Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch
Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (<i>PNAS</i>, 110: 7182–7187, 2013) and to con...
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MDPI AG
2023-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/19/14452 |
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| author | Yuanyuan Zhang Yapeng Li Hui Lin Guotao Mao Xiang Long Xinyu Liu Hongge Chen |
| author_facet | Yuanyuan Zhang Yapeng Li Hui Lin Guotao Mao Xiang Long Xinyu Liu Hongge Chen |
| author_sort | Yuanyuan Zhang |
| collection | DOAJ |
| description | Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (<i>PNAS</i>, 110: 7182–7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from <i>Clostridium thermocellum</i> (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin’s phosphorolysis. |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T21:43:24Z |
| publishDate | 2023-09-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-23b01dff66a24d2d9a4f5a08492eb5762023-11-19T14:26:46ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-09-0124191445210.3390/ijms241914452Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to StarchYuanyuan Zhang0Yapeng Li1Hui Lin2Guotao Mao3Xiang Long4Xinyu Liu5Hongge Chen6College of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCollege of Life Sciences, Henan Agricultural University, Zhengzhou 450046, ChinaCellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (<i>PNAS</i>, 110: 7182–7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from <i>Clostridium thermocellum</i> (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin’s phosphorolysis.https://www.mdpi.com/1422-0067/24/19/14452cellobiose phosphorylasecellodextrin phosphorylasesubstrate specificitycellodextrinamylose |
| spellingShingle | Yuanyuan Zhang Yapeng Li Hui Lin Guotao Mao Xiang Long Xinyu Liu Hongge Chen Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch International Journal of Molecular Sciences cellobiose phosphorylase cellodextrin phosphorylase substrate specificity cellodextrin amylose |
| title | Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch |
| title_full | Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch |
| title_fullStr | Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch |
| title_full_unstemmed | Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch |
| title_short | Broadening the Substrate Specificity of Cellobiose Phosphorylase from <i>Clostridium thermocellum</i> for Improved Transformation of Cellodextrin to Starch |
| title_sort | broadening the substrate specificity of cellobiose phosphorylase from i clostridium thermocellum i for improved transformation of cellodextrin to starch |
| topic | cellobiose phosphorylase cellodextrin phosphorylase substrate specificity cellodextrin amylose |
| url | https://www.mdpi.com/1422-0067/24/19/14452 |
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