Location: Bioenergy ResearchTitle: Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756
|DE SOUZA, ANGELICA - Sao Paulo State University (UNESP)|
|DE ARAUJO, GABRIELA - Sao Paulo State University (UNESP)|
|ZANPHORLIN, LETICIA - Brazilian Bioethanol Science & Technology Laboratory|
|RULLER, ROBERTO - Brazilian Bioethanol Science & Technology Laboratory|
|FRANCO, FERNANDA - Sao Paulo State University (UNESP)|
|TORRES, FERNANDO - University Of Brasilia|
|GOMES, ELENI - Sao Paulo State University (UNESP)|
|DA SILVA, ROBERTO - Sao Paulo State University (UNESP)|
Submitted to: International Journal of Biological Macromolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2016
Publication Date: 9/1/2016
Publication URL: http://handle.nal.usda.gov/10113/5519533
Citation: de Souza, A.R., de Araujo, G.C., Zanphorlin, L.M., Ruller, R., Franco, F.C., Torres, F.A.G., Mertens, J.A., Bowman, M.J., Gomes, E., Da Silva, R. 2016. Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756. International Journal of Biological Macromolecules. 93:20-26. doi: 10.1016/j.ijbiomac.2016.08.056.
Interpretive Summary: Agricultural biomass utilization will be required to meet future fuel and specialty chemical needs. Biomass residues are recalcitrant and are typically broken down by chemical pre-treatment and enzymes into useful simple sugars. While there are some issues associated with pretreatment processes, one of the more significant costs associated with biomass utilization is enzyme cost. This is due in part to low temperature stability and therefore reduced activity of these enzymes in pretreated biomass slurries. In this work we have determined amino acids which increase the temperature stability of a xylanase enzyme from the fungus Thermoascus aurantiacus. Xylans are a major component of plant biomass and improved xylanase enzymes are needed to break down xylan to simple sugars. This work also leads to additional knowledge of enzyme structure and function as it relates to the engineering of more stable xylanase enzymes that will ultimately lead to a decrease in the cost of using agricultural crops for the production of high value products.
Technical Abstract: The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis, using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. At 70°C and 75°C, respectively, the three most thermostable mutants (Q158R, H209N, and N257D) retained an average of 31-22%, 46-41%, and 24-10% higher activity than the wild type (WT). Q158R and N257D were stable in the pH range 5.0 – 10.0, while WT and H209N were alkali-thermostable (pH values 8, 9, and 10). Circular dichroism analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular and dynamics modeling analyses suggest that the increase in H209N thermostability is attributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.