, Sanjay -
item Heng, Chamroeun
item Braker, Jay
item Chan, Victor
item Lee, Charles
item Jordan, Douglas
item Yuan, Ling -
item Wagschal, Kurt

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 24, 2013
Publication Date: November 29, 2013
Repository URL: http://dx.doi.org/10.1007/s10295-013-1377-0
Citation: Singh, S.K., Heng, C., Braker, J.D., Chan, V.J., Lee, C.C., Jordan, D.B., Yuan, L., Wagschal, K.C. 2013. Directed evolution of GH43 ß-xylosidase XylBH43 thermal stability and L186 saturation. Journal of Industrial Microbiology and Biotechnology. 41(3):489-498. DOI: 10.1007/s10295-013-1377-0.

Interpretive Summary: Hemicellulose is a sugar polymer that is a major component of the woody part of plants, and represents a significant renewable source of chemical energy. It is necessary to break hemicellulose polymers down into simpler component sugars that can be converted by other processes to more useable forms, e.g. bioethanol. To do so enzymatically requires a suite of different enzymes, a critical one of which is termed a xylosidase. The naturally occurring xylosidase XylBH43 is able to efficiently hydrolyze its natural substrate xylobiose, but loses activity due to thermal inactivation at temperatures above 50 ºC. We describe here the use of in-vitro evolution as an enzyme engineering tool to generate XylBH43 mutants with up to 8.8'C±0.3 ºC increases in a thermal stability parameter which measures the temperature at which the enzyme loses 50% of its activity in an hour.

Technical Abstract: Directed evolution of ß-xylosidase XylBH43 using DNA family shuffling identified three mutations R45K, M69P, and L186Y that affect thermal stability parameter Kt0.5 by -1.8±0.1 º C, 1.7±0.3 º C, and 3.2±0.4 º C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved Kt0.5 by ~3'C; none of the individual amino acid changes measurably affect Kt0.5. Saturation mutagenesis of L186 identified variant L186K as having Kt0.5 most improved, by 8.1±0.3 ºC. The L186Y mutation was found to be additive, resulting in Kt0.5 increasing by up to 8.8'C±0.3 ºC when several beneficial mutations were combined. While kcat of xylobiose and 4-nitrophenyl-'-D-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, Km, Kss (substrate inhibition), and Ki (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.

Last Modified: 9/22/2014
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