The Experts below are selected from a list of 16347 Experts worldwide ranked by ideXlab platform
Yu-guo Zheng - One of the best experts on this subject based on the ideXlab platform.
-
enhanced catalytic efficiency and enantioselectivity of epoxide hydrolase from agrobacterium radiobacter ad1 by iterative saturation mutagenesis for r Epichlorohydrin synthesis
Applied Microbiology and Biotechnology, 2018Co-Authors: Shuping Zou, Yu-guo Zheng, Zhicai Wang, Yaping Xue, Zhi-qiang LiuAbstract:Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-Epichlorohydrin. Six amino acid residues, I108, A110, D131, I133, T247, and G245, were selected for site saturation mutagenesis, and a sequential combination of positive mutants using ISM was constructed. Targeted mutagenesis generated five mutants (T247K, I108L, D131S, T247K/I108L, and T247K/I108L/D131S) with improved activity and enantioselectivity. Kinetics analysis showed that the best mutant, T247K/I108L/D131S, exhibited a 4.5-fold higher catalytic efficiency (k cat/K m) value and a 2.1-fold higher enantioselectivity (E value) towards Epichlorohydrin than the wild-type (WT) enzyme. Molecular docking computations support the source of notably improved enantioselectivity. In addition, the triple mutant also displayed a significantly enhanced thermostability, with > 8-fold longer half-life at 50 °C than WT. A gram-scale kinetic resolution of (R,S)-Epichlorohydrin was performed using T247K/I108L/D131S mutant as biocatalyst, affording a (R)-Epichlorohydrin yield of 40.2% (> 99.9% enantiomeric excess) and an average productivity of 1410 g L−1 d−1. The engineered T247K/I108L/D131S variant is a promising biocatalyst for the enzymatic synthesis of (R)-Epichlorohydrin.
-
Biosynthesis of (R)‐Epichlorohydrin at high substrate concentration by kinetic resolution of racemic Epichlorohydrin with a recombinant epoxide hydrolase
Engineering in Life Sciences, 2013Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:The substrate concentration and yield were shown to be very low in the production of (R)-Epichlorohydrin by hydrolysis of racemic Epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)-Epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic Epichlorohydrin was obtained. However, the ee of (R)-Epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)-3-chloro-1,2-propanediol displayed non-competitive inhibition in the conversion of (S)-Epichlorohydrin but non-significant inhibition for (R)-Epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)-Epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of (R)-Epichlorohydrin. This is the first report of high-yield production of (R)-Epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.
-
production of r Epichlorohydrin from 1 3 dichloro 2 propanol by two step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase in two phase system
Biochemical Engineering Journal, 2013Co-Authors: Zhongce Hu, Yu-guo ZhengAbstract:Abstract Recombinant Escherichia coli cells harbouring haloalcohol dehalogenase and epoxide hydrolase were successfully immobilized by adsorption onto perlite and used to prepare (R)-Epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis in a specially designed reactor. Two-phase solution was used as the reaction system in order to improve the yield of Epichlorohydrin. In the two-phase system containing 40% (v/v) cyclohexane, the yield of racemic Epichlorohydrin formed in the first step was 73%, and the yield of (R)-Epichlorohydrin with enantiomeric excess (ee) ≥99% increased from 19.2% to 25.1% in the second step. Ultimately, the yield of (R)-Epichlorohydrin reached 26.4% by optimization of the flow rate of air and amount of immobilized cells. To our knowledge, this was the first report on production of (R)-Epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase.
-
Enantioselective hydrolysis of Epichlorohydrin using whole Aspergillus niger ZJB-09173 cells in organic solvents
Journal of biosciences, 2012Co-Authors: Huo-xi Jin, Yu-guo ZhengAbstract:The enantioselective hydrolysis of racemic Epichlorohydrin for the production of enantiopure (S)-Epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB-09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic Epichlorohydrin using A. niger ZJB-09173. The racemic Epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-Epichlorohydrin with 98% enantiomeric excess from 153.6 mM of racemic Epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-Epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.
