Scientific Reports
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2017
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IF 4.259
Kunpeng Li,Rongzhen Zhang,Yan Xu,Zhimeng Wu,Jing Li,Xiaotian Zhou,Jiawei Jiang,Haiyan Liu,Rong Xiao
(S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular "stapler" for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat /K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (T m ) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved T m s and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.
研究领域:
生物化学
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酶工程
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蛋白质工程
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DOI
PubMed
高影响因子
FOOD RESEARCH INTERNATIONAL
·
2024
·
IF 7
Chenyu Song,Wei Xu,Cuie Guang,Ting Xue,Wanmeng Mu
Patulin (PAT) is a mycotoxin commonly found in fruits and vegetables, prompting the need for effective removal and detoxification methods, which have garnered significant research attention in recent years. Among these methods, the utilization of microbial-derived enzymes stands out due to their mild operating conditions, specificity in targeted functional groups, and the production of non-toxic by-products, making it a preferred degradation approach. In this study, a novel PAT-degrading enzyme derived from Cyberlindnera fabianii ( Cyfa -SDR) was identified, demonstrating its highest catalytic activity at pH 7.0 and 80 °C against PAT. This temperature tolerance level represents the highest reported for PAT-degrading enzymes to date. The enzyme was further characterized as a short-chain dehydrogenase through analysis of its amino acid composition, conserved GXXXGXG motif, and dependency on NADPH. Moreover, the study evaluated the efficiency of PAT degradation by Cyfa -SDR at varying substrate and enzyme concentrations, surpassing the performance of other PAT-degrading enzymes, thus highlighting its substantial potential for the biological control of PAT. In conclusion, the enzymatic treatment using the PAT-degrading enzyme Cyfa -SDR presents a viable and promising solution for enhancing the quality and safety of fruit juice.
研究领域:
食品科学
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微生物学
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酶学
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DOI
PubMed
高影响因子
ACS Catalysis
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2019
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IF 12.221
Bing-Mei Su,Ze-Hui Shao,Ai-Peng Li,Muhammad Naeem,Juan Lin,Li-Dan Ye,Hong-Wei Yu
Inspired by the conformational change of the enzyme–substrate complex in molecular dynamics (MD) simulation with distance restriction, we propose a strategy for identifying the engineering targets based on the comparative analysis of enzyme-/substrate- binding modes in MD simulations with and without distance restriction (prereaction-state simulation and free-state simulation). Taking the short-chain dehydrogenase/reductase (SDR) mutant EbSDR8-G94A/S153L (Mu0) with poor activity toward bulky aryl ketones as an example, H145 and Y188 were identified as the engineering targets due to the distinct conformation difference in the two simulation modes. To break the “beam” structure formed by these residues at the entry of cavity C2 in free-state simulation, the substrate-binding pocket was reconstructed, and meanwhile the relative size of cavities C1 and C2 was modulated to improve the enantioselectivity. In this way, mutants for efficient asymmetric reduction of o-halogenated acetophenones, propiophenones, aromatic ketoesters, and diaryl ketones were designed, delivering chiral alcohols with >99% conversion and >98% ee. The effectiveness of this design strategy was also validated by the successful redesign of PpYSDR, generating a variant for efficient reduction of (4-chlorophenyl) 2-pyridyl ketone into the S-product with >99% conversion and 96% ee. MD simulations suggested a suitable binding pocket with proper energy contribution as the ubiquitous mechanism for the improvement of activity and enantioselectivity toward substrates with varied structures. The success in this study provides hints for the rational design of alcohol dehydrogenases/reductases with both a broad substrate spectrum and high enantioselectivity.
研究领域:
生物化学
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计算生物学
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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
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IF 8.2
Xiaoxiang Hu,Wenjing Liu,Yi Yan,Huaxiang Deng,Yujie Cai
Short-chain dehydrogenase/reductase (SDR) acts as a biocatalyst in the synthesis of chiral alcohols with high optical purity. Herein, we achieved immobilization via crosslinking on novel magnetic metal–organic framework nanoparticles with a three-layer shell structure (Fe 3 O 4 @PDA@Cu (PABA)). The results of scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy confirmed the morphology and cross-linking property of immobilized SDR, which was more durable, stable, and reusable and exhibited better kinetic performance than free enzyme . The SDR and glucose dehydrogenase (GDH) were co-immobilized and then used for the asymmetric reduction of COBE and ethyl 2-oxo-4-phenylbutanoate (OPBE). These finding suggest that enzymes immobilized on novel MOF nanoparticles can serve as promising biocatalysts for asymmetric reduction prochiral ketones into chiral alcohols.
