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Yu-guo Zheng - One of the best experts on this subject based on the ideXlab platform.
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Upscale production of (R)-mandelic Acid with a stereospecific nitrilase in an aqueous system.
Bioprocess and biosystems engineering, 2020Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Chuyan Wang, Xue Cai, Yu-guo ZhengAbstract:(R)-Mandelic Acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity of the Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6 U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500 mM R,S-mandelonitrile (R,S-MN) was resolved into 426 mM (64.85 g/L) R-MA within 8 h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.
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Production of R -Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine
Applied biochemistry and biotechnology, 2017Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yuan-shan Wang, Yu-guo ZhengAbstract:Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-Mandelic Acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L−1 day−1 and the space-time productivity of 143.2 mmol L−1 h−1 g−1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.
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R-Mandelic Acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor
Biocatalysis and Biotransformation, 2016Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yu-guo ZhengAbstract:AbstractA recirculating packed bed reactor (RPBR) was used for efficient production of R-Mandelic Acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52 mM/min) and yield (100%) by recycling 100 mL of substrate solution (70 mM) through RPBR packed with 6.0 g immobilized cells at a substrate-feeding rate of 19 mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12 g/L/h for R-MA with >99% of enantiomeric excess.
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improvement of alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of r mandelic Acid
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
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Improvement of Alcaligenes faecalis Nitrilase by Gene Site Saturation Mutagenesis and Its Application in Stereospecific Biosynthesis of (R)-(−)-Mandelic Acid
Journal of agricultural and food chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
Tadayuki Imanaka - One of the best experts on this subject based on the ideXlab platform.
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efficient production of r mandelic Acid by isopropanol permeabilized recombinant e coli cells expressing alcaligenes sp nitrilase
Biochemical Engineering Journal, 2015Co-Authors: Zhijun Zhang, Tadayuki ImanakaAbstract:Resting cells or immobilized cells are preferentially used as biocatalyst for biocatalytic processes due to the easy preparation and operation, however, the catalytic efficiency of biocatalyst is usually limited by the transport barrier of cell membrane. In this work, the transport barrier of recombinant Escherichia coli cells expressing the nitrilase from Alcaligenes sp. ECU0401 was relieved by isopropanol permeabilization. Under the optimal permeabilization conditions, the nitrilase activity of the permeabilized cells was 4.6-fold that of the cells without permeabilization. The permeabilized cells were evaluated for the enantioselective hydrolysis of mandelonitrile, which showed an initial reaction rate of about 2-fold that of the cells without permeabilization. The permeabilization process did not impair the stability of the cells and after simple immobilization by glutaraldehyde cross-linking, the immobilized permeabilized cells were successfully recycled for 15 runs without significant activity loss, suggesting the potential application of the immobilized permeabilized cells for the production of (R)-(−)-mandelic Acid. The technology reported in this work might also be extended to other bioprocesses facing cell membrane transport barriers.
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Efficient production of (R)-(−)-mandelic Acid by isopropanol-permeabilized recombinant E. coli cells expressing Alcaligenes sp. nitrilase
Biochemical Engineering Journal, 2015Co-Authors: Zhijun Zhang, Tadayuki ImanakaAbstract:Resting cells or immobilized cells are preferentially used as biocatalyst for biocatalytic processes due to the easy preparation and operation, however, the catalytic efficiency of biocatalyst is usually limited by the transport barrier of cell membrane. In this work, the transport barrier of recombinant Escherichia coli cells expressing the nitrilase from Alcaligenes sp. ECU0401 was relieved by isopropanol permeabilization. Under the optimal permeabilization conditions, the nitrilase activity of the permeabilized cells was 4.6-fold that of the cells without permeabilization. The permeabilized cells were evaluated for the enantioselective hydrolysis of mandelonitrile, which showed an initial reaction rate of about 2-fold that of the cells without permeabilization. The permeabilization process did not impair the stability of the cells and after simple immobilization by glutaraldehyde cross-linking, the immobilized permeabilized cells were successfully recycled for 15 runs without significant activity loss, suggesting the potential application of the immobilized permeabilized cells for the production of (R)-(−)-mandelic Acid. The technology reported in this work might also be extended to other bioprocesses facing cell membrane transport barriers.