-
Nitrite‐mediated synthesis of chiral Epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1
Biotechnology and applied biochemistry, 2012Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni–nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral Epichlorohydrin in the presence of NO2−. The optimal pH, temperature, and NO2− concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)-Epichlorohydrin are 714.3 µmol min−1 mg−1 and 17.2 mM, respectively, whereas those for (R)-Epichlorohydrin are 166.8 µmol min−1 mg−1 and 29.0 mM, respectively. Under optimal conditions, (R)-Epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral Epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)-Epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3-dichloro-2-propanol by the ring opening of racemic Epichlorohydrin in the presence of NO2− after the ring closure of 1,3-dichloro-2-propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of Epichlorohydrin with NO2− and synthesis of chiral Epichlorohydrin with 99% enantiomeric excess from 1,3-dichloro-2-propanol by combining ring closure of 1,3-dichloro-2-propanol and ring opening of racemic Epichlorohydrin.
Zhi-qiang Liu - One of the best experts on this subject based on the ideXlab platform.
-
enhanced catalytic efficiency and enantioselectivity of epoxide hydrolase from agrobacterium radiobacter ad1 by iterative saturation mutagenesis for r Epichlorohydrin synthesis
Applied Microbiology and Biotechnology, 2018Co-Authors: Shuping Zou, Yu-guo Zheng, Zhicai Wang, Yaping Xue, Zhi-qiang LiuAbstract:Enantioselective hydrolysis of epoxides by epoxide hydrolase (EH) is one of the most attractive approaches for the synthesis of chiral epoxides. So far, attempts to develop an efficient epoxide hydrolase -mediated biotransformation have been limited by either the low activity or insufficient enantioselectivity of epoxide hydrolase. In this study, iterative saturation mutagenesis (ISM) of epoxide hydrolase from Agrobacterium radiobacter AD1 (ArEH) was performed for efficient production of (R)-Epichlorohydrin. Six amino acid residues, I108, A110, D131, I133, T247, and G245, were selected for site saturation mutagenesis, and a sequential combination of positive mutants using ISM was constructed. Targeted mutagenesis generated five mutants (T247K, I108L, D131S, T247K/I108L, and T247K/I108L/D131S) with improved activity and enantioselectivity. Kinetics analysis showed that the best mutant, T247K/I108L/D131S, exhibited a 4.5-fold higher catalytic efficiency (k cat/K m) value and a 2.1-fold higher enantioselectivity (E value) towards Epichlorohydrin than the wild-type (WT) enzyme. Molecular docking computations support the source of notably improved enantioselectivity. In addition, the triple mutant also displayed a significantly enhanced thermostability, with > 8-fold longer half-life at 50 °C than WT. A gram-scale kinetic resolution of (R,S)-Epichlorohydrin was performed using T247K/I108L/D131S mutant as biocatalyst, affording a (R)-Epichlorohydrin yield of 40.2% (> 99.9% enantiomeric excess) and an average productivity of 1410 g L−1 d−1. The engineered T247K/I108L/D131S variant is a promising biocatalyst for the enzymatic synthesis of (R)-Epichlorohydrin.
-
Biosynthesis of (R)‐Epichlorohydrin at high substrate concentration by kinetic resolution of racemic Epichlorohydrin with a recombinant epoxide hydrolase
Engineering in Life Sciences, 2013Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:The substrate concentration and yield were shown to be very low in the production of (R)-Epichlorohydrin by hydrolysis of racemic Epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)-Epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic Epichlorohydrin was obtained. However, the ee of (R)-Epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)-3-chloro-1,2-propanediol displayed non-competitive inhibition in the conversion of (S)-Epichlorohydrin but non-significant inhibition for (R)-Epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)-Epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of (R)-Epichlorohydrin. This is the first report of high-yield production of (R)-Epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.
