INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
·
IF 8.5
Zhe Dou,Pei-Ru Hu,Lin-Lin Song,Yi-Cheng Wang,Hao-Nan Tang,Zhen-Qi Weng,Ya-Jun Wang,Yuan-Qiang Cai
Carbonyl reductases (CRs) are promising biological macromolecules for the asymmetric synthesis of chiral secondary alcohols. A novel CR was evolved for efficient conversion of d -xylose into anti-aging ( S )-Pro-Xylane. Through extensive gene mining, an active carbonyl reductase Zm CR was identified from Zygofabospora marxiana with high reductive activity towards d -xylose-derived 1-C-(β- d -xylopyranosyl)-acetone (XPSA) and in situ cofactor regeneration capability. Using a three-round of concise engineering strategy, a hextuple mutant C85V/V128C/S129A/V161S/Y166H/T217A (M6) was identified from merely 157 mutants with specific activity of 46.00 U·mg −1 , about 306 folds of WT. The Shannon-Wiener index of M6 was 2.21, much higher than WT, indicating its extended substrate scope. M6 could efficiently catalyze the asymmetric reduction of 190 g·L −1 XPSA into ( S )-XPSP. The k cat / K M XPSA of M6 was 203.84 s −1 ·mM −1 , about 910 folds of WT. More non-bonded interactions were monitored in M6, contributing to the stabilization of reactive catalytic conformation. This study provides a promising biocatalyst for the chemoenzymatic synthesis of value-added anti-aging ( S )-Pro-Xylane from lignocellulosic d -xylose.
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
Catalysts
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2022
·Vol. 12
·期 10
·1086
·
IF 4.501
Byu Ri Sim,Jie Gu,Yvette Ley,Shenggan Luo,Yihan Liu,Qin Chen,Yao Nie,Yi-Lei Zhao
Candida parapsilosiscarbonyl reductases (CpRCR) have been widely used for the reductive conversion of ketone precursors and chiral alcohol products in pharmaceutical industries. The enzymatic enantioselectivity is believed to be related to the shape complementation between the cavities in the enzymes and the substitutions of the ketone substrates. In this work, we reported an unexpected enantioselectivity in the enzyme reductions of dihydrofuran-3(2H)-one (DHF) to (S)-tetrahydrofuran-3-ol (DHF-ol, enantiomeric excess: 96.4%), while dihydrothiophen-3(2H)-one substrate (DHT) was unproductive under the same experimental conditions. To rationalize the exclusiveS-configuration and the specific reactivity ofDHF, we carried out molecular dynamics simulations for the reacting complexations ofDHFwithCpRCR, andDHTwithCpRCR. Our calculations indicate thatDHFpreferentially binds to the small cavity near L119, F285, and W286, while the large cavity near theα1helix was mainly occupied by solvent water molecules. Moreover, the pre-reaction state analysis suggests that thepro-Sconformations were more abundant than thepro-R, in particular forDHF. This suggests that the non-polar interaction of substrate C4-C5 methylene contacting the hydrophobic side-chains of L119-F285-W286, and the polar interaction of funanyl oxygen exposing the solvent environment play important roles in the enantioselectivity and reactivity. The phylogenetic tree ofCpRCR homologues implies that a variety of amino acid combinations at positions 285 and 286 were available and thereby potentially useful for redesigning enantioselective reductions of 5-membered-ring heterocyclic ketones.Keywords:carbonyl reductases;enantioselectivity;heterocyclic ketone;molecular modelling
研究领域:
生物化学
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酶工程
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产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2023
·
IF 8.2
Mengnan Han,Xiaozheng Li,Xuming Wang,Dexu Liu,Shuangqing Fu,Wenzhi Xu,Wei Li,Honglei Zhang
A polyhydroxyalkanoate (PHA) magnetic microsphere was designed for one-step purification and immobilization of a novel carbonyl reductase (RLSR5) from recombinant Escherichia coli lysate. The hydrophobic core of this microsphere was composed of a highly biocompatible polymer, poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) (PHBHHx), in which magnetic Fe 3 O 4 particles were embedded during solvent evaporation. The hydrophilic shell of the fusion protein formed by PHA particle-binding protein (PhaP) and RLSR5 (PR) was expressed in recombinant E. coli . The magnetic core of Fe 3 O 4 @PHBHHx directly purified the hydrophilic shell from the E. coli lysate, and the two self-assembled to form Fe 3 O 4 @PHBHHx-PR through hydrophobic and hydrophilic interactions, eliminating the separation of the fusion protein. The microstructure, magnetic properties, morphology, size, and dispersion of Fe 3 O 4 @PHBHHx-PR were investigated by XRD, VSM, SEM, TEM, elemental mapping and DLS. It was found that Fe 3 O 4 @PHBHHx-PR correctly assembled, with a well dispersed spherical structure at the nanoscale and superparamagnetism properties. The amount of RLSR5 immobilized on PHA microspheres reached 121.9 mg/g. The Fe 3 O 4 @PHBHHx-PR was employed to synthesize (R )-tolvaptan with 99 % enantiomeric excess and 97 % bioconversion efficiency, and the catalyst maintained 78.6 % activity after 10 recovery cycles. These PHA magnetic microspheres are versatile carriers for enzyme immobilization and demonstrate improved stability and reusability of the free enzyme .