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efficient production of r mandelic Acid with highly substrate product tolerant and enantioselective nitrilase of recombinant alcaligenes sp
Process Biochemistry, 2010Co-Authors: Zhijun Zhang, Liming Ouyang, Youyan Liu, Tadayuki ImanakaAbstract:Abstract For efficient production of ( R )-(−)-mandelic Acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli . After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of ( R )-(−)-mandelic Acid after only 4 h of transformation reached 197 mM with an enantiomeric excess ( ee p ) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of ( R )-(−)-mandelic Acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure ( R )-(−)-mandelic Acid.
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Efficient production of (R)-(−)-mandelic Acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.
Process Biochemistry, 2010Co-Authors: Zhijun Zhang, Liming Ouyang, Youyan Liu, Tadayuki ImanakaAbstract:Abstract For efficient production of ( R )-(−)-mandelic Acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli . After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of ( R )-(−)-mandelic Acid after only 4 h of transformation reached 197 mM with an enantiomeric excess ( ee p ) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of ( R )-(−)-mandelic Acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure ( R )-(−)-mandelic Acid.
Zhijun Zhang - One of the best experts on this subject based on the ideXlab platform.
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efficient production of r mandelic Acid by isopropanol permeabilized recombinant e coli cells expressing alcaligenes sp nitrilase
Biochemical Engineering Journal, 2015Co-Authors: Zhijun Zhang, Tadayuki ImanakaAbstract:Resting cells or immobilized cells are preferentially used as biocatalyst for biocatalytic processes due to the easy preparation and operation, however, the catalytic efficiency of biocatalyst is usually limited by the transport barrier of cell membrane. In this work, the transport barrier of recombinant Escherichia coli cells expressing the nitrilase from Alcaligenes sp. ECU0401 was relieved by isopropanol permeabilization. Under the optimal permeabilization conditions, the nitrilase activity of the permeabilized cells was 4.6-fold that of the cells without permeabilization. The permeabilized cells were evaluated for the enantioselective hydrolysis of mandelonitrile, which showed an initial reaction rate of about 2-fold that of the cells without permeabilization. The permeabilization process did not impair the stability of the cells and after simple immobilization by glutaraldehyde cross-linking, the immobilized permeabilized cells were successfully recycled for 15 runs without significant activity loss, suggesting the potential application of the immobilized permeabilized cells for the production of (R)-(−)-mandelic Acid. The technology reported in this work might also be extended to other bioprocesses facing cell membrane transport barriers.
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Efficient production of (R)-(−)-mandelic Acid by isopropanol-permeabilized recombinant E. coli cells expressing Alcaligenes sp. nitrilase
Biochemical Engineering Journal, 2015Co-Authors: Zhijun Zhang, Tadayuki ImanakaAbstract:Resting cells or immobilized cells are preferentially used as biocatalyst for biocatalytic processes due to the easy preparation and operation, however, the catalytic efficiency of biocatalyst is usually limited by the transport barrier of cell membrane. In this work, the transport barrier of recombinant Escherichia coli cells expressing the nitrilase from Alcaligenes sp. ECU0401 was relieved by isopropanol permeabilization. Under the optimal permeabilization conditions, the nitrilase activity of the permeabilized cells was 4.6-fold that of the cells without permeabilization. The permeabilized cells were evaluated for the enantioselective hydrolysis of mandelonitrile, which showed an initial reaction rate of about 2-fold that of the cells without permeabilization. The permeabilization process did not impair the stability of the cells and after simple immobilization by glutaraldehyde cross-linking, the immobilized permeabilized cells were successfully recycled for 15 runs without significant activity loss, suggesting the potential application of the immobilized permeabilized cells for the production of (R)-(−)-mandelic Acid. The technology reported in this work might also be extended to other bioprocesses facing cell membrane transport barriers.
