Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase

Autor(es)

Ribeiro, Lucas Ferreira ^{[1, 2]} ; Nicholes, Nathan ^[1] ; Tullman, Jennifer ^[3] ; Costa Ribeiro, Liliane Fraga ^{[2, 4]} ; Fuzo, Carlos Alessandro ^[5] ; Vieira, Davi Serradella ^[6] ; Furtado, Gilvan Pessoa ^[2] ; Ostermeier, Marc ^[1] ; Ward, Richard John ^{[5, 7]}

Afiliação do(s) autor(es)

^[1] Johns Hopkins Univ, Baltimore, MD - USA

^[2] Univ Sao Paulo, Dept Bioquim & Imunol, FMRP, BR-14049 Ribeirao Preto, SP - Brazil

^[3] Inst Biosci & Biotechnol Res, Rockville, MD - USA

^[4] UMBC, Baltimore, MD - USA

^[5] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, BR-14040901 Ribeirao Preto, SP - Brazil

^[6] Univ Fed Rio Grande do Norte, BR-59072970 Natal, RN - Brazil

^[7] Brazilian Bioethanol Sci & Technol Lab CTBE CNPEM, Campinas, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNOLOGY FOR BIOFUELS; v. 8, AUG 15 2015.
• Citações Web of Science: 11

Resumo

Background: Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme. Results: A semi-rational protein fusion strategy was used to create a protein fusion library where the Bacillus subtilis GH11 xylanase A (XynA) was inserted at 144 surface positions of the Escherichia coli xylose binding protein (XBP). Two XynA insertions at XBP positions 209 ({[}209]XBP-Xyn-XBP) and 262 ({[}262]XBP-Xyn-XBP) showed a 20% increased xylanolytic activity in the presence of xylose, conditions where native XynA is inhibited. Random linkers of 1-4 Gly/Ala residues were inserted at the XynA N- and C-termini in the {[}209]XBP and {[}262]XBP, and the chimeras 2091A and 2621B were isolated, showing a twofold increased xylanolytic activity in the presence of xylose and k(cat) values of 200 and 240 s(-1) in the 2091A and 2621B, respectively, as compared to 70 s-1 in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the similar to 3-to 3.5-fold stimulation of catalytic efficiency by xylose was the result of allosteric coupling between the XBP and XynA domains. Molecular dynamics simulations of the chimeras suggested conformation alterations in the XynA on xylose binding to the XBP resulted in exposure of the catalytic cavity and increased mobility of catalytic site residues as compared to the native XynA. Conclusions: These results are the first report of engineered glycosyl hydrolase showing allosteric product stimulation and suggest that the strategy may be more widely employed to overcome enzyme product inhibition and to improve catalytic performance. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático