JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
·
2016
·
IF 2.189
Xiqian Sheng,Ming Yan,Lin Xu,Miao Wei
We identified and characterized YqiG, a novel Old Yellow Enzyme (OYE) from Bacillus subtilis str.168 , as a member of the “thermophilic-like” subfamily. It is most related to XenA from Pseudomonas putida , with 37.9% identity, but it exhibits certain differences in sequence and enzyme properties. The YqiG can reduce various activated alkenes and exhibits high temperature (60 °C for 12 h) and pH stability (pH 4.0 and 9.0 for 12 h), which indicates that it has a great potential for biocatalysis. However, it shows low tolerance toward organic solvents. Also the YqiG shows high activities towards maleimide, dimethyl maleate and acyclic ketones (55.5 U/mg, 6.22 U/mg and 2.97 U/mg, respectively). Besides, The YqiG can catalyze citral to (S)-citronellal with a low rate but high ee% (>99%).
研究领域:
生物化学
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酶学
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微生物学
✓ 已验证
DOI
高影响因子
Methods in Enzymology
·
2025
·
IF 1.682
Hui Wang,Yuxuan Ye
Flavin-dependent old yellow enzymes (OYEs) have been widely employed as biocatalysts for the stereoselective anti -hydrogenations of activated C C bonds through a “Ping-Pong” bi-bi mechanism. Over the past decade, studies have expanded their reactivity to new-to-nature radical transformations by exploiting their catalytic promiscuity and adaptability to protein engineering. Recently, our group repurposed OYEs to enable asymmetric carbonyl desaturation by inverting their native activity, addressing key limitations of traditional desaturation approaches—such as harsh reaction conditions and inadequate stereochemical control. By directed evolution of an OYE from Geobacillus kaustophilus ( Gk OYE), we achieved desymmetrizing desaturation of diverse cyclohexanones to form the corresponding cyclohexenones featuring a remote quaternary stereogenic center under mild conditions with excellent stereoselectivity. This chapter provides a comprehensive description of the development of Gk OYE-based desymmetrizing desaturation system, including high-throughput enzyme engineering, protein expression, analytical and scalable reaction set-ups, offering a roadmap for adapting OYEs to challenging desaturative transformations.
研究领域:
生物催化
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有机化学
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酶工程
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合成生物学
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不对称合成
✓ 已验证
DOI
PubMed
高影响因子
Nature Communications
·
2024
·Vol. 15
·期 1
·1-16
·
IF 14.7
Wang Lei,Wu Yaoyun,Hu Jun,Yin Dejing,Wei Wanqing,Wen Jian,Chen Xiulai,Gao Cong,Zhou Yiwen,Liu Jia,Hu Guipeng,Li Xiaomin,Wu Jing,Zhou Zhi,Liu Liming,Song Wei
Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of Gk OYE in both MBH and reduction reactions. By blocking H - and H + transfer pathways, we engineer Gk OYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by Gk OYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants Gk OYE.11 and Gk OYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.
研究领域:
生物化学
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合成生物学
✓ 已验证
DOI
PubMed
高影响因子
Green Synthesis and Catalysis
·
2022
·
IF 0
Yongxing Li,Pengqian Luan,Lele Dong,Jianqiao Liu,Luying Jiang,Jing Bai,Fufeng Liu,Yanjun Jiang
Asymmetric reduction of the conjugated C C bonds by the old yellow enzymes (OYEs) presents a promising field in the synthesis of chiral chemicals. Nevertheless, few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability. Herein, a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase (GDH) from Bacillus megaterium was constructed to stereoselectively reduce the conjugated C C bonds in a self-sufficient continuous process. The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation. The best fusion YqjM_G_GDH gave 98% conversion of 100 mmol/L 2-methylcyclopentenone with an excellent ee value (>99%) in 3 h, while the mixture of individual enzymes only obtained 68% conversion after more than 8 h. The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated. A one-pot process was designed to purify and immobilize the fusion on the Ni 2+ -nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers. The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β -unsaturated ketones (20 mmol/L) continuously with only 50 μmol/L NADP + to initiate the whole process, but also retained more than 82% of the initial activity after seven cycles, serving as a good candidate for the industrial applications.
研究领域:
生物催化
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酶工程
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有机化学
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材料科学
✓ 已验证
DOI
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
·
2025
·
IF 1.1
Wu S. J.,Chen F.,Ma X. J.,Sun S. Y.,Yan H. D.,Li T. T.,Qiu L. Q.