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Efficient production of (R)-(−)-mandelic Acid using glutaraldehyde cross-linked Escherichia coli cells expressing Alcaligenes sp. nitrilase
Bioprocess and biosystems engineering, 2013Co-Authors: Zhijun Zhang, Jiang Pan, Gao-wei ZhengAbstract:Recombinant Escherichia coli cells expressing Alcaligenes sp. nitrilase were simply immobilized by direct cross-linking using glutaraldehyde. About 85 % of the total nitrilase activity was recovered under the optimal cross-linking conditions. The thermal stabilities of the cross-linked cells measured at 30, 40 and 50 °C were 4.5-, 5.3-, and 5.1-fold those of the free cells, respectively. The concentration of (R)-(−)-mandelic Acid reached 280 mM after merely 2 h transformation with the immobilized cells using 300 mM mandelonitrile as substrate, affording an extremely high productivity of 510.7 g L−1 d−1. In addition, operational stability of the immobilized cells was obviously superior to that of free cells, without significant activity loss after 15 cycles of batch reactions or 8 cycles of repeated fed-batch reactions. Therefore, the easy preparation and robust characteristics of the immobilized biocatalyst make it a very promising biocatalyst for high-performance and low-cost production of optically pure (R)-(−)-mandelic Acid.
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significant enhancement of r mandelic Acid production by relieving substrate inhibition of recombinant nitrilase in toluene water biphasic system
Journal of Biotechnology, 2011Co-Authors: Zhijun Zhang, Jiang Pan, Junfeng Liu, Youyan LiuAbstract:The enantioselective hydrolysis of mandelonitrile with whole cells of a recombinant Escherichia coli expressing nitrilase activity was severely inhibited by the substrate at high concentrations (>300mM), which resulted in a low yield of the target product (R)-(-)-mandelic Acid. To relieve the substrate inhibition and to enhance the (R)-(-)-mandelic Acid productivity, eight water-organic solvent biphasic systems were attempted in this work. Toluene was found to be the most suitable solvent as the organic phase among the solvents tested. Various parameters were systematically examined and optimized in shake flasks. The phase volume ratio, buffer pH and reaction temperature were shown to be sensitive parameters affecting both the yield and the enantiopurity of product in the biphasic system. Under the optimized conditions, significant enhancement of substrate tolerance from 200mM to 500mM and average productivity from 179.6gl(-1)d(-1) to 352.6gl(-1)d(-1) were achieved. Subsequently, the biocatalytic hydrolysis of mandelonitrile was successfully carried out in a stirred reactor (2-l scale) by repeated use of the calcium alginate entrapped cells for 5 batches, affording 110.7g (R)-(-)-mandelic Acid in 98.0% ee (enantiomeric excess) and a specific production of 13.8g (mandelic Acid) g(-1) (cell), respectively.
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cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from alcaligenes sp ecu0401 and its potential for r mandelic Acid production
Bioprocess and Biosystems Engineering, 2011Co-Authors: Zhijun Zhang, Liming Ouyang, Youyan LiuAbstract:A nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli BL21 (DE3) in a soluble form. The encoded protein with a His6-tag was purified to nearly homogeneity as revealed by SDS-PAGE with a molecular weight of approximately 38.5 kDa, and the holoenzyme was estimated to be composed of 10 subunits of identical size by size exclusion chromatography. The V max and K m parameters were determined to be 27.9 μmol min−1 mg−1 protein and 21.8 mM, respectively, with mandelonitrile as the substrate. The purified enzyme was highly thermostable with a half life of 155 h at 30 °C and 94 h at 40 °C. Racemic mandelonitrile (50 mM) could be enantioselectively hydrolyzed to (R)-(−)-mandelic Acid by the purified nitrilase with an enantiomeric excess of 97%. The extreme stability, high activity and enantioselectivity of this nitrilase provide a solid base for its practical application in the production of (R)-(−)-mandelic Acid.
Zhi-qiang Liu - One of the best experts on this subject based on the ideXlab platform.
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Upscale production of (R)-mandelic Acid with a stereospecific nitrilase in an aqueous system.
Bioprocess and biosystems engineering, 2020Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Chuyan Wang, Xue Cai, Yu-guo ZhengAbstract:(R)-Mandelic Acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity of the Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6 U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500 mM R,S-mandelonitrile (R,S-MN) was resolved into 426 mM (64.85 g/L) R-MA within 8 h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.