研究领域:
生物化学
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材料科学
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DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
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2017
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IF 5.651
Guochao Xu,Yaping Zhang,Yue Wang,Ye Ni
In this work, genome hunting strategy was adopted in screening for reductases from Candida glabrata . A total of 37 putative reductases were successfully expressed in E. coli BL21(DE3). A substrate library containing 32 substrates was established for characterization of each reductase by average specific activity and Shannon-Wiener index. Among them, Cg26 was identified with the highest efficiency and wider substrate spectrum in the reduction of prochiral ketones, with average activity and Shannon-Wiener index of 8.95 U·mg −1 and 2.82. Cg26 is a member of ‘extended’ short chain dehydrogenase/reductase superfamily. Ni 2+ could improve its activity. As much as 150 g·L −1 ethyl 2-oxo-4-phenylbutyrate could be completely converted by 10 g·L −1 Cg26. This study provides evidence for this newly identified Cg26 in the preparation of chiral secondary alcohols.
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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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2018
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IF 4.946
Ying Zhou,Qiyuan Peng,Ling Zhang,Sihua Cheng,Lanting Zeng,Fang Dong,Ziyin Yang
1-Phenylethanol is a chiral flavor compound that has enantiomers, ( R )- and ( S )-1-phenylethanol, with different flavor properties. Given that isolating these enantiomers from plants is low yielding and costly, enzymatic synthesis presents an alternative approach. However, the genes/enzymes that specifically produce ( R )- and ( S )-1-phenylethanol in plants are unknown. To identify these enzymes in tea ( Camellia sinensis ) flowers, 21 short chain dehydrogenase (SDR) genes were isolated from tea flowers, cloned, and functionally characterized. Several recombinant SDRs in Escherichia coli exhibited activity for converting acetophenone to ( S )-1-phenylethanol (CsSPESs, >99.0%), while only one SDR produced ( R )-1-phenylethanol (CsRPES, 98.6%). A pair of homologue enzymes (CsSPES and CsRPES) showed a strong preference for NADPH cofactor, with optimal enzymatic reaction conditions of 45–55 °C and pH 8.0. Identification of the tea flower-derived gene responsible for specific synthesis of ( R )- and ( S )-1-phenylethanolsuggests enzymatic synthesis of enantiopure 1-phenylethanol is possible using a plant-derived gene.
研究领域:
生物化学
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酶学
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植物科学
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产品关联
DOI
PubMed
高影响因子
ChemCatChem
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2024
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IF 3.8
Yuhao Zhang,Shuhui Huang,Wan-Qiu Liu,Jian Li
Graphical 4′-O-Methylnorbelladine (4′-MB) is a key precursor for the synthesis of Amaryllidaceae plant-derived alkaloids. In this study, an efficient biosynthetic system for the production of 4′-MB was developed through a comprehensive optimization strategy that includes protein engineering, enzyme immobilization, by-product recycling, and cofactor regeneration. This approach paves the way for the large-scale production of pharmaceuticals derived from Amaryllidaceae plants. Amaryllidaceae alkaloids (AAs) are plant-derived natural compounds with diverse bioactivities. To biosynthesize AAs, 4′-O-methylnorbelladine (4′-MB) is a key precursor that is first formed by C–N coupling between tyramine and 3,4-dihydroxybenzaldehyde catalyzed by a NADPH-dependent reductase. However, 3,4-dihydroxybenzaldehyde is oxygen sensitive and not stable due to its catechol moiety when constructing an in vitro enzymatic system for 4′-MB synthesis. To address this issue, we design an artificial biosynthetic route to synthesize 4′-MB. First, we choose isovanillin instead of 3,4-dihydroxybenzaldehyde as an alternative substrate. Then, a short-chain dehydrogenase/reductase (SDR) from Zephyranthes treatiae is utilized to couple tyramine and isovanillin. After demonstrating the synthesis of 4′-MB, we optimize the whole biocatalytic system with enhanced performance. The final enzymatic system can be reused for multiple cycles with the help of a crystalline inclusion protein (CipB)-mediated enzyme immobilization. Additionally, we scale up the reaction system from 50 µL to 10 mL, generating approximately 800 µM of 4′-MB. We envision that our work will provide an efficient and sustainable approach to produce 4′-MB, which could support the large-scale production of Amaryllidaceae plant-originated natural products.
研究领域:
植物化学
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生物合成
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药用植物学
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DOI
高影响因子
Nature Communications
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2021
·Vol. 12
·期 1
·1-11
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IF 14.919
Wen Wan-Hong,Zhang Yue,Zhang Ying-Ying,Yu Qian,Jiang Chu-Chu,Tang Man-Cheng,Pu Jin-Yue,Wu Lian,Zhao Yi-Lei,Shi Ting,Zhou Jiahai,Tang Gong-Li
Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.
