Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance
Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved...
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.924447/full |
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| author | Xiaolong Cen Xiaolong Cen Xiaolong Cen Xiaolong Cen Rui Zhang Rui Zhang Rui Zhang Rui Zhang Limei He Limei He Limei He Limei He Xianghua Tang Xianghua Tang Xianghua Tang Xianghua Tang Qian Wu Qian Wu Qian Wu Qian Wu Junpei Zhou Junpei Zhou Junpei Zhou Junpei Zhou Zunxi Huang Zunxi Huang Zunxi Huang Zunxi Huang |
| author_facet | Xiaolong Cen Xiaolong Cen Xiaolong Cen Xiaolong Cen Rui Zhang Rui Zhang Rui Zhang Rui Zhang Limei He Limei He Limei He Limei He Xianghua Tang Xianghua Tang Xianghua Tang Xianghua Tang Qian Wu Qian Wu Qian Wu Qian Wu Junpei Zhou Junpei Zhou Junpei Zhou Junpei Zhou Zunxi Huang Zunxi Huang Zunxi Huang Zunxi Huang |
| author_sort | Xiaolong Cen |
| collection | DOAJ |
| description | Inulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment. |
| first_indexed | 2024-04-13T16:54:33Z |
| format | Article |
| id | doaj.art-f0a2c87431d14c85bdfabcd5d356f284 |
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| issn | 1664-302X |
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| last_indexed | 2024-04-13T16:54:33Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-f0a2c87431d14c85bdfabcd5d356f2842022-12-22T02:38:51ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-06-011310.3389/fmicb.2022.924447924447Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol ToleranceXiaolong Cen0Xiaolong Cen1Xiaolong Cen2Xiaolong Cen3Rui Zhang4Rui Zhang5Rui Zhang6Rui Zhang7Limei He8Limei He9Limei He10Limei He11Xianghua Tang12Xianghua Tang13Xianghua Tang14Xianghua Tang15Qian Wu16Qian Wu17Qian Wu18Qian Wu19Junpei Zhou20Junpei Zhou21Junpei Zhou22Junpei Zhou23Zunxi Huang24Zunxi Huang25Zunxi Huang26Zunxi Huang27Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaEngineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming, ChinaCollege of Life Sciences, Yunnan Normal University, Kunming, ChinaKey Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Normal University, Kunming, ChinaKey Laboratory of Yunnan Provincial Education Department for Plateau Characteristic Food Enzymes, Yunnan Normal University, Kunming, ChinaInulin is the rich water-soluble storage polysaccharide after starch in nature, and utilization of inulin through hydrolysis of exo-inulinases has attracted much attention. Thermo-halo-alcohol tolerance is essential for exo-inulinase applications, while no report reveals the molecular basis involved in halo-alcohol tolerance of exo-inulinases via experimental data. In this study, two loops of exo-inulinase InuAMN8, including the loop built with 360GHVRLGPQP368 linking domains of Glyco_hydro_32N and Glyco_hydro_32C and another loop built with 169GGAG172 in the catalytic domain, were deleted to generate mutants MutG360Δ9 and MutG169Δ4, respectively. After heterologous expression, purification, and dialysis, InuAMN8, MutG169Δ4, and MutG360Δ9 showed half-lives of 144, 151, and 7 min at 50°C, respectively. InuAMN8 and MutG169Δ4 were very stable, while MutG360Δ9 showed a half-life of approximately 60 min in 5.0% (w/v) NaCl, and they showed half-lives of approximately 60 min in 25.0, 25.0, and 5.0% (w/v) ethanol, respectively. Structural analysis indicated that two cation-π bonds, which contributed to thermal properties of InuAMN8 at high temperatures, broke in MutG360Δ9. Four basic amino acid residues were exposed to the structural surface of MutG360Δ9 and formed positive and neutral electrostatic potential that caused detrimental effects on halo-alcohol tolerance. The study may provide a better understanding of the loop-function relationships that are involved in thermo-halo-alcohol adaptation of enzymes in extreme environment.https://www.frontiersin.org/articles/10.3389/fmicb.2022.924447/fullinulinaseloopthermostabilitysaltalcoholstructure |
| spellingShingle | Xiaolong Cen Xiaolong Cen Xiaolong Cen Xiaolong Cen Rui Zhang Rui Zhang Rui Zhang Rui Zhang Limei He Limei He Limei He Limei He Xianghua Tang Xianghua Tang Xianghua Tang Xianghua Tang Qian Wu Qian Wu Qian Wu Qian Wu Junpei Zhou Junpei Zhou Junpei Zhou Junpei Zhou Zunxi Huang Zunxi Huang Zunxi Huang Zunxi Huang Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance Frontiers in Microbiology inulinase loop thermostability salt alcohol structure |
| title | Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance |
| title_full | Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance |
| title_fullStr | Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance |
| title_full_unstemmed | Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance |
| title_short | Deletion of the Loop Linking Two Domains of Exo-Inulinase InuAMN8 Diminished the Enzymatic Thermo-Halo-Alcohol Tolerance |
| title_sort | deletion of the loop linking two domains of exo inulinase inuamn8 diminished the enzymatic thermo halo alcohol tolerance |
| topic | inulinase loop thermostability salt alcohol structure |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.924447/full |
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