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Production of R -Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine
Applied biochemistry and biotechnology, 2017Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yuan-shan Wang, Yu-guo ZhengAbstract:Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-Mandelic Acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L−1 day−1 and the space-time productivity of 143.2 mmol L−1 h−1 g−1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.
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R-Mandelic Acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor
Biocatalysis and Biotransformation, 2016Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yu-guo ZhengAbstract:AbstractA recirculating packed bed reactor (RPBR) was used for efficient production of R-Mandelic Acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52 mM/min) and yield (100%) by recycling 100 mL of substrate solution (70 mM) through RPBR packed with 6.0 g immobilized cells at a substrate-feeding rate of 19 mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12 g/L/h for R-MA with >99% of enantiomeric excess.
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improvement of alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of r mandelic Acid
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
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Improvement of Alcaligenes faecalis Nitrilase by Gene Site Saturation Mutagenesis and Its Application in Stereospecific Biosynthesis of (R)-(−)-Mandelic Acid
Journal of agricultural and food chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
Ya-ping Xue - One of the best experts on this subject based on the ideXlab platform.
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Upscale production of (R)-mandelic Acid with a stereospecific nitrilase in an aqueous system.
Bioprocess and biosystems engineering, 2020Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Chuyan Wang, Xue Cai, Yu-guo ZhengAbstract:(R)-Mandelic Acid (R-MA) is a key precursor for the synthesis of semi-synthetic penicillin, cephalosporin, anti-obesity drugs, antitumor agents, and chiral resolving agents for the resolution of racemic alcohols and amines. In this study, an enzymatic method for the large-scale production of R-MA by a stereospecific nitrilase in an aqueous system was developed. The nitrilase activity of the Escherichia coli BL21(DE3)/pET-Nit whole cells reached 138.6 U/g in a 20,000-L fermentor. Using recombinant E. coli cells as catalyst, 500 mM R,S-mandelonitrile (R,S-MN) was resolved into 426 mM (64.85 g/L) R-MA within 8 h, and the enantiomeric excess (ee) value of R-MA reached 99%. During the purification process, pure R-MA with a recovery rate of 78.8% was obtained after concentration and crystallization. This study paved the foundation for the upscale production of R-MA using E. coli whole cells as biocatalyst.
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Production of R -Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine
Applied biochemistry and biotechnology, 2017Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yuan-shan Wang, Yu-guo ZhengAbstract:Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-Mandelic Acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L−1 day−1 and the space-time productivity of 143.2 mmol L−1 h−1 g−1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.
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R-Mandelic Acid production with immobilized recombinant Escherichia coli cells in a recirculating packed bed reactor
Biocatalysis and Biotransformation, 2016Co-Authors: Xinhong Zhang, Ya-ping Xue, Zhi-qiang Liu, Bo Yang, Yu-guo ZhengAbstract:AbstractA recirculating packed bed reactor (RPBR) was used for efficient production of R-Mandelic Acid (R-MA) by kinetic resolution of racemic R,S-mandelonitrile (R,S-MN) using the recombinant E. coli cells crosslinked with diatomite (DA)/glutaraldehyde (GA)/polyethyleneimine (PEI). The performance and productivity of RPBR were evaluated by several parameters, including cell load, substrate feeding rate, height diameter (H/D) ratio, reactor structures, and operation stability. The kinetic resolution process showed higher initial reaction rate (1.52 mM/min) and yield (100%) by recycling 100 mL of substrate solution (70 mM) through RPBR packed with 6.0 g immobilized cells at a substrate-feeding rate of 19 mL/min while the H/D ratio was 2.8. The immobilized cells were successfully applied into kinetic resolution of R,S-MN in the RPBR for 50 batches with an average productivity of 4.12 g/L/h for R-MA with >99% of enantiomeric excess.
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improvement of alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of r mandelic Acid
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
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Improvement of Alcaligenes faecalis Nitrilase by Gene Site Saturation Mutagenesis and Its Application in Stereospecific Biosynthesis of (R)-(−)-Mandelic Acid
Journal of agricultural and food chemistry, 2014Co-Authors: Zhi-qiang Liu, Ya-ping Xue, Xinhong Zhang, Yu-guo ZhengAbstract:Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic Acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic Acid after 7.5 h of conversion reached 693 mM w...