研究领域:
生物技术
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酶工程
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材料科学
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DOI
PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
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2016
·
IF 2.667
Ping Wei,Jia-Xin Gao,Gao-Wei Zheng,Hong Wu,Min-Hua Zong,Wen-Yong Lou
The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9 U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0 U/mg-protein in the recombinant E. coli . As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet- gaccr - gdh ) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for ( S )-enantiomer, and the initial rate and product yield reached 8.04 μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.
研究领域:
生物工程
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合成生物学
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DOI
PubMed
高影响因子
Bioresources and Bioprocessing
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2017
·Vol. 4
·期 1
·1-12
·
IF 0
Wei Ping,Cui Yu-Han,Zong Min-Hua,Xu Pei,Zhou Jian,Lou Wen-Yong
Background
Acetobacter sp. CCTCC M209061 could catalyze carbonyl compounds to chiral alcohols following anti-Prelog rule with excellent enantioselectivity. Therefore, the enzymatic characterization of carbonyl reductase (CR) from Acetobacter sp. CCTCC M209061 needs to be investigated. Results A CR from Acetobacter sp. CCTCC M209061 (AcCR) was cloned and expressed in E. coli . AcCR was purified and characterized, finding that AcCR as a dual coenzyme-dependent short-chain dehydrogenase/reductase (SDR) was more preferred to NADH for biocatalytic reactions. The AcCR was activated and stable when the temperature was under 35 °C and the pH range was from 6.0 to 8.0 for the reduction of 4′-chloroacetophenone with NADH as coenzyme, and the optimal temperature and pH were 45 °C and 8.5, respectively, for the oxidation reaction of isopropanol with NAD + . The enzyme showed moderate thermostability with half-lives of 25.75 h at 35 °C and 13.93 h at 45 °C, respectively. Moreover, the AcCR has broad substrate specificity to a range of ketones and ketoesters, and could catalyze to produce chiral alcohol with e.e. >99% for the majority of tested substrates following the anti-Prelog rule. Conclusions The recombinant AcCR exhibited excellent enantioselectivity, broad substrate spectrum, and highly stereoselective anti-Prelog reduction of prochiral ketones. These results suggest that AcCR is a powerful catalyst for the production of anti-Prelog alcohols. The biocatalytic reactions conducted with the recombinant AcCR
研究领域:
生物化学
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酶工程
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工业微生物学
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DOI
PubMed
RSC Advances
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2015
·Vol. 5
·期 29
·22703-22711
·
IF 3.84
Guo-Chao Xu,Hui-Lei Yu,Yue-Peng Shang,Jian-He Xu
Two robust stereocomplementary carbonyl reductases (DhCR and CgCR) were identified through rescreening the carbonyl reductase toolbox. Five reductases were returned through the activity and enantioselectivity assay for α-chloro-1-acetophenone and ethyl 4-chloro-3-oxo-butanate (COBE). Three reductases were stable at elevated substrate loading. Enzymatic characterization revealed that DhCR and CgCR were more thermostable. As much as 330 g COBE in 1 L biphasic reaction mixture was reduced to (S)- and (R)-3-hydroxy-4-chlorobutyrate by DhCR and CgCR (coexpressed with glucose dehydrogenase), with 92.5% and 93.0% yields, >99% ee, and total turnover numbers of 53 800 and 108 000, respectively. Six other α-halohydrins were asymmetrically reduced to optically pure forms at a substrate loading of 100 g L−1. Our results indicate the potential of these two stereocomplementary reductases in the synthesis of valuable α-halohydrins for pharmaceuticals.
