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Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
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synthesis of optically pure ethyl s 4 Chloro 3 hydroxybutanoate by escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes
Applied Microbiology and Biotechnology, 2001Co-Authors: Noriyuki Kizaki, Michihiko Kataoka, Masaru Wada, Yoshihiko Yasohara, Junzo Hasegawa, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-Chloro-3-oxobutanoate (COBE) to ethyl (S)-4-Chloro-3-hydroxybutanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous mono-phase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.
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molecular cloning and overexpression of the gene encoding an nadph dependent carbonyl reductase from candida magnoliae involved in stereoselective reduction of ethyl 4 Chloro 3 oxobutanoate
Bioscience Biotechnology and Biochemistry, 2000Co-Authors: Yoshihiko Yasohara, Michihiko Kataoka, Masaru Wada, Noriyuki Kizaki, Junzo Hasegawa, Sakayu ShimizuAbstract:An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-Chloro-3-oxobutanoate (COBE) to ethyl (S)-4-Chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.
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cloning overexpression and mutagenesis of the sporobolomyces salmonicolor aku4429 gene encoding a new aldehyde reductase which catalyzes the stereoselective reduction of ethyl 4 Chloro 3 oxobutanoate to ethyl s 4 Chloro 3 hydroxybutanoate
Applied and Environmental Microbiology, 1999Co-Authors: Keiko Kita, Takanobu Fukura, Kohichi Nakase, Kenji Okamoto, Hideshi Yanase, Michihiko Kataoka, Sakayu ShimizuAbstract:Aldehyde reductase (EC 1.1.1.2), aldose reductase (EC 1.1.1.21), and carbonyl reductase (EC 1.1.1.184) catalyze NADPH-dependent reduction of a variety of carbonyl compounds and are widely distributed in mammalian and plant tissues. These enzymes are members of the aldo-keto reductase superfamily (4, 8); however, their physiological functions are not well understood. The amino acid sequences of aldose reductases and aldehyde reductases exhibit significant levels of similarity, but the amino acid sequences of carbonyl reductases do not (32). In previous papers, we described purification and characterization of three NADPH-dependent aldehyde reductases (ARI, ARII, and ARIII) of the red yeast Sporobolomyces salmonicolor AKU4429 (9, 14, 34). ARI is the most abundant aldehyde reductase in this yeast and catalyzes asymmetric reduction of ethyl 4-Chloro-3-oxobutanoate (4-COBE) to ethyl (R)-4-Chloro-3-hydroxybutanoate (4-CHBE) {enantiomeric excess for (R) = [(R − S)/(R + S] × 100 and vice versa}, a promising chiral building block for organic synthesis. In contrast, ARII is produced in considerably smaller amounts but reduces 4-COBE to the (S) enantiomer (92.7% enantiomeric excess), which is also a useful chiral building block for chemical synthesis of pharmaceuticals. In addition to the stereoselectivity of activity against 4-COBE, the N-terminal amino acid sequences of these two aldehyde reductases are quite different. Based on the amino acid sequence deduced from the cDNA sequence, ARI belongs to the aldo-keto reductase superfamily (13). Recently, an NADPH-dependent aldehyde reductase (S1), which reduces 4-COBE to the (S) enantiomer (100% enantiomeric excess), was purified from Candida magnoliae AKU4643 (31). The substrate specificities, subunit structures, and N-terminal amino acid sequences of ARII and S1 are not similar. This indicates that the two enzymes belong to the different groups. In this study, we cloned and analyzed a cDNA clone of the aldehyde reductase gene (ARII) in order to compare the catalytic mechanisms of ARI and ARII and to understand the molecular basis of the stereospecific reduction of 4-COBE.
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stereoselective reduction of ethyl 4 Chloro 3 oxobutanoate by escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes
Applied Microbiology and Biotechnology, 1999Co-Authors: Michihiko Kataoka, Keiko Kita, Hideshi Yanase, Masaru Wada, Hiroshi Kawabata, Kazuhiko Yamamoto, Sakayu ShimizuAbstract:The asymmetric reduction of ethyl 4-Chloro-3-oxobutanoate (COBE) to ethyl (R)-4-Chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
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purification and characterization of nadph dependent carbonyl reductase involved in stereoselective reduction of ethyl 4 Chloro 3 oxobutanoate from candida magnoliae
Bioscience Biotechnology and Biochemistry, 1998Co-Authors: Masaru Wada, Michihiko Kataoka, Hiroshi Kawabata, Yoshihiko Yasohara, Noriyuki Kizaki, Junzo Hasegawa, Sakayu ShimizuAbstract:A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-Chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced alpha-, beta-keto esters and conjugated diketones in addition to ethyl 4-Chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
Hanjie Ying - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a novel nadh dependent carbonyl reductase from pichia stipitis involved in biosynthesis of optically pure ethyl s 4 Chloro 3 hydroxybutanoate
Bioresource Technology, 2011Co-Authors: Hou Cao, Ming Yan, Yan Wang, Jian Xiong, Pingkai Ouyang, Yueyuan Zhang, Guanglou Zang, Hanjie YingAbstract:A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-Chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the Km of PsCRII for ethyl 4-Chloro-3-oxobutanoate (COBE) was 3.3 mM, and the corresponding Vmax was 224 μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study.
