ENZYME AND MICROBIAL TECHNOLOGY
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2021
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IF 3.493
Qinghua Shi,Yutian Jia,Huibin Wang,Shang Li,Hengyu Li,Jiyang Guo,Tong Dou,Bin Qin,Song You
The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens , Pseudomonas fluorescens , and Pseudomonas veronil . The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2026
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IF 6.7
Lulu Zhang,Zehao Li,Wanying Wang,Yingjie Feng,Jinchu Yang,Yongming Xu,Zhenzhen Huang,Nannan Wang,Kaijing Yin
Dihydrocarvone is an important flavor compound and chiral building block obtained through the ene reductase (ER)-mediated reduction of carvone. In this study, a new ER, KlebER1, from Klebsiella sp. O852 was identified and characterized. Optimal enzyme activity occurred at 40 °C and pH 7, with approximately 80% activity retained after incubation at 20–40 °C for 3 h. Based on sequence alignment, alanine scanning, and molecular docking analyses, a site-directed mutation (A303N) was introduced, resulting in a 1.73-fold enhancement in activity. Finally, an enzymatic cascade system incorporating the A303N mutant and glucose dehydrogenase for coenzyme regeneration was constructed both in vivo and in vitro for dihydrocarvone production. This biocatalytic system achieved a dihydrocarvone concentration of 5.88 mmol/L and a conversion rate of 99%, representing a significant improvement compared to the single use of A303N mutant. This study lays the foundation for investigating the function and modification of ERs and promoting the efficient biosynthesis of dihydrocarvone.
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Zehao Li,Wanying Wang,Lulu Zhang,Jinchu Yang,Yongming Xu,Zhenzhen Huang,Wanzeng Li,Shiying Wang,Jingwen Yang,Zhifeng Zhang
Dihydrocarvone is extensively utilized in food, pharmaceutical, and cosmetic fields because of its pleasant odor, high economic value, and diverse pharmacological properties. In this study, an ene reductase KlebER3, which catalyzed the conversion of carvone to dihydrocarvone using NAD(P)H as a cofactor, was identified from Klebsiella sp. O852 through genome sequencing. This enzyme displayed optimal activity at 30 °C and pH 7 and maintained appreciable stability at 20–50 °C and pH 6–8. Besides, KlebER3 had good resistance to dimethyl sulfoxide and glycerine and exhibited wide-ranging substrate acceptance for α,β-unsaturated compounds. Furthermore, a Q26A mutant with enhanced activity, stability, and stereoselectivity was obtained by molecular docking, alanine scanning, and site-directed mutagenesis. Molecular dynamics simulations revealed that these improvements may be attributed to increased nonpolar interactions, elevated flexibility of the active pocket, reduced binding free energy, and strengthened rigidity of protein conformation. This study established a basis for further investigations of the function and modification of ene reductase as well as the efficient biosynthesis of dihydrocarvone.
研究领域:
酶学
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生物催化
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微生物生物技术
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分子生物学
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计算生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）