Home Journals Articles Updates About
CN
image
收藏切换
Discovery of a novel epoxide hydrolase and elucidation of its catalytic mechanism
收藏切换
PDF
Meinan ZHU1, #, Xiao GU1, #, Dekai LIU1, Lingzhi ZHANG1, Shuyan ZHAO1, Lijuan ZHANG2, Guangya ZHANG1, Wei JIANG1, *
Acta Microbiologica Sinica | 2025, 65(1) : 389 - 401
Less
收藏切换
Acta Microbiologica Sinica | 2025, 65(1): 389-401
• Research Articles •
Discovery of a novel epoxide hydrolase and elucidation of its catalytic mechanism
Full
Meinan ZHU1, #, Xiao GU1, #, Dekai LIU1, Lingzhi ZHANG1, Shuyan ZHAO1, Lijuan ZHANG2, Guangya ZHANG1, Wei JIANG1, *
Affiliations
  • 1 College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian, China
  • 2 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, Shandong, China
Published: 2025-01-04 doi: 10.13343/j.cnki.wsxb.20240554
Outline
收藏切换

[Objective] Epoxide hydrolases (EHs) play a key role in the synthesis of chiral pharmaceuticals. We explored new EHs by engineering or gene retrieval, aiming to enrich and discover more high-performance EHs. [Methods] A novel epoxide hydrolase (Aspergillus carlsbadensis epoxide hydrolase, AcEH) from Aspergillus carlsbadensis was identified by gene retrieval technology. We then used AutoDock2 to predict the key hydrolysis sites of AcEH and employed computational design to clarify the influences of important sites on the structure and catalytic mechanism of AcEH. [Results] The primary structure of the novel EH had three characteristic α/β EH motifs: HGWP, GYTFS, and GGDIGS. AcEH exhibited high activity and could completely hydrolyze styrene oxide (SO) within 15 min, with a specific activity of 13 951 U/g. The Km, Vmax, and kcat/Km of AcEH were (107.07±57.98) mmol/L, (37.22±17.85) μmol/(min·mg), and 1.17 mmol/(L·s), respectively. The key hydrolysis sites of AcEH were Asp192-His372-Glu346, which catalyzed the triad, and two conserved tyrosine residues, Tyr251/314. The mutations R49L and R49Y caused enzyme inactivation, while the mutation Y45L resulted in the formation of inactive inclusion bodies. The interaction network revealed that changes in the 49th amino acid residue disrupted the interactions between key active site residues, leading to enzyme inactivation. On the other hand, the alteration of the 45th amino acid residue destabilized the enzyme structure, leading to the formation of inclusion bodies. [Conclusion] This study discovered a novel EH and analyzed its hydrolysis mechanism. The findings provide valuable insights for further research and engineering on this enzyme.

biocatalysis  /  epoxide hydrolase  /  chiral pharmaceutical  /  molecular dynamics simulation  /  molecular docking  /  hydrolysis mechanism
Meinan ZHU, Xiao GU, Dekai LIU, Lingzhi ZHANG, Shuyan ZHAO, Lijuan ZHANG, Guangya ZHANG, Wei JIANG. Discovery of a novel epoxide hydrolase and elucidation of its catalytic mechanism[J]. Acta Microbiologica Sinica, 2025 , 65 (1) : 389 -401 . DOI: 10.13343/j.cnki.wsxb.20240554
Full Text
Less
Chiral epoxides and their adjacent diols play an important role in the fields of fine chemicals, materials and pharmaceuticals[1]. They are valuable intermediates that can be used to synthesize important drugs and novel materials[2-5]. For example, chiral epoxide precursors can be used for the synthesis of important drugs and materials such as anti-obesity drugs (l-carnitine), anti-HIV drugs (lopinavir), calcium channel blockers (diltiazem), and chiral polyesters[6-7]. (R)-octane-1, 2-diol was used for the synthesis of mniopetal C, a compound inhibiting the reverse transcriptase of HIV-1, and (R)-7-octene-1, 2-diol for greensporone C having anticancer activity, and so on[8-9]. Although most chiral drugs can be synthesized using chemical methods, there are many issues associated with these methods, such as expensive chiral ligands[10], environmentally unfriendly metal catalysts, and harsh reaction conditions[11-12]. In contrast, biocatalysts not only exhibit excellent catalytic performance but are also environmentally friendly[13]. Therefore, biocatalysis is considered a promising alternative or complement to chemical catalysis.