-
Nitrite‐mediated synthesis of chiral Epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1
Biotechnology and applied biochemistry, 2012Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni–nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral Epichlorohydrin in the presence of NO2−. The optimal pH, temperature, and NO2− concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)-Epichlorohydrin are 714.3 µmol min−1 mg−1 and 17.2 mM, respectively, whereas those for (R)-Epichlorohydrin are 166.8 µmol min−1 mg−1 and 29.0 mM, respectively. Under optimal conditions, (R)-Epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral Epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)-Epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3-dichloro-2-propanol by the ring opening of racemic Epichlorohydrin in the presence of NO2− after the ring closure of 1,3-dichloro-2-propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of Epichlorohydrin with NO2− and synthesis of chiral Epichlorohydrin with 99% enantiomeric excess from 1,3-dichloro-2-propanol by combining ring closure of 1,3-dichloro-2-propanol and ring opening of racemic Epichlorohydrin.
-
characterization of a newly synthesized epoxide hydrolase and its application in racemic resolution of r s Epichlorohydrin
Catalysis Communications, 2011Co-Authors: Zhi-qiang Liu, Yu-guo Zheng, Liping Zhang, Feng Cheng, Litao Ruan, Yinchu ShenAbstract:Abstract In the current study, an epoxide hydrolase (EH) gene from Rhodosporidium toruloides was synthesized and expressed in Escherichia coli. After purification, we found that the optimal pH and temperature of this enzyme were 7.5 and 35 °C, respectively. The recombinant EH obtained in this study was temperature-sensitive and the activity decreased significantly above 45 °C. The values of apparent Km and Vmax were 0.5953 mol/l and 0.0105 mol/(L·min). In addition, enantiomeric excesses value of (R)-Epichlorohydrin could reach 100% after 40-min reaction. Moreover, this EH showed a broad substrates specificity toward epoxides. To the best of our knowledge, this is the first time to report the application of R. toruloides EH in racemic resolution of (R,S)-Epichlorohydrin to produce (R)-Epichlorohydrin.
Huo-xi Jin - One of the best experts on this subject based on the ideXlab platform.
-
Enzymatic approaches to the preparation of chiral Epichlorohydrin
RSC Advances, 2015Co-Authors: Huo-xi Jin, Xiao-kun OuyangAbstract:Enantiomerically pure Epichlorohydrin is a key chiral synthon in the preparation of 4-chloro-3-hydroxybutyrate, pheromones, L-carnitine, and β-adrenergic blockers. Various methods are known for obtaining the enantiomerically pure epoxides, including chemical and enzymatic approaches, but a clear understanding of the synthesis process in case of chiral Epichlorohydrin is unavailable. This review gives an overview of the enzymatic approaches for preparation of the chiral Epichlorohydrin, highlighting the synthetic routes using haloalcohol dehalogenase and epoxide hydrolase as biocatalysts.
-
Biosynthesis of (R)‐Epichlorohydrin at high substrate concentration by kinetic resolution of racemic Epichlorohydrin with a recombinant epoxide hydrolase
Engineering in Life Sciences, 2013Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:The substrate concentration and yield were shown to be very low in the production of (R)-Epichlorohydrin by hydrolysis of racemic Epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)-Epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic Epichlorohydrin was obtained. However, the ee of (R)-Epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)-3-chloro-1,2-propanediol displayed non-competitive inhibition in the conversion of (S)-Epichlorohydrin but non-significant inhibition for (R)-Epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)-Epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of (R)-Epichlorohydrin. This is the first report of high-yield production of (R)-Epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.
-
Enantioselective hydrolysis of Epichlorohydrin using whole Aspergillus niger ZJB-09173 cells in organic solvents
Journal of biosciences, 2012Co-Authors: Huo-xi Jin, Yu-guo ZhengAbstract:The enantioselective hydrolysis of racemic Epichlorohydrin for the production of enantiopure (S)-Epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB-09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic Epichlorohydrin using A. niger ZJB-09173. The racemic Epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-Epichlorohydrin with 98% enantiomeric excess from 153.6 mM of racemic Epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-Epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.
