Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol
© 2021. The Author(s)..
Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. KEY POINTS: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:105 |
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| Enthalten in: |
Applied microbiology and biotechnology - 105(2021), 19 vom: 09. Okt., Seite 7309-7319 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Xia, Yuanyuan [VerfasserIn] |
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| Links: |
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| Themen: |
α-D-glucosylglycerol |
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| Anmerkungen: |
Date Completed 08.10.2021 Date Revised 31.05.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1007/s00253-021-11551-0 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM330845802 |
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| 100 | 1 | |a Xia, Yuanyuan |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol |
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| 520 | |a © 2021. The Author(s). | ||
| 520 | |a Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. KEY POINTS: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Molecular dynamics simulations | |
| 650 | 4 | |a Semi-rational design | |
| 650 | 4 | |a Sucrose phosphorylase | |
| 650 | 4 | |a Thermostability | |
| 650 | 4 | |a α-D-glucosylglycerol | |
| 650 | 7 | |a Glucosides |2 NLM | |
| 650 | 7 | |a glucosylglycerol |2 NLM | |
| 650 | 7 | |a Glucosyltransferases |2 NLM | |
| 650 | 7 | |a EC 2.4.1.- |2 NLM | |
| 650 | 7 | |a sucrose phosphorylase |2 NLM | |
| 650 | 7 | |a EC 2.4.1.7 |2 NLM | |
| 700 | 1 | |a Li, Xiaoyu |e verfasserin |4 aut | |
| 700 | 1 | |a Yang, Linli |e verfasserin |4 aut | |
| 700 | 1 | |a Luo, Xiaozhou |e verfasserin |4 aut | |
| 700 | 1 | |a Shen, Wei |e verfasserin |4 aut | |
| 700 | 1 | |a Cao, Yu |e verfasserin |4 aut | |
| 700 | 1 | |a Peplowski, Lukasz |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Xianzhong |e verfasserin |4 aut | |
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