Epoxide hydrolases (EHs) are widely distributed in animals, plants and microorganisms[14]. As a class of biocatalysts that do not require cofactors, they can selectively hydrolyze chiral epoxides to synthesize chiral epoxides and vicinal diols[15]. EHs can be classified into α/β-fold hydrolases, leukotriene A4 hydrolases, and limonene 1, 2-epoxide hydrolases based on their structural organization[16]. The majority of characterized EHs belong to the α/β-hydrolase fold superfamily, which is characterized by an α/β domain consisting of a β-sheet surrounded by a cluster of α-helices, and a cap domain that contains a variable cap-loop[9]. The substrate binding pocket is located between the two aforementioned structural domains. Its hydrolytic function is carried out by a catalytic triad (Asp-His-Asp/Glu) and two Tyr residues, Figure 1 depicts the hydrolysis process of EH as summarized from previous studies and the analysis of this article. Firstly, the substrate enters the active pocket and forms hydrogen bonds with two tyrosines, aiding in ring opening. Then, aspartate performs a nucleophilic attack on the α or β carbon atom of the epoxide ring, forming an intermediate. Finally, the activated water molecule by histidine and aspartic acid interacts with the intermediate, ultimately leading to the formation of vicinal diols[17-18]. Currently, with the advancement of genomics, an increasing number of epoxy hydrolases from various sources have been discovered. However, almost none of these enzymes exhibit satisfactory catalytic performance. Therefore, it is highly necessary to engineer or genetically explore new epoxy hydrolases[19].
To supplement and discover more high-performance EHs, this study identified an enzyme (Aspergillus carlsbadensis epoxide hydrolase, AcEH) belonging to the α/β hydrolase family from Aspergillus carlsbadensis. The enzyme was successfully cloned and expressed in Escherichia coli BL21(DE3), demonstrating EH activity. The study then analyzed the basic enzymatic properties and hydrolysis mechanism. Additionally, during the process of modifying the thermal stability, two key sites were discovered that were of significant importance for enzyme activity and stability. Possible reasons for these findings were explored through analysis. The exceptional activity exhibited by AcEH suggests its potential as an industrial biocatalyst. Furthermore, the study of its hydrolysis mechanism and exploration of thermal stability modification can serve as a reference for future research.
1 Materials and Methods
Less
1.1 Experimental materials
Escherichia coli DH5α plasmid was cloned and heterologously expressed by BL21(DE3). They were purchased from Golden Bio Co. Ltd. (Golden Biotechnology Corporation, Beijing). AcEH was heterologously expressed with plasmid pET-28a. The original strains are kept in the laboratory. Kanamycin and IPTG were purchased from TaKaRa (TaKaRa Biomedical Technology Co., Ltd., Xiamen). The plasmid extraction kit and SDS-PAGE gel preparation kit were purchased from Omega (Omega Engineering). Other chemical reagents were purchased from Aladdin (Shanghai Aladdin Biochemical Technology Co., Ltd.) and were of chromatographic or analytical purity.
1.2 Database search and sequence analysis
Through gene retrieval on the NCBI server (http://www.ncbi.nlm.nih.gov/), protein sequences with homology lower than 80% to AnEH1 (CAB59813, PDB: 1QO7) and unknown function were searched. Unreported proteins were selected from these sequences, with homology ranging from 60% to 80%, for cloning and expression, aiming to discover new EHs. Furthermore, the selected proteins were subjected to sequence alignment using the online tool ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/). Subsequently, sequences from different Aspergillus species were selected, and the MEGA 11 software (https://megasoftware.net/) was used for processing and constructing a phylogenetic tree.