研究领域:
生物化学
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酶工程
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制药工业
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DOI
高影响因子
CHEMBIOCHEM
·
2021
·Vol. 23
·期 5
·e202100589
·
IF 3.164
Yunfeng Cui,Liangyan Zhu,Xi Chen,Jinhui Feng,Qiaqing Wu,Dunming Zhu
Graphical Rational engineering of the carbonyl reductase PhADH resulted in a double mutant H93C/A139L, which enhanced the enantioselectivity toward the reduction of 3-cyclopentyl-3-ketopropanenitrile 1 a from 85 % to 99 %, as well as a 6-fold improvement in the specific activity. ( S )-3-Cyclopentyl-3-hydroxypropanenitrile (( S )- 1 b ) was prepared at 200 g/L (1.5 M) substrate concentration. ( S )-3-Cyclopentyl-3-hydroxypropanenitrile is the key precursor for the synthesis of ruxolitinib. The bioreduction of 3-cyclopentyl-3-ketopropanenitrile ( 1 a ) offers an attractive method to access this important compound. A carbonyl reductase (PhADH) from Paraburkholderia hospita catalyzed the reduction of 1 a giving the ( S )-alcohol ( 1 b ) with 85 % ee. Rational engineering of PhADH resulted in a double mutant H93C/A139L, which enhanced the enantioselectivity from 85 % to >98 %, as well as a 6.3-fold improvement in the specific activity. The bioreduction of 1 a was performed at 200 g/L (1.5 M) substrate concentration, leading to isolation of ( S )- 1 b in 91 % yield. Similarly, using this mutant enzyme, 3-cyclohexyl-3-ketopropanenitrile ( 2 a ) and 3-phenyl-3-ketopropanenitrile ( 3 a ) were reduced at high concentration affording the corresponding alcohols in >99 % ee, and 90 % and 92 % yield, respectively. The results showed that the variant H93C/A139L was a powerful biocatalyst for reduction of β-substituted-β-ketonitriles.
研究领域:
生物化学
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药物化学
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合成生物学
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DOI
高影响因子
Molecular Catalysis
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2019
·
IF 2.938
Ping Wei,Ze-Wang Guo,Xiao-Ling Wu,Shan Liang,Xiao-Yang Ou,Pei Xu,Min-Hua Zong,Ji-Guo Yang,Wen-Yong Lou
Chiral alcohols and their derivatives are vital building blocks to synthesize pharmaceutical drugs and high-valued chemicals. Wild-type carbonyl reductase Ac CR from Acetobacter sp. has ideal enantioselectivity toward 11 prochiral substrates ( e.e. >99%) but poor activity. In this work, a semi-rational engineering was performed to enhance the activity of Ac CR. Fortunately, three positive double-mutants (mut-E144A/G152 L, mut-G152 L/Y189 N, and mut-I147 V/G152 L) with specific activity 17–61 folds higher than that of enzyme without modified were achieved. Kinetic studies suggested that the catalytic efficiencies ( k cat / K m ) of these mutants were also well enhanced. Finally, these modified mut- Ac CRs were successfully applied in asymmetric reductions of 11 structurally diverse prochiral substrates (200 mM) with excellent product yields (76.8%–99.1%) and enantiomeric excess ( e.e. >99%), which provides an alternative strategy for efficient synthesis of chiral alcohols for pharmaceuticals industry with ideal yield and enantioselectivity.
研究领域:
生物化学
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酶工程
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制药工业
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高影响因子
ChemCatChem
·
2019
·Vol. 11
·期 11
·2600-2606
·
IF 4.495
Meng Chen,Xiao-Yan Zhang,Chen-Guang Xing,Chao Zhang,Yu-Cong Zheng,Jiang Pan,Jian-He Xu,Yun-Peng Bai
Graphical Great engineering ! In this work, structurally diverse γ- and δ-lactones were efficiently stereoselectively synthesized using an engineered carbonyl reductase from Serratia marcescens ( Sm CR V4 ). Sm CR V4 exhibited improved activity and thermostability toward 14 γ-/δ-keto acids and esters compared with the wild-type enzyme, with 110-fold enhancement in catalytic efficiency ( k cat / K m ) toward methyl 4-oxodecanoate. The preparative synthesis of alkyl and aromatic γ- and δ-lactones with 95 %–>99 % ee was demonstrated. The highest space-time yield, 1175 g L −1 d −1 , was achieved for ( R )-γ-decalactone. Structurally diverse γ- and δ-lactones were efficiently synthesized stereoselectively using an engineered carbonyl reductase from Serratia marcescens ( Sm CR V4 ). Sm CR V4 exhibited improved activity (up to 500-fold) and thermostability toward 14 γ-/δ-keto acids and esters, compared with the wild-type enzyme, with 110-fold enhancement in catalytic efficiency ( k cat / K m ) toward methyl 4-oxodecanoate. The preparative synthesis of alkyl and aromatic γ- and δ-lactones with 95 %–>99 % ee and 78 %–90 % yields was demonstrated. The highest space-time yield, 1175 g L −1 d −1 , was achieved for ( R )-γ-decalactone.
