Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides

Ye, Hui-Hua; Hu, Die; Shi, Xiao-Ling; Wu, Minchen; Deng, Chao; Li, Jianfang

doi:10.1016/j.catcom.2016.08.036

Cited by 21 publications

(8 citation statements)

References 15 publications

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“…According to the research of Ye et al [10] mutant Pv EH1 Y3 displayed a limited enantioconvergence towards m CSO ( ee p = 69.7%). But it still did not meet the requirement of production of ( R )- m CPED.…”

Section: Resultsmentioning

confidence: 99%

“…Among all tested mutants of Pv EH1, Pv EH1 L105I/M160A/M175I (renamed Pv EH1 Y3 ), was selected (Additional file 1: Table S1). Its enantioconvergence towards rac - m CSO increased to 69.7% from 1.0% ee p of Pv EH1 [10]. In our present work, the SBP of Pv EH1, identified and analyzed using a CAVER 3.0 software (http://www.caver.cz/), was divided into three zones.…”

Section: Introductionmentioning

confidence: 98%

“…To completely and quickly hydrolyze rac -epoxide, EH applied in enantioconvergent hydrolysis also have to possess a low enantioselectivity (i.e., enantiomeric ratio, E ). In view of the merits of mono-enzymatic catalysis and the shortage of highly enantioconvergent EHs, it is necessary to excavate novel EHs or to modify specific local configurations of the existing EHs by protein engineering [10]. Among the different gene mutagenesis techniques, saturation mutagenesis (SM) at sites lining the enzyme’s binding pocket has emerged as a particularly viable approach to improve selectivity [11, 12].…”

Section: Introductionmentioning

confidence: 99%

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Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Zong

et al. 2019

Microb Cell Fact

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BackgroundEpoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO).ResultsPvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield.ConclusionsIn summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Zong

et al. 2019

Microb Cell Fact

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“…In our previous studies, a Pv EH1-encoding gene ( pveh 1, GenBank accession no: KR604729) was cloned and expressed in Escherichia coli BL21(DE3) ( Ye et al, 2016 ). Then, its mutant Pv EH1 Z4X4-59 towards rac - p CSO was studied.…”

Section: Introductionmentioning

confidence: 99%

Gram-Scale Synthesis of (R)-P-Chlorophenyl-1,2-Ethanediol at High Concentration by a Pair of Epoxide Hydrolases

Zhang

Lei

Wang

et al. 2022

Front. Bioeng. Biotechnol.

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(R)-p-chlorophenyl-1,2-ethanediol (pCPED) is an important intermediate for the synthesis of (R)-eliprodil that is widely applied in the treatment of ischemic stroke. To prepare (R)-pCPED with high enantiomeric excess (eep) and yield via the enantioconvergent hydrolysis of racemic styrene oxide (rac-pCSO) at high concentration, the bi-enzymatic catalysis was designed and investigated by a pair of epoxide hydrolases, a mutant (PvEH1Z4X4-59) of Phaseolus vulgaris EH1 and a mutant (RpEHF361V) of Rhodotorula paludigena RpEH. Firstly, the maximum allowable concentration of rac-pCSO was confirmed. Subsequently, the addition mode and the weight ratio of two Escherichia coli cells were optimized. Finally, under the optimized reaction conditions—the cell weight ratio 20:1 of E. coli/pveh1z4x4-59 to E. coli/rpehF361V, a simultaneous addition mode, and reaction temperature at 25°C—300 mM rac-pCSO in the 100 ml 4% (v/v) Tween-20/phosphate buffer system (100 mM, pH 7.0) was completely hydrolyzed within 5 h, affording (R)-pCPED with 87.8% eep, 93.4% yield, and 8.63 g/L/h space–time yield (STY). This work would be an efficient technical strategy for the preparation of chiral vicinal diols at industrial scale.

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“…Recently, limonene EH (LEH) mutants catalyzed the kinetic resolution of racemic styrene oxides and obviously increased the ee p and yield of product ( R )- or ( S )-phenyl glycol compared with wild-type LEH; among them, ee p of ( R )-phenyl glycol catalyzed by variant SZ649 increased from 21 to 79% ee p . , Comparatively, the enantioconvergent hydrolysis can give diols with high ee p and 100% theoretical yield . The convergent hydrolysis by a single EH can be considered as an ideal bioprocess, but only a few EHs had the high and opposite regioselectivity toward two enantiomers of epoxide . For plant EHs, only a Solanum tuberosum EH ( St EH) had the high and complementary regioselectivity toward ( R )- and ( S )- m -chlorostyrene oxides, producing ( R )- m -chlorophenyl glycol with 97% ee p and 88% yield .…”

Section: Introductionmentioning

confidence: 99%

Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

Hu¹,

Tang

Li³

et al. 2017

J. Agric. Food Chem.

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To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 °C and high stability at pH 4.5-7.5 and 55 °C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a-3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 °C. These afforded (R)-1b with >99% ee and 78.7% overall yield after recrystallization and (R)-9a with >99% ee, 38.7% overall yield, and 12.7 g/L/h space-time yield.

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Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides

Cited by 21 publications

References 15 publications

Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Gram-Scale Synthesis of (R)-P-Chlorophenyl-1,2-Ethanediol at High Concentration by a Pair of Epoxide Hydrolases

Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

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