1.3 Cloning and expression of AcEH
The E. coli strain containing the EH gene was inoculated in LB medium supplemented with 100 μg/mL kanamycin and incubated overnight at 37 ℃ as the seed culture. Subsequently, 2% (V/V) of the seed culture was inoculated into fresh LB medium and grown until reaching an OD600 of 0.6−0.8 at 37 ℃. The culture was then induced with 0.1 mmol/L IPTG and incubated for 20 h at 18 ℃. The cells were harvested, resuspended in a K2HPO4·KH2PO4 buffer (100 mmol/L, pH 7.0), and subjected to sonication for disruption. The resulting mixture was centrifuged at 11 000 r/min for 30 minutes at 4 ℃, and the supernatant was collected. The supernatant was filtered through a 0.22 μm membrane and purified using nickel Ni-NTA affinity chromatography to obtain the target protein enzyme as a catalyst.
1.4 Determination of protein and enzymatic properties
SDS-PAGE was used to analyze the expression of AcEH. Standard protein was used as a reference to estimate its apparent molecular weight using the ExPASy (https://web.expasy.org/protparam/) software. The concentration standard curves of SO, (S)-SO and (R)-SO, the activity of AcEH, pH stability, temperature stability and enzyme kinetics were determined using UV/Vis spectrophotometry with 4-(p-nitrobenzyl) pyridine (NBP) as previously described[20-21]. Additionally, the effects of different metal ions and buffer solutions on enzyme activity were also measured.
To calculate the half-life of AcEH, the purified enzyme protein is incubated at 30 ℃. Then, the enzyme activity of AcEH is measured at regular intervals using the untreated enzyme solution as a reference under optimal reaction conditions. The deactivation of AcEH follows a first-order kinetic model, and the formula to calculate the half-life is as follows formula (1).
In this context, Kc is the decay constant, E0 represents the initial enzyme activity, while E represents the remaining enzyme activity at a given temperature (t).
The enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol of substrate per minute under specified conditions. The specific enzyme activity (U/g) can be calculated using the formula (2).
Where C0 is the initial substrate concentration (20 mmol/L), v is the reaction volume, c is the substrate conversion rate, t is the reaction time (min), and m is the protein mass (g).
1.5 The hydrolysis mechanism of AcEH
Due to the low homology (64.12%) between the protein sequences of AcEH and the reported crystal structure (PDB ID: 1QO7), a combination of de novo modeling using AlphaFold2 (https://alphafold.ebi.ac.uk/) and homology modeling using SWISS-MODEL (https://swissmodel.expasy.org/interactive) is considered to predict its possible structure. Discovery Studio (DS2019) can be used for structural alignment and to evaluate its reliability using a Ramachandran plot (PyMOL: https://pymol.org.edu/2/). After obtaining the model, molecular docking of the protein-ligand complex can be performed using AutoDock (http://www.autodock.scripps.edu). The models with better overall scores can be selected to explain the hydrolysis mechanism of the enzyme. Finally, the results can be visualized using PyMOL. SO, (S)-SO, and (R)-SO ligands can be drawn using InDraw (https://www.integle.com/static/indraw). DS2019 originates from the servers of the Third Institute of Oceanography, Xiamen, Fujian.
1.6 Attempts to improve the thermal stability of AcEH
The modeling results of AcEH were input into FoldX (https://foldxsuite.crg.eu/) for comprehensive alanine scanning mutagenesis of its entire sequence, and the protein folding free energy changes were calculated. Amino acid residues that had a detrimental effect on thermal stability were identified, and active pocket positions and conserved amino acids were filtered out. The remaining residues were subjected to amino acid saturation mutagenesis using I-Mutant 3.0 (https://biofold.org/) and FoldX. The selected variants from both methods were then input into GROMACS software (https://www.gromacs.org/) and Discovery Studio for molecular dynamics simulations and thermal stability analysis (Figures S2 and S3, Data has been submitted to the National Microbial Science Data Center, number: NMDCX0001726). Key mutants that significantly improved thermal stability were identified. Subsequently, primers were designed for the mutated primers (Table S1, Data has been submitted to the National Microbial Science Data Center, number: NMDCX0001726), and the mutations were verified by PCR amplification followed by 1% agarose gel electrophoresis. The PCR products were then digested overnight with Dpn I restriction enzyme. The PCR products were transformed into Escherichia coli DMT cells, and after incubation, plasmids were extracted and sent to a Sangon Biotechnology (Shanghai) Co., Ltd. The successfully sequenced plasmids were then transformed into Escherichia coli BL21(DE3) cells and stored in 30% glycerol.
