Engineering increased thermostability in the GH-10 endo-1,4-beta-xylanase from Thermoascus aurantiacus CBMAI 756

Author(s)

de Souza, Angelica R. ; de Araujo, Gabriela C. ; Zanphorlin, Leticia M. ; Ruller, Roberto ; Franco, Fernanda C. ; Torres, Fernando A. G. ; Mertens, Jeffrey A. ; Bowman, Michael J. ; Gomes, Eleni ; Da Silva, Roberto

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 93, p. 7-pg., 2016-12-01.

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 degrees C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70 degrees C and 75 C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD 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 degrees C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure. (C) 2016 Elsevier B.V. All rights reserved. (AU)

• FAPESP's process: 14/04272-8 - Engineering increased thermostability in the thermostable GH-10 xylanase from Thermoascus aurantiacus, by epPCR and site-directed mutagenesis
• Grantee: Angelica Rodrigues de Souza
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 10/12624-0 - Application of physical-chemical and enzymatic methods in the saccharification of sugar cane bagasse: studies on microorganisms, fermentative processes and hydrolyses methods
• Grantee: Eleni Gomes
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 11/17658-3 - Computational studies on protein folding and applications in the study of enzymes involved in bioethanol production
• Grantee: Vitor Barbanti Pereira Leite
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/02080-4 - Production of ethanol from bagasse sugarcane pretreated with ozone: study of inhibitors, fermentation and industrial yeasts
• Grantee: Roberto da Silva
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants