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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme

Texto completo
Autor(es):
Ruller, Roberto [1, 2] ; Alponti, Juliana [2] ; Deliberto, Laila Aparecida [2] ; Zanphorlin, Leticia Maria [1] ; Machado, Carla Botelho [2, 1] ; Ward, Richard John [1, 2]
Número total de Autores: 6
Afiliação do(s) autor(es):
[1] CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol CTBE, Campinas, SP - Brazil
[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040901 Ribeirao Preto, SP - Brazil
Número total de Afiliações: 2
Tipo de documento: Artigo Científico
Fonte: PROTEIN ENGINEERING DESIGN & SELECTION; v. 27, n. 8, p. 255-262, AUG 2014.
Citações Web of Science: 12
Resumo

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 degrees 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 degrees C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80 degrees C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80 degrees 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 (t(50)) of 60 min at 80 degrees C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase. (AU)

Processo FAPESP: 08/05181-5 - Estratégias Metagenômicas na Bioprospecção de Celulases para o uso na Biorefinaria de Biomassa
Beneficiário:Carla Botelho Machado
Linha de fomento: Bolsas no Brasil - Doutorado
Processo FAPESP: 05/59592-8 - Evolução dirigida de endoxilanases e a aplicação metagenômica na biotecnologia industrial
Beneficiário:Roberto Ruller
Linha de fomento: Bolsas no Brasil - Pós-Doutorado
Processo FAPESP: 05/56608-0 - Produção e caracterização de mutantes termoestáveis de endoxilanase recombinante de Bacillus subtilis 1 a 1
Beneficiário:Laila Aparecida Deliberto Leonelo
Linha de fomento: Bolsas no Brasil - Iniciação Científica
Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
Beneficiário:Richard John Ward
Linha de fomento: Auxílio à Pesquisa - Temático