Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric Acid Monoamide, a Key Intermediate in Pregabalin Production.
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| Format: | Artículo científico |
| Langue: | en |
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Microbial biotechnology
2026
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| _version_ | 1868266071864115200 |
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| author | Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng |
| author_facet | Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng |
| collection | PubMed - marine biology |
| contents | Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric Acid Monoamide, a Key Intermediate in Pregabalin Production. Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng Escherichia coli Pregabalin Amidohydrolases Stereoisomerism Glutarates Enzyme Stability Temperature Hydrogen-Ion Concentration Substrate Specificity Gene Expression Recombinant Proteins Pregabalin is a widely used clinical pharmaceutical agent for treatment of neuropathic pain and as an adjunctive therapy for epilepsy. The efficient synthesis of (R)-3-isobutylglutaric acid monoamide (R-IBM), a critical intermediate, is crucial for pregabalin manufacturing. This present study focused on identifying R-selective and robust D-hydantoinases with R-selectivity and robustness for the stereoselective synthesis of R-IBM, a key intermediate of pregabalin, from 3-isobutylglutarimide (IBI). Three D-hydantoinases, namely GsDHTase (GenBank No. PX317655), AcDHTase (GenBank No. PX317656) and BhDHTase (GenBank No. PX317657) with unique catalytic advantages were successfully isolated from a marine gene resource library. These enzymes exhibited exceptional robustness under extreme conditions (pH 10, 70°C or 80°C) and inherent high stereoselectivity for R-IBM. Wild-type and mutant forms of the three D-hydantoinases were heterologously expressed in E. coli BL21(DE3) using the pET-28a vector, followed by whole-cell catalysis experiments. The optimal IPTG induction temperature was determined to be 28°C. Maximum catalytic activity, corresponding to the highest conversion rate of IBI to R-IBM, was achieved at 50°C and pH 10.0. Notably, triple amino acid mutations (M63A, F65H and C317T) significantly enhanced stereoselectivity with only a slight reduction in enzymatic activity. The enantiomeric excess (ee%) of R-IBM improved from 73.74% to 97.55% for GsDHTase, 56.08% to 97.59% for AcDHTase and 78.32% to 96.85% for BhDHTase after mutation. Owing to their inherent robustness, these enzymes hold great potential for developing a theoretical 100% atom-economical route for the eco-friendly and cost-effective industrial production of pregabalin. |
| format | Artículo científico |
| id | pubmed_41846534 |
| institution | PubMed |
| language | en |
| publishDate | 2026 |
| publisher | Microbial biotechnology |
| record_format | pubmed |
| spellingShingle | Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric Acid Monoamide, a Key Intermediate in Pregabalin Production. Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng Escherichia coli Pregabalin Amidohydrolases Stereoisomerism Glutarates Enzyme Stability Temperature Hydrogen-Ion Concentration Substrate Specificity Gene Expression Recombinant Proteins Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric Acid Monoamide, a Key Intermediate in Pregabalin Production. Zhang, Jiujiuzi Zhi, Bin Xie, Zhenyu Zhang, Beibei Wang, Ning Wang, Wanpeng Escherichia coli Pregabalin Amidohydrolases Stereoisomerism Glutarates Enzyme Stability Temperature Hydrogen-Ion Concentration Substrate Specificity Gene Expression Recombinant Proteins Pregabalin is a widely used clinical pharmaceutical agent for treatment of neuropathic pain and as an adjunctive therapy for epilepsy. The efficient synthesis of (R)-3-isobutylglutaric acid monoamide (R-IBM), a critical intermediate, is crucial for pregabalin manufacturing. This present study focused on identifying R-selective and robust D-hydantoinases with R-selectivity and robustness for the stereoselective synthesis of R-IBM, a key intermediate of pregabalin, from 3-isobutylglutarimide (IBI). Three D-hydantoinases, namely GsDHTase (GenBank No. PX317655), AcDHTase (GenBank No. PX317656) and BhDHTase (GenBank No. PX317657) with unique catalytic advantages were successfully isolated from a marine gene resource library. These enzymes exhibited exceptional robustness under extreme conditions (pH 10, 70°C or 80°C) and inherent high stereoselectivity for R-IBM. Wild-type and mutant forms of the three D-hydantoinases were heterologously expressed in E. coli BL21(DE3) using the pET-28a vector, followed by whole-cell catalysis experiments. The optimal IPTG induction temperature was determined to be 28°C. Maximum catalytic activity, corresponding to the highest conversion rate of IBI to R-IBM, was achieved at 50°C and pH 10.0. Notably, triple amino acid mutations (M63A, F65H and C317T) significantly enhanced stereoselectivity with only a slight reduction in enzymatic activity. The enantiomeric excess (ee%) of R-IBM improved from 73.74% to 97.55% for GsDHTase, 56.08% to 97.59% for AcDHTase and 78.32% to 96.85% for BhDHTase after mutation. Owing to their inherent robustness, these enzymes hold great potential for developing a theoretical 100% atom-economical route for the eco-friendly and cost-effective industrial production of pregabalin. |
| title | Thermotolerant D-Hydantoinases for Efficient and Stereoselective Intermediate Synthesis of (R)-3-Isobutylglutaric Acid Monoamide, a Key Intermediate in Pregabalin Production. |
| topic | Escherichia coli Pregabalin Amidohydrolases Stereoisomerism Glutarates Enzyme Stability Temperature Hydrogen-Ion Concentration Substrate Specificity Gene Expression Recombinant Proteins |
| url | https://pubmed.ncbi.nlm.nih.gov/41846534/ |