Biotechnology for Biofuels | |
Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase | |
Lucas Ferreira Ribeiro5  Nathan Nicholes4  Jennifer Tullman3  Liliane Fraga Costa Ribeiro2  Carlos Alessandro Fuzo6  Davi Serradella Vieira1  Gilvan Pessoa Furtado5  Marc Ostermeier4  Richard John Ward6  | |
[1] Universidade Federal do Rio Grande do Norte, Natal, Brazil | |
[2] University of Maryland Baltimore County-UMBC, Baltimore, MD, USA | |
[3] Institute for Bioscience and Biotechnology Research, Rockville, MD, USA | |
[4] Johns Hopkins University, Baltimore, MD, USA | |
[5] Departamento de Bioquímica e Imunologia, FMRP, Universidade de São Paulo-USP, Ribeirão Preto, SP, Brazil | |
[6] Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo-USP, Av. Bandeirantes, 3900, Ribeirão Preto 14040-901, SP, Brazil | |
关键词: Semi-rational design; Non-homologous genes; Heterotropic allosteric regulation; Enzyme engineering; | |
Others : 1228587 DOI : 10.1186/s13068-015-0293-0 |
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received in 2015-01-29, accepted in 2015-07-24, 发布年份 2015 |
【 摘 要 】
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 kcatvalues of 200 and 240 s −1in the 2091A and 2621B, respectively, as compared to 70 s −1in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the ~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.
【 授权许可】
2015 Ribeiro et al.
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