title: Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme.
abstract: As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase.
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) - CNPEM, Campinas, SP, Brazil Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Juliana
authors.initials: J
authors.lastname: Alponti
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Laila Aparecida
authors.initials: LA
authors.lastname: Deliberto
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) - CNPEM, Campinas, SP, Brazil.
authors.forename: Letícia Maria
authors.initials: LM
authors.lastname: Zanphorlin
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) - CNPEM, Campinas, SP, Brazil Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Carla Botelho
authors.initials: CB
authors.lastname: Machado
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) - CNPEM, Campinas, SP, Brazil Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil rjward@ffclrp.usp.br.
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D000468:Alkalies; D043364:Endo-1,4-beta Xylanases
keywords: DNA shuffling; alkali-tolerant enzyme; directed evolution; endo-xylanase; error-prone PCR