研究领域:
生物化学
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酶工程
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有机合成
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DOI
高影响因子
BIOPROCESS AND BIOSYSTEMS ENGINEERING
·
2025
·
IF 3.6
Zhang Xiao-Jian,Chen Kai-Li,Li Xiang-Yang,Yuan Qi-Qi,Lin Chao-Ping,Liu Zhi-Qiang,Zheng Yu-Guo
Duloxetine, a prominent 5-hydroxytryptamine norepinephrine reuptake inhibitor, is deployed mainly in the management of adult depression, showcasing minimal side effects, swift therapeutic onset, and a robust safety profile. Ethyl ( S )-3-hydroxy-3-(2-thienyl)propionate (( S )-HEES) is the crucial chiral intermediate for duloxetine production. Asymmetric synthesis of ( S )-HEES using carbonyl reductase as the biocatalyst has exhibited advantages including mild reaction conditions, high catalytic efficiency and environmental friendliness. In the present study, a loop region alteration strategy was developed to screen for a carbonyl reductase for ( S )-HEES synthesis and Ea SDR6 from Exiguobacterium sp. s126 was identified with considerable catalytic performance and broad substrate adaptability. Site-directed mutagenesis was subsequently performed, Mut-R142A/N204A was identified with a 3.6-fold enhancement in activity relative to the wild-type Ea SDR6. The mutant k cat value was 52.5 s −1 , 2.9-fold compared to the wild type, and the total catalytic efficiency ( k cat / K M ) was 24.9 mM −1 s −1 , 1.9-fold higher than the wild type. The n -butyl acetate–aqueous biphasic bioreaction system was established for the asymmetric synthesis of ( S )-HEES with the conversion of ethyl 3-oxo-3-(2-thienyl)propionate (KEES) of 90.2%, the product e.e. of > 99% after 8 h reaction at a substrate concentration of 200 g/L. The spatiotemporal yield reached 22.5 g/(L·h), which was higher than the ever reports about ( S )-HEES biosynthesis. The present research provides new knowledge and technology for the construction of stereoselective carbonyl reductase and the green biosynthesis of chiral alcohol pharmaceutical intermediates. Graphical abstract
研究领域:
生物化学
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制药工程
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酶工程
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产品关联
DOI
PubMed
高影响因子
Science Advances
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2025
·Vol. 11
·期 6
·
IF 12.5
Feng Liu,Jianyu Dong,Ruofei Cheng,Shuang-Feng Yin,Lang Chen,Lebin Su,Renhua Qiu,Yongbo Zhou,Li-Biao Han,Chao-Jun Li
Reductive functionalization of aldehydes and ketones is one of the most challenging but ultimately rewarding areas in synthetic chemistry and related sciences. We report a simple and extremely versatile carbonyl reductive functionalization strategy achieving direct, highly selective, and efficient reductive amination, etherification, esterification, and phosphinylation reactions of (hetero)aryl aldehydes and ketones, which are extremely challenging or unattainable to achieve by traditional strategies, using only diphenylphosphine oxide and an inorganic base. It enables modular synthesis of functionally and structurally diverse tertiary amines, ethers, esters, phosphine oxides, etc., as well as related pesticides, drug intermediates, and pharmaceuticals. Compared to phosphorus-mediated name reactions, this strategy firstly transformed C═O bonds into C-element single bonds. Mechanistically, phosphinates are formed as intermediates, which undergo unconventional nucleophilic substitution at the C atom within their C─O─P unit. Thus, this work provides important strides in the field of reductive functionalization of aldehydes/ketones, phosphorus-mediated transformation, and various fundamental reactions.
研究领域:
有机化学
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合成化学
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DOI
PubMed
高影响因子
CHINESE JOURNAL OF CHEMICAL ENGINEERING
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2020
·
IF 2.627
Liqun Shen,Ran Cang,Guang Yang,Anqi Zeng,He Huang,Zhigang Zhang
The microbial production of either ester/lactones or enantio-enriched alcohols through Baeyer–Villiger oxidation or stereoselective reduction of ketones, respectively, is possible by using whole cells of A. subglaciale F134 as a bifunctional biocatalyst. The chemoselective pattern of acetophenone biotransformation catalyzed by these cells can be regulated through reaction temperature, directing the reaction either towards oxidation or reduction products. The Baeyer–Villiger oxidation activity of A. subglaciale F134 whole cells is particularly dependent on reaction temperature. Acetophenone was transformed efficiently to phenol via the primary Baeyer–Villiger product phenyl acetate at 20 °C after 48 h with 100% conversion. In contrast, at 35 °C, enantio-enriched ( S )-1-phenylethanol was obtained as the sole product with 64% conversion and 89% ee . In addition, A. subglaciale F134 cells also catalyze the selective reduction of various structurally different aldehydes and ketones to alcohols with 40% to 100% yield, indicating broad substrate spectrum and good enantioselectivity in relevant cases. Our study provides a bifunctional biocatalyst system that can be used in Baeyer–Villiger oxidation as well as in asymmetric carbonyl reduction, setting the stage for future work concerning the identification and isolation of the respective enzymes.
研究领域:
生物化学
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微生物学
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DOI
高影响因子
危险品化学品经营许可证（带存储）