2 Results and Discussion
Less
2.1 Structure analysis of the AcEH
Through phylogenetic tree analysis, the result observed that the AcEH is distantly related to EH enzymes from other Aspergillus sources, indicating its novelty as an EH enzyme (Figure 2). Additionally, the amino acid sequence of AcEH shares more than 50% similarity with several EH enzymes from different Aspergillus sources (AnEH1, AnEH2, AnEH3, AfEH, AfEH-Z5, and AfEH-293). A sequence alignment was performed on these amino acids, and three characteristic regions were identified, as shown in Figure 2. Results revealed that AcEH contains a characteristic region specific to α/β hydrolases[22-23], namely SmXNuXSmSm (GGDIGS, amino acid position 190−195, where Sm, X, and Nu represent small amino acids, any amino acid, and nucleophilic amino acids, respectively), confirming its classification within the α/β hydrolase family. Furthermore, both AcEH and the aforementioned EHs share two conserved regions[24]: the HGXP motif (HGWP, amino acid position 115−118), which has been shown to form an oxyanion hole stabilizing the anion formed during nucleophilic attack, and the GXSmXS/T motif (GYTFS, amino acid position 151−155). These findings indicate that the AcEH is an α/β EH.
2.2 Heterologous expression of the AcEH
By cloning and induced expression, it was discovered that only EH from Aspergillus carlsbadensis (referred to as AcEH) formed soluble expression. The purified protein of AcEH was obtained. The size of AcEH was analyzed using SDS-PAGE gel, and it has 400 amino acids in length. According to predictions from the ExPASy website, the estimated size of AcEH is around 41 kDa. Additionally, based on predictions from native PAGE, the non-denatured size and position of AcEH were larger than BSA (66 kDa). Considering that the template 1QO7 is a dimeric protein, it is speculated that AcEH may also be a dimeric protein (Figure S1, Data has been submitted to the National Microbial Science Data Center, number: NMDCX0001726).
2.3 Enzymatic properties of the AcEH
The enzymatic properties of the AcEH were determined, and the results are shown in Figure 3 and Table 1. Figure 3A indicates that the optimal pH for enzyme activity is 7.0, and the enzyme remains stable within the pH range of 6.0−7.5. Figure 3B demonstrates that the enzyme exhibits high activity at temperatures of 20−30 ℃. In Figure 3C, it is observed that the enzyme remains stable when incubated at temperatures between 20 to 35 ℃ for 5 h, with enzyme activity maintained at over 80%. While the enzymatic reaction conditions of AcEH demand mild conditions, it exhibits remarkable stability, thereby conferring a distinct advantage in comparison to other counterparts[25-27]. Figure 3D shows that the majority of metal ions significantly inhibit the enzyme activity, except for Ca2+ at a concentration of 50 mg/L, which enhances the enzyme activity. Under optimal conditions, with SO as the substrate, AcEH has a half-life of 136.5 h. This is significantly higher than the reported EHs[28-29]. In a 15 min reaction, it can achieve a maximum specific activity of 13 952 U/g, and it is capable of completely hydrolyzing both (S)-SO and (R)-SO. Its hydrolytic activity exhibits a substantial advantage compared to the reported EHs[30].
The enzymatic kinetic parameters, as shown in Table 1, reveal that the enzyme exhibits excellent enzymatic activity and fast reaction rates towards SO, (S)-SO, and (R)-SO. The Vmax values are (37.22±17.85), (137.91±7.00), and (185.28±20.24) μmol/(min·mg), respectively, which are significantly higher than those reported for EHs[31]. Additionally, AcEH demonstrates high substrate affinity for both (S)- and (R)-configurations, with Km values of (4.00±0.65) and (6.87±1.58) mmol/L, respectively. Unlike most EHs discovered so far, AcEH exhibits the ability to rapidly hydrolyze both enantiomers of chiral epoxides[32-34], indicating its potental for enantioselective hydrolysis to produce pure racemic epoxides.
