Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP
In order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database min...
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The editorial department of Science and Technology of Food Industry
2024-11-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023120312 |
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| author | Yali CHEN Qian WANG Han LIU Zhiyou ZONG Shuli MAN Yuanxia SUN Peng CHEN |
| author_facet | Yali CHEN Qian WANG Han LIU Zhiyou ZONG Shuli MAN Yuanxia SUN Peng CHEN |
| author_sort | Yali CHEN |
| collection | DOAJ |
| description | In order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database mining and sequence analysis. After confirming that the C-terminal of TsGP was a non-catalytic domain in the three-dimensional structure predicted by AlphaFold2, a C-terminal truncated mutant ΔTsGP was constructed. Heterologous recombinant expression of TsGP and ΔTsGP was performed in E.coli BL21(DE3), and their enzymatic properties were characterized. The results showed that the expression level of ΔTsGP in E.coli was 3.76 times higher than that of TsGP. At the optimal reaction temperature 70 ℃, the specific enzyme activity of ΔTsGP was 23.87 U/mg, which was 1.3 times higher than that of TsGP. Under 50~65 ℃ which was widely applied as industrial catalytic temperature, the thermal stability of ΔTsGP was equivalent to that of TsGP, and the enzyme activity was higher. In addition, ΔTsGP and TsGP had the same substrate specificity, their smallest substrate was maltotriose, and as the substrate chain length increased, their specific enzyme activity gradually increased. Using maltoheptaose as the substrate, the kcat value of ΔTsGP was 37.09 s−1, which was 1.21 times higher than that of TsGP, but its substrate affinity (Km=3.30 mmol/L) was reduced by 2.77 times. Through structural analysis and non-catalytic domain truncation strategies, the study successfully improved the expression level of TsGP in E. coli and the specific enzyme activity under industrial enzyme catalytic conditions. The study would provide guidance for the efficient expression and application of αGPase protein and lays the foundation for further optimizing the performance of this enzyme through engineering modification. |
| format | Article |
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| issn | 1002-0306 |
| language | zho |
| publishDate | 2024-11-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
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| series | Shipin gongye ke-ji |
| spelling | doaj-art-9460fa9c2f7d462c95c91ae5791853202025-08-20T02:32:38ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062024-11-01452213114010.13386/j.issn1002-0306.20231203122023120312-22Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGPYali CHEN0Qian WANG1Han LIU2Zhiyou ZONG3Shuli MAN4Yuanxia SUN5Peng CHEN6School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, ChinaSchool of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaSchool of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaIn order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database mining and sequence analysis. After confirming that the C-terminal of TsGP was a non-catalytic domain in the three-dimensional structure predicted by AlphaFold2, a C-terminal truncated mutant ΔTsGP was constructed. Heterologous recombinant expression of TsGP and ΔTsGP was performed in E.coli BL21(DE3), and their enzymatic properties were characterized. The results showed that the expression level of ΔTsGP in E.coli was 3.76 times higher than that of TsGP. At the optimal reaction temperature 70 ℃, the specific enzyme activity of ΔTsGP was 23.87 U/mg, which was 1.3 times higher than that of TsGP. Under 50~65 ℃ which was widely applied as industrial catalytic temperature, the thermal stability of ΔTsGP was equivalent to that of TsGP, and the enzyme activity was higher. In addition, ΔTsGP and TsGP had the same substrate specificity, their smallest substrate was maltotriose, and as the substrate chain length increased, their specific enzyme activity gradually increased. Using maltoheptaose as the substrate, the kcat value of ΔTsGP was 37.09 s−1, which was 1.21 times higher than that of TsGP, but its substrate affinity (Km=3.30 mmol/L) was reduced by 2.77 times. Through structural analysis and non-catalytic domain truncation strategies, the study successfully improved the expression level of TsGP in E. coli and the specific enzyme activity under industrial enzyme catalytic conditions. The study would provide guidance for the efficient expression and application of αGPase protein and lays the foundation for further optimizing the performance of this enzyme through engineering modification.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023120312thermophilic alpha-glucan phosphorylaseglucose-1-phosphatenon-catalytic domainprotein truncationenzymatic properties |
| spellingShingle | Yali CHEN Qian WANG Han LIU Zhiyou ZONG Shuli MAN Yuanxia SUN Peng CHEN Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP Shipin gongye ke-ji thermophilic alpha-glucan phosphorylase glucose-1-phosphate non-catalytic domain protein truncation enzymatic properties |
| title | Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP |
| title_full | Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP |
| title_fullStr | Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP |
| title_full_unstemmed | Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP |
| title_short | Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP |
| title_sort | effect of the c terminal non catalytic domain on the enzymatic properties of thermophilic α glucan phosphorylase tsgp |
| topic | thermophilic alpha-glucan phosphorylase glucose-1-phosphate non-catalytic domain protein truncation enzymatic properties |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2023120312 |
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