( R )-citronellal is a key intermediate in the synthesis of L-menthol, one of the world’s three most important spices. It can be obtained by asymmetric reduction of ( E / Z )-citral using old yellow enzymes (OYEs), but catalysis by the wild-type enzyme suffers from poor enantioselectivity as well as low efficient conversion. A protein-directed evolution technique was used to enhance the activity and enantioselectivity of Ct OYE from Chroococcidiopsis thermalis . Upon the sequence alignment and molecular docking results, two amino acid residues at positions 103 and 351 were selected as saturation mutation sites. Finally, three single substitution variants of Ct OYE-W103E, Ct OYE-W103M, Ct OYE-W103P with improved enantioselectivity ( ee > 99%) and moderate conversion (>71%), and one double substitution variant Ct OYE-Y351Q/W103E with high enantioselectivity ( ee > 99%) and high conversion (83.2%) for reduction of ( E / Z )-citral were obtained.
✓ 已验证
产品关联
DOI
高影响因子
Bioresources and Bioprocessing
·
2018
·Vol. 5
·期 1
·1-12
·
IF 0
Zheng Liandan,Lin Jinping,Zhang Baoqi,Kuang Yuyao,Wei Dongzhi
A NAD(P)H-dependent enoate reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 was discovered by genome data mining for asymmetric reduction of (E/Z)-citral to (R)-citronellal with high enantioselectivity.This enzyme was heterologously expressed in E. coli and characterized for its biocatalytic properties. OYE2p was identified with reduction activities toward a diverse range of ɑ,β-unsaturated compounds bearing conjugated aldehyde, ketone, imide, carboxylic acid and ester.OYE2p showed the highest specific activity at 40 °C and a pH optimum at 7.0–8.0. The stability of OYE2p was rather pH-independent, and the half-life time values of the enzyme at pH 6.0–8.0 were more than 257 h. With regard to the reduction of (E)-citral and (Z)-citral, OYE2p exhibited different selectivity patterns. (E)-citral was exclusively reduced to (R)-citronellal by OYE2p in ≥ 99% ee, which was independent on pH. OYE2p produced both enantiomers of citronellal from (Z)-citral, but showed (R)-citronellal formation tendency, and the ee value of (R)-citronellal was affected by pH in the reaction system. Accordingly, the ee values for (R)-citronellal formation increased with the increasing levels of E-isomer in the (E/Z)-citral mixture as well as the increase of pH. Under the reaction conditions (30 °C and pH 8.6), using purified OYE2p as catalyst, 200 mM (E/Z)-citral (an approximately 10:9 mixture of geometric E-isomer and Z-isomer) was efficiently converted to (R)-citronellal with 88.8% ee and 87.2% yield.All these positive features demonstrate high potential of OYE2p for practical synthesis of (R)-citronellal and in asymmetric reduction of activated alkenes.
研究领域:
生物化学
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酶工程
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合成生物学
✓ 已验证
产品关联
DOI
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2022
·Vol. 194
·期 11
·4999-5016
·
IF 3.094
Ma Teng,Kong Weixi,Liu Yunting,Zhao Hao,Ouyang Yaping,Gao Jing,Zhou Liya,Jiang Yanjun
The application of immobilized enzymes in pharmaceutical and bulk chemical production has been shown to be economically viable. We demonstrate the exceptional performance of a method that immobilizes the old yellow enzyme YqjM and glucose dehydrogenase (GDH) on resin for the asymmetric hydrogenation (AH) of C = C bonds in a SpinChem reactor. When immobilized YqjM and GDH are reused 10 times, the conversion of 2-methylcyclopentenone could reach 78%. Which is because the rotor of the SpinChem reactor effectively reduces catalyst damage caused by shear force in the reaction system. When the substrate concentration is 175 mM, an 87% conversion of 2-methylcyclopentenone is obtained. The method is also observed to perform well for the AH of C = C bonds in other unsaturated carbonyl compounds with the SpinChem reactor. Thus, this method has great potential for application in the enzymatic production of chiral compounds.
研究领域:
生物催化
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酶工程
·
药物化学
·
有机合成
✓ 已验证
DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2022
·
IF 3.705
Liya Zhou,Yaping Ouyang,Weixi Kong,Teng Ma,Hao Zhao,Yanjun Jiang,Jing Gao,Li Ma
In this study, a novel kind of Ni-NTA modified monodispersed SiO 2 nanoflowers (Ni-NTA@SiO 2 nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO 2 nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)− 3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO 2 nanoflowers was 40.17 mg/g support and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of C C bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.
研究领域:
生物化学
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酶工程
·
纳米材料科学
✓ 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）