2.4 Hydrolysis mechanism of the AcEH
The results of the comparative analysis between the de novo modeling by AlphaFold and the homology modeling by SWISS-MODEL are shown in Figure 4A, with an RMSD (root mean square deviation) value of 0.530. This indicates a high similarity between the two modeling results. The Ramachandran plots evaluation of the two modeling results shows that 91.5% and 92.4% of the amino acids are located in the most favoured regions (Figure 4B). Generally, if more than 90.0% of the amino acid residues fall within the allowed and generously allowed regions, it can be considered that the conformation complies with the rules of stereochemistry[35]. Therefore, the 3D structure of AcEH is considered reasonable and can be further analyzed.
The molecular docking of the protein-ligand complex was performed using AutoDock2, and the model with a better overall score was selected to explain the hydrolysis mechanism of the enzyme[17]. The results are shown in Figure 5. Figure 5A depicts the 3D structure of the AcEH monomer, with the alpha helices shown in cyan, beta sheets in purple, loops in pink, and the solvent-accessible surface of the active pocket of AcEH in white. The green substance inside represents the SO. Figures 5B and 5C provide detailed views of the active pocket and the catalytic mechanism. The active pocket includes the classical catalytic triad (Asp192-His372-Glu346) and two conserved tyrosine residues (Tyr251, Tyr314). The hydrolysis mechanism is consistent with the previously reported typical hydrolysis pathway. It is speculated that the hydrolysis steps inferred and summarized in Figure 1 are consistent with the hydrolysis steps mentioned: the active hydroxyl groups of the two Tyr251/314 residues form hydrogen bonds with the oxygen atoms in the epoxide ring, stabilizing the substrate. Simultaneously, the negatively charged Asp192 selectively nucleophilically attacks the two carbon atoms in the epoxide ring. Then, a water molecule activated by histidine also nucleophilically attacks the substrate, opening the three-membered ring to form a tetrahedral intermediate. Finally, a water molecule donates a proton to release the resulting vicinal diol from the active site, completing the hydrolysis process.
2.5 Single point mutation modification and further mechanism exploration
After calculations, two optimal theoretical mutation sites were identified, namely position 45 and position 49. A total of three mutant variants were constructed, namely Y45L, R49L, and R49Y. The results revealed that the mutation at position 45 resulted in the formation of an inclusion body with no enzymatic activity, while the mutation at position 49 led to soluble expression of the enzyme but loss of enzymatic activity. Based on these findings, protein-protein interaction network analysis (PPI) of single-point mutants was carried out using the RING (Regulation of Interaction Networks in Genomes: https://ring.biocomputingup.it/help) website tool and Cytoscape to elucidate potential reasons[36].
Upon analysis, it was found that in the wild-type PPI as shown in Figure 6A, the key amino acids involved in the interaction of the wild-type protein are Trp284, His255, Tyr307/251, Trp117, and Phe154. In the Y45L mutant, as depicted in Figure 6B, the interaction between key amino acids Trp117 and Tyr251 is reduced. By comparing the degree values of key sites in the wild-type and Y45L mutant, as shown in Table 2, it was observed that the degree value of the Tyr45 node in the Y45L mutant is zero, indicating the complete disappearance of interactions with other amino acids. Protein structure analysis revealed that Tyr45 is located on a loop in the AcEH protein folding structure. It is speculated that Tyr45 on the loop plays a vital role in connecting the two α-helices, leading to the formation of inactive inclusion body enzyme in the Y45L mutant. In the PPI of the R49L and R49Y mutant variants, shown in Figures 6C and 6D, the key amino acid interactions have also changed. Since both mutants lack enzymatic activity, the main analysis focused on the changes in the degree values of their interactions with the wild-type catalytic site. The results, as shown in Table 3, revealed a decrease of one degree value for each of the catalytic triad Asp192-His372-Glu346, including the disappearance of the Ionic bond between Glu346 and His372. It is speculated that this may be the main reason for the loss of enzyme activity, as thehydrolytic action of the enzyme requires the joint action of Glu346-His372 to activate the water molecule for nucleophilic attack and proton transfer in the three-membered ring opening.