-
Nitrite‐mediated synthesis of chiral Epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1
Biotechnology and applied biochemistry, 2012Co-Authors: Huo-xi Jin, Zhi-qiang Liu, Yu-guo ZhengAbstract:In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni–nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral Epichlorohydrin in the presence of NO2−. The optimal pH, temperature, and NO2− concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)-Epichlorohydrin are 714.3 µmol min−1 mg−1 and 17.2 mM, respectively, whereas those for (R)-Epichlorohydrin are 166.8 µmol min−1 mg−1 and 29.0 mM, respectively. Under optimal conditions, (R)-Epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral Epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)-Epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3-dichloro-2-propanol by the ring opening of racemic Epichlorohydrin in the presence of NO2− after the ring closure of 1,3-dichloro-2-propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of Epichlorohydrin with NO2− and synthesis of chiral Epichlorohydrin with 99% enantiomeric excess from 1,3-dichloro-2-propanol by combining ring closure of 1,3-dichloro-2-propanol and ring opening of racemic Epichlorohydrin.
-
Enantioselective hydrolysis of Epichlorohydrin using whole
2012Co-Authors: Huo-xi Jin, Yu-guo ZhengAbstract:The enantioselective hydrolysis of racemic Epichlorohydrin for the production of enantiopure (S)-Epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic Epichlorohydrin using A. niger ZJB-09173. The racemic Epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-Epichlorohydrin with 98% enantiomeric excess from 153.6 mM of racemic Epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-Epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.
Borislav Miličević - One of the best experts on this subject based on the ideXlab platform.
-
Isolation of starch from two wheat varieties and their modification with Epichlorohydrin
Carbohydrate Polymers, 2010Co-Authors: Đurđica Ačkar, Jurislav Babić, Drago Šubarić, Mirela Kopjar, Borislav MiličevićAbstract:The aim of this research was determination of influence of wheat variety and modification with Epichlorohydrin on starch properties. Starch was isolated from two wheat varieties: “Golubica” and “Srpanjka” and modified with different concentrations of Epichlorohydrin (0.1%, 0.3% and 0.5% v/w). Both native and modified starches were characterised. Results showed similar characteristics of native wheat starches. Modification with Epichlorohydrin increased temperature of gelatinisation, but gelatinisation and retrogradation enthalpy were affected by the extent of the chemical reaction between starch and Epichlorohydrin, due to differences in reactivity between wheat starch varieties. Maximum viscosity decreased as well as breakdown and setback values. Swelling power and solubility were also decreased by modification, while paste clarity and freeze–thaw stability were influenced differently, due to different extent of the chemical reaction between different starch varieties and Epichlorohydrin. Colour of starch was not significantly altered by modification. Digestibility of starches can be reduced by proper selection of Epichlorohydrin concentration used for modification.
Cha Yong Choi - One of the best experts on this subject based on the ideXlab platform.
-
Biocatalytic production of chiral Epichlorohydrin in organic solvents.
Journal of bioscience and bioengineering, 1999Co-Authors: Won Jae Choi, Eun Yeol Lee, Sung Jun Yoon, Seung-taek Yang, Cha Yong ChoiAbstract:Enantioselective hydrolysis of racemic Epichlorohydrin was accomplished for the production of enantiopure Epichlorohydrin using the whole cells of an isolated Aspergillus niger spps. To overcome the spontaneous chemical degradation of Epichlorohydrin that occurs in aqueous buffer, organic solvents were employed in the reaction medium. The enantioselectivity was highly dependent on the solvent structure, water content of the medium, and the initial Epichlorohydrin concentration. (S)-Epichlorohydrin could be obtained from its racemates (60 mM) with an optical purity of 100% enantiomeric excess (ee) and 20% yield in cyclohexane supplemented with 2.0% (v/v) water.