title: Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering.
abstract: Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis (XynA), five single mutants (S22E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed in Escherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (Topt) of 55°C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55°C (t50) decreased in the order S22E/N32D>N181R>S22E>Wild-type>S27E=N32D≈N54E. The values of the van't Hoff denaturation enthalpy change (ΔHND), melting temperature (Tm) and heat capacity at constant pressure (ΔCp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328K (ΔG328) S22E/N32D>N181R>S22E>Wild-type>S27E≈N54E>N32D correlates well with the thermotolerance results, and is dominated by changes in ΔHND which is consistent with increased in hydrogen bonding in the thermostable mutants.
authors.affiliation: Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, CEP 14040-901 São Paulo, Brazil.
authors.forename: Juliana Sanchez
authors.initials: JS
authors.lastname: Alponti
authors.affiliation: Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Raquel
authors.initials: R
authors.lastname: Fonseca Maldonado
authors.affiliation: Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, CEP 14040-901 São Paulo, Brazil; Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, São Paulo, Brazil. Electronic address: rjward@ffclrp.usp.br.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D013696:Temperature
chemical_list: D043364:Endo-1,4-beta Xylanases
keywords: Combinatorial mutagenesis; Protein thermodynamics; Site-directed mutagenesis