title: Engineering increased thermostability in the GH-10 endo-1,4-β-xylanase from Thermoascus aurantiacus CBMAI 756.
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. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°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°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.
authors.affiliation: UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil.
authors.forename: Angelica R
authors.initials: AR
authors.lastname: de Souza
authors.affiliation: UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil.
authors.forename: Gabriela C
authors.initials: GC
authors.lastname: de Araújo
authors.affiliation: CTBE (Brazilian Bioethanol Science and Technology LaboratoryatNational Center for Research in Energy and Materials), Campinas, SP 13083-100, Brazil.
authors.forename: Letícia M
authors.initials: LM
authors.lastname: Zanphorlin
authors.affiliation: CTBE (Brazilian Bioethanol Science and Technology LaboratoryatNational Center for Research in Energy and Materials), Campinas, SP 13083-100, Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.affiliation: UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil.
authors.forename: Fernanda C
authors.initials: FC
authors.lastname: Franco
authors.affiliation: University of Brasília, Department of Cell Biology - Brasília, Distrito Federal, Brazil.
authors.forename: Fernando A G
authors.initials: FA
authors.lastname: Torres
authors.affiliation: Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA - ARS, 1815 N, University St, Peoria, IL 61604, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Mertens
authors.affiliation: Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA - ARS, 1815 N, University St, Peoria, IL 61604, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bowman
authors.affiliation: UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil.
authors.forename: Eleni
authors.initials: E
authors.lastname: Gomes
authors.affiliation: UNESP (São Paulo State University - Júlio de Mesquita Filho), Biochemistry and Applied Microbiology Laboratory, São José do Rio Preto, SP 15054-000, Brazil. Electronic address: dasilva@ibilce.unesp.br.
authors.forename: Roberto
authors.initials: R
authors.lastname: Da Silva
mesh_terms: D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D055327:Thermoascus / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Endo-xylanase; Protein engineering; Rational design; Site-directed mutagenesis; Thermostability