研究领域:
微生物学
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药理学
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结构生物学
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DOI
PubMed
高影响因子
ORGANIC PROCESS RESEARCH & DEVELOPMENT
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2022
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IF 3.858
Ling He,Wenjie Ye,Youyu Xie,Qinghai Liu,Hualei Wang,Dongzhi Wei
Bioreductions catalyzed by ketoreductases play an important role in the synthesis of chiral alcohols. However, the synthesis of (R)-2-chloro-1-(3,4-difluorophenyl)ethanol (24b), an important chiral intermediate for the synthesis of the anticoagulant ticagrelor, poses significant challenges in terms of high substrate concentration requirements that limit its production. In this study, a novel NADH-dependent, short-chain dehydrogenase, PpKR8, from Paraburkholderia phymatum STM815, exhibited excellent enantioselectivity and high activity for the production of (R)-CFPL (24b) through the reduction of 2-chloro-1-(3,4-difluorophenyl)ethanone (24a). The coexpression of PpKR8 and glucose dehydrogenase from Bacillus subtilis in Escherichia coli allowed up to 300 g/L (1.57 M) CFPO (24a) to be completely converted into 24b with 99.9% enantiomeric excess and a high space–time yield (728 g/(L day)). Substrate specificity assays demonstrated a broad substrate spectrum for PpKR8, which included 35 α/β-ketoesters, aromatic ketones, and heterocyclic ketones. Moreover, three additional optically pure chiral alcohols that are used as important drug intermediates were synthesized at high substrate concentrations (150–330 g/L), demonstrating the excellent industrial potential of PpKR8-mediated bioreductions.
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
Green Synthesis and Catalysis
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2020
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IF 0
Bingmei Su,Lian Xu,Xinqi Xu,Lichao Wang,Aipeng Li,Juan Lin,Lidan Ye,Hongwei Yu
As an important building block for the synthesis of angiotensin-converting enzyme inhibitors, ethyl ( R )-2-hydroxyl-4-phenylbutanoate [( R )-HPBE] has attracted increasing attention. The key to industrial biosynthesis of ( R )-HPBE is a biocatalyst that efficiently reduces ethyl 2-oxo-4-phenylbutanoate (OPBE) with high R -enantioselectivity. This paper proposed a strategy for identifying key residues involved in enantioselectivity control based on per-residue free energy decomposition and sequence conservatism analysis. Using this strategy, 4 nonconservative sites with high energy contribution to binding of OPBE were chosen as engineering targets, generating variant Mu27 with 99% conversion and 98% ( R ) ee value at substrate loading of up to 500 mmol/L. MD simulations suggested the higher stability and formation probability of Mu27-OPBE proR prereaction state as key reasons for the excellent R -enantioselectivity of Mu27 towards OPBE. The success in this study provides a viable approach for rational design of alcohol dehydrogenases with high enantioselectivity towards unnatural substrates.
研究领域:
生物化学
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酶工程
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计算生物学
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DOI
GREEN CHEMISTRY
·
2022
·Vol. 24
·期 22
·8691-8704
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IF 11.034
Xin Pan,Xue Wang,Sihua Wu,Lei Xu,Leilei Zhang,Zhan Zhang,Bingfeng Li,Xuejun He,Siyuan Chang
Upgrading of the bio-based platform chemical 5-hydroxymethylfurfural (HMF) into high-value derivatives is an important research topic, particularly in green and sustainable chemistry. Herein, we applied a new, highly HMF-tolerant strain, Enterobacter ludwigii YYP3, as a whole-cell biocatalyst for efficient reduction of HMF to 2,5-bis(hydroxymethyl)furan (BHMF). Upon process optimization, within only 3 h, BHMF was produced with a yield >99% and 98.5% selectivity using 100 mM HMF, resulting in the highest space time yield (4.2 g L−1 h−1) among the currently reported HMF bioreduction processes. In a fed-batch conversion, E. ludwigii YYP3 achieved large-scale production of 290 mM BHMF within 9 h and retained its high catalytic activity for three runs (27 h), suggesting an excellent cycling stability. Based on genome and transcriptome analysis, the molecular mechanism underlying the high HMF tolerance of E. ludwigii YYP3 was explored, primarily through the downregulation of genes related to amino acid biosynthetic and metabolic processes and upregulation of genes associated with DNA replication, recombination, and repair; biofilm formation; and redox homeostasis. Meanwhile, two novel short-chain dehydrogenase/reductase family oxidoreductases ElSDR-ykvO and ElSDR-SSP1627 were identified as target enzymes responsible for conversion of HMF to less toxic BHMF in E. ludwigii YYP3. Combined with structure and mutation analysis, the catalytic mechanisms of target enzymes were determined to be based on the active sites Ser, Tyr, and Lys. Our work not only confirms that E. ludwigii YYP3 has promising application prospects in large-scale production of BHMF, but also provides novel insights into understanding the molecular mechanism of HMF reduction.
研究领域:
生物催化
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生物化学工程
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分子生物学
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绿色化学
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）