3 Conclusion
Less
In this study, AcEH was extracted by gene extraction and successfully expressed in E. coli. A new EH with high hydrolytic activity, long half-life and enantiomeric hydrolysis was successfully found. At the same time, its hydrolysis principle was explained by bioinformatics technology, which provided a theoretical basis for the further study of the hydrolysis mechanism of the enzyme.
This study also constructed three mutants, although failed to achieve the expected results, but explained the possible reasons, but also provided some warnings and references for the transformation of the enzyme. The completion of this task is of great significance for further optimization and industrial application of AcEH.
Funding
Less
  • National Natural Science Foundation of China(21808073)
  • Natural Science Foundation of Fujian Province(2021J05058)
  • State Key Laboratory of Microbial Technology Open Projects Fund(M2022-02)
  • Fundamental Research Funds for the Central Universities(ZQN-814)
  • High-level Personnel Activation Fee of Huaqiao University(600005-Z17Y0072)
  • Quanzhou City Science & Technology Program(2018C008)
References
Less
[1]
HWANG S, CHOI YC, LEE YE.Bio- and chemo-catalytic preparations of chiral epoxides Journal of Industrial and Engineering Chemistry 2010,16(1):1-6.
[2]
DUAN M, DÍAZ-OVIEDO CD, ZHOU Y, CHEN XY, YU PY, LIST B, HOUK KN, LAN Y.Chiral phosphoric acid catalyzed conversion of epoxides into thiiranes: mechanism, stereochemical model, and new catalyst design Angewandte Chemie International Edition 2022,61(9):e202113204.
[3]
LI J, LIU Y, REN WM, LU XB.Enantioselective terpolymerization of racemic and meso-epoxides with anhydrides for preparation of chiral polyesters Proceedings of the National Academy of Sciences of the United States of America 2020,117(27):15429-15436.
[4]
HU FL, CHEN ZL, TAN Y, XU D, HUANG SL, JIA SQ, GONG XN, QIN WL, YAN HL.Organocatalytic enantioselective γ-elimination: applications in the preparation of chiral peroxides and epoxides Organic Letters 2020,22(5):1934-1940.
[5]
LI J, REN BH, WAN ZQ, CHEN SY, LIU Y, REN WM, LU XB.Enantioselective resolution copolymerization of racemic epoxides and anhydrides: efficient approach for stereoregular polyesters and chiral epoxides Journal of the American Chemical Society 2019,141(22):8937-8942.
[6]
MOSCHONA F, SAVVOPOULOU I, TSITOPOULOU M, TATARAKI D, RASSIAS G.Epoxide syntheses and ring-opening reactions in drug development Catalysts 2020,10(10):1117.
[7]
YANG H, YU HX, STOLARZEWICZ IA, TANG WJ.Enantioselective transformations in the synthesis of therapeutic agents Chemical Reviews 2023,123(15):9397-9446.
[8]
MAYNERT EW, FOREMAN RL, WATABE T.Epoxides as obligatory intermediates in the metabolism of olefins to glycols The Journal of Biological Chemistry 1970,245(20):5234-5238.
[9]
HU B, HU D, ZHANG D, WEN Z, ZANG J, WU MC.Manipulating the regioselectivity of a Solanum lycopersicum epoxide hydrolase for the enantioconvergent synthesis of enantiopure alkane- and alkene-1, 2-diols Catalysis Science & Technology 2020,10(17):5886-5895.
[10]
JIANG JJ, WONG MK.Recent advances in the development of chiral gold complexes for catalytic asymmetric catalysis Chemistry-An Asian Journal 2021,16(5):364-377.
