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Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance
BACKGROUND: The Bacillus subtilis endo-β-1,4-glucanase (BsCel5A) hydrolyzes β-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds β-1,4-linked glucan. METHODS: Here we investigate the BsCel5A β-1,3-1,4 glucanase activity after exchanging the CBM3 d...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686792/ https://www.ncbi.nlm.nih.gov/pubmed/29163671 http://dx.doi.org/10.1186/s13068-017-0964-0 |
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author | Fonseca-Maldonado, Raquel Meleiro, Luana P. Mendes, Luís F. S. Alves, Luana F. Carli, Sibeli Morero, Lucas D. Basso, Luis G. M. Costa-Filho, Antonio J. Ward, Richard J. |
author_facet | Fonseca-Maldonado, Raquel Meleiro, Luana P. Mendes, Luís F. S. Alves, Luana F. Carli, Sibeli Morero, Lucas D. Basso, Luis G. M. Costa-Filho, Antonio J. Ward, Richard J. |
author_sort | Fonseca-Maldonado, Raquel |
collection | PubMed |
description | BACKGROUND: The Bacillus subtilis endo-β-1,4-glucanase (BsCel5A) hydrolyzes β-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds β-1,4-linked glucan. METHODS: Here we investigate the BsCel5A β-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having β-1,3-1,4 glucan affinity. RESULTS: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley β-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with β-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with β-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the β-1,3-1,4 glucan. CONCLUSIONS: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-017-0964-0) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5686792 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-56867922017-11-21 Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance Fonseca-Maldonado, Raquel Meleiro, Luana P. Mendes, Luís F. S. Alves, Luana F. Carli, Sibeli Morero, Lucas D. Basso, Luis G. M. Costa-Filho, Antonio J. Ward, Richard J. Biotechnol Biofuels Research BACKGROUND: The Bacillus subtilis endo-β-1,4-glucanase (BsCel5A) hydrolyzes β-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds β-1,4-linked glucan. METHODS: Here we investigate the BsCel5A β-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having β-1,3-1,4 glucan affinity. RESULTS: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley β-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with β-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with β-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the β-1,3-1,4 glucan. CONCLUSIONS: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-017-0964-0) contains supplementary material, which is available to authorized users. BioMed Central 2017-11-14 /pmc/articles/PMC5686792/ /pubmed/29163671 http://dx.doi.org/10.1186/s13068-017-0964-0 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Fonseca-Maldonado, Raquel Meleiro, Luana P. Mendes, Luís F. S. Alves, Luana F. Carli, Sibeli Morero, Lucas D. Basso, Luis G. M. Costa-Filho, Antonio J. Ward, Richard J. Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title | Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title_full | Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title_fullStr | Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title_full_unstemmed | Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title_short | Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
title_sort | lignocellulose binding of a cel5a-rtcbm11 chimera with enhanced β-glucanase activity monitored by electron paramagnetic resonance |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686792/ https://www.ncbi.nlm.nih.gov/pubmed/29163671 http://dx.doi.org/10.1186/s13068-017-0964-0 |
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