[11]
ETAYO P, VIDAL-FERRAN A.Rhodium-catalysed asymmetric hydrogenation as a valuable synthetic tool for the preparation of chiral drugs Chemical Society Reviews 2013,42(2):728-54.
[12]
YOON M, SRIRAMBALAJI R, KIM K.Homochiral metal-organic frameworks for asymmetric heterogeneous catalysis Chemical Reviews 2012,112(2):1196-1231.
[13]
ROSSINO G, ROBESCU MS, LICASTRO E, TEDESCO C, MARTELLO I, MAFFEI L, VINCENTI G, BAVARO T, COLLINA S.Biocatalysis: a smart and green tool for the preparation of chiral drugs Chirality 2022,34(11):1403-1418.
[14]
WIDERSTEN M, GURELL A, LINDBERG D.Structure-function relationships of epoxide hydrolases and their potential use in biocatalysis Biochimica et Biophysica Acta 2010,1800(3):316-326.
[15]
GODASE VP, KUMBHAR NM, GOVINDWAR SP, RAVI KUMAR A.Characterization reveals a putative epoxide hydrolase from Yarrowia lipolytica with the ability to convert rac-1, 2-epoxyhexane to (R)-diol Process Biochemistry 2023,125:248-258.
[16]
BUČKO M, KANIAKOVÁ K, HRONSKÁ H, GEMEINER P, ROSENBERG M.Epoxide hydrolases: multipotential biocatalysts International Journal of Molecular Sciences 2023,24(8):7334.
[17]
ZOU JY, HALLBERG BM, BERGFORS T, OESCH F, ARAND M, MOWBRAY SL, JONES TA.Structure of Aspergillus niger epoxide hydrolase at 1.8 Å resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases Structure 2000,8(2):111-122.
[18]
CHOI WJ, CHOI CY.Production of chiral epoxides: epoxide hydrolase-catalyzed enantioselective hydrolysis Biotechnology and Bioprocess Engineering 2005,10(3):167-179.
[19]
HUANG R, LI C, ZHAO SG, LIU QT, LIU Y, XUE ZL.Enantioconvergent hydrolysis of racemic 1, 2-epoxypentane and 1, 2-epoxyhexane by an engineered Escherichia coli strain overexpressing a novel Streptomyces fradiae epoxide hydrolase Enzyme and Microbial Technology 2023,166:110228.
[20]
ZOCHER F, ENZELBERGER MM, BORNSCHEUER UT, HAUER B, SCHMID RD.A colorimetric assay suitable for screening epoxide hydrolase activity Analytica Chimica Acta 1999,391(3):345-351.
[21]
MATEO C, ARCHELAS A, FURSTOSS R.A spectrophotometric assay for measuring and detecting an epoxide hydrolase activity Analytical Biochemistry 2003,314(1):135-141.
[22]
HOLMQUIST M.Alpha/beta-hydrolase fold enzymes: structures, functions and mechanisms Current Protein & Peptide Science 2000,1(2):209-235.
[23]
KUAN J, TSAI CH, CHOU CC, WU C, WU WF.Enzymatic characterization of a novel HSL family IV esterase EstD04 from Pseudomonas sp. D01 in mealworm gut microbiota Molecules 2023,28(14):5410.
[24]
STOJANOVSKI G, DOBRIJEVIC D, HAILES HC, WARD JM.Identification and catalytic properties of new epoxide hydrolases from the genomic data of soil bacteria Enzyme and Microbial Technology 2020,139:109592.
[25]
BELOTI LL, COSTA BZ, TOLEDO MAS, SANTOS CA, CRUCELLO A, FÁVARO MT, SANTIAGO AS, MENDES JS, MARSAIOLI AJ, SOUZA AP.A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization Protein Expression and Purification 2013,91(2):175-183.
[26]
XU W, XU JH, PAN J, GU Q, WU XY.Enantioconvergent hydrolysis of styrene epoxides by newly discovered epoxide hydrolases in mung bean Organic Letters 2006,8(8):1737-40.
[27]
VAN LOO B, KINGMA J, ARAND M, WUBBOLTS MG, JANSSEN DB.Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis Applied and Environmental Microbiology 2006,72(4):2905-2917.
[28]
JIN HX, LI Y, ZHANG QW, LIN SJ, YANG ZS, DING GF.Enantioselective hydrolysis of styrene oxide and benzyl glycidyl ether by a variant of epoxide hydrolase from Agromyces mediolanus Marine Drugs 2019,17(6):367.
[29]
ZOU SP, ZHENG YG, WU Q, WANG ZC, XUE YP, LIU ZQ.Enhanced catalytic efficiency and enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis for (R)-epichlorohydrin synthesis Applied Microbiology and Biotechnology 2018,102(2):733-742.
[30]
HU D, TANG CD, LI C, KAN TT, SHI XL, FENG L, WU MC.Stereoselective hydrolysis of epoxides by reVrEH3, a novel Vigna radiata epoxide hydrolase with high enantioselectivity or high and complementary regioselectivity Journal of Agricultural and Food Chemistry 2017,65(45):9861-9870.
[31]
SAINI P, KUMAR N, WANI SI, SHARMA S, CHIMNI SS, SAREEN D.Bioresolution of racemic phenyl glycidyl ether by a putative recombinant epoxide hydrolase from Streptomyces griseus NBRC 13350 World Journal of Microbiology & Biotechnology 2017,33(5):82.
[32]
HU D, WANG R, SHI XL, YE HH, WU Q, WU MC, CHU JJ.Kinetic resolution of racemic styrene oxide at a high concentration by recombinant Aspergillus usamii epoxide hydrolase in an n-hexanol/buffer biphasic system Journal of Biotechnology 2016,236:152-158.
[33]
XUE F, LIU ZQ, ZOU SP, WAN NW, ZHU WY, ZHU Q, ZHENG YG.A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: cloning, characterization and application Process Biochemistry 2014,49(3):409-417.
[34]
WU SK, LI AT, CHIN YS, LI Z.Enantioselective hydrolysis of racemic and Meso-epoxides with recombinant Escherichia coli expressing epoxide hydrolase from Sphingomonas sp. HXN-200: preparation of epoxides and vicinal diols in high ee and high concentration ACS Catalysis 2013,3(4):752-759.
[35]
JANSON G, PAIARDINI A.PyMod 3: a complete suite for structural bioinformatics in PyMOL Bioinformatics 2020,37(10):1471-1472.
[36]
CLEMENTEL D, del CONTE A, MONZON AM, CAMAGNI GF, MINERVINI G, PIOVESAN D, TOSATTO SCE.RING 3.0: fast generation of probabilistic residue interaction networks from structural ensembles Nucleic Acids Research 2022,50(W1):W651-W656.
Appendix
Less
Year 2025 volume 65 Issue 1
PDF
113
40
Cite this Article
BibTeX
Article Info
doi: 10.13343/j.cnki.wsxb.20240554
  • Receive Date：2024-09-08
  • Online Date：2026-03-21
  • Published：2025-01-04
Article Data
Affiliations
History
  • Received：2024-09-08
  • Accepted：2024-11-02
Funding
National Natural Science Foundation of China(21808073)
Natural Science Foundation of Fujian Province(2021J05058)
State Key Laboratory of Microbial Technology Open Projects Fund(M2022-02)
Fundamental Research Funds for the Central Universities(ZQN-814)
High-level Personnel Activation Fee of Huaqiao University(600005-Z17Y0072)
Quanzhou City Science & Technology Program(2018C008)
Affiliations
    1 College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian, China
    2 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, Shandong, China

Corresponding:

JIANG Wei, E-mail:
References
Share
https://castjournals.cast.org.cn/joweb/wswxb/EN/10.13343/j.cnki.wsxb.20240554
Share to
QR

Scan QR to access full text

Cite this article
BibTeX
Citations
表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
关闭全屏
  • BibTeX
  • EndNote
  • RefWorks
  • TxT
Links
Articles
Latest Articles
Most Read
Collections
Updates
Events
News
Multimedia
About
About Us
Contact
No. 86 Xueyuan South Road, Haidian District, Beijing
100081
010-62199257
qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House