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biosynthesis of s 4 Chloro 3 hydroxybutanoate ethyl using escherichia coli co expressing a novel nadh dependent carbonyl reductase and a glucose dehydrogenase
Bioresource Technology, 2010Co-Authors: Hou Cao, Ming Yan, Yong Chen, Yan Wang, Jian Xiong, Pingkai Ouyang, Hanjie YingAbstract:Abstract A novel NADH-dependent carbonyl reductase (PsCR II) gene with an open reading frame of 855 bp encoding 285 amino acids was cloned from Pichia stipitis . Analysis of the amino acid sequence of PsCR II revealed less than 55% identity to known reductases that produce ( S )-4-Chloro-3-hydroxybutanoates ethyl [( S )-CHBE]. When NADH was provided as an electron donor, Escherichia coli with pET-22b-PsCRII exhibited an activity of 15 U/mg protein using 4-Chloro-3-oxobutanoate ethyl (COBE) as a substrate. This activity was the highest ever reported for reductases, with the exception of PsCR I, which in our previous analysis required NADPH for catalysis. Biocatalysis of COBE to ( S )-CHBE was investigated using E. coli with a polycistronic plasmid pET-BP II co-expressing PsCR II and a glucose dehydrogenase in a water/butyl acetate system for 24 h. The transformants gave a molar yield of 91%, and an optical purity of the ( S )-isomer of higher than 99% enantiomeric excess.
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construction and co expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl s 4 Chloro 3 hydroxybutanoate
Bioresource Technology, 2010Co-Authors: Hou Cao, Ming Yan, Yong Chen, Jian Xiong, Pingkai Ouyang, Fei Cao, Yueyuan Zhang, Hanjie YingAbstract:Abstract Biocatalysis of ethyl 4-Chloro-3-oxobutanoate (COBE) to ethyl ( S )-4-Chloro-3-hydroxybutanoate [( S )-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium . An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine–Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to ( S )-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles ( S )-CHBE formed per mole NADP + , was 12,600, which was more than 10-fold higher than in aqueous systems.
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a new member of the short chain dehydrogenases reductases superfamily purification characterization and substrate specificity of a recombinant carbonyl reductase from pichia stipitis
Bioresource Technology, 2009Co-Authors: Ming Yan, Hou Cao, Yong Chen, Hanjie Ying, Jian Xiong, Zhong Yao, Jianxin Bai, Pingkai OuyangAbstract:Abstract A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl ( S )-4-Chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K m value of PsCR for ethyl 4-Chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.
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a novel carbonyl reductase from pichia stipitis for the production of ethyl s 4 Chloro 3 hydroxybutanoate
Biotechnology Letters, 2009Co-Authors: Ming Yan, Hou Cao, Yong Chen, Hanjie YingAbstract:An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-Chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-Chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.
Yuguo Zheng - One of the best experts on this subject based on the ideXlab platform.
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efficient biosynthesis of ethyl r 4 Chloro 3 hydroxybutyrate using a stereoselective carbonyl reductase from burkholderia gladioli
BMC Biotechnology, 2016Co-Authors: Xiang Chen, Zhiqiang Liu, Chaoping Lin, Yuguo ZhengAbstract:Background Ethyl (R)-4-Chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-Chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently.
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upscale production of ethyl s 4 Chloro 3 hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous organic solvent system
Applied Microbiology and Biotechnology, 2015Co-Authors: Zhiqiang Liu, Yuguo Zheng, Zhenyang Shen, Huabin Hong, Jianbo Yan, Yi Lin, Zhengxu Chen, Yinchu ShenAbstract:(S)-4-Chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the (S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65 U/L and 13.90 g DCW/L which was 3.24 and 2.60-folds compared with those in the 50 L fermentor. The biocatalytic process for the synthesis of (S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-Chloro-3-oxobutanoate concentration reached to 1.7 M, and the (S)-CHBE with yield of 97.2 % and enantiomeric excess (e.e.) of 99 % was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17 mM/h and 27.55 mM/h/g DCW for (S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, (S)-CHBE was extracted from the reaction mixture with 82 % of yield and 95 % of purity. This study paved the foundation for the upscale production of (S)-CHBE by biocatalysis method.
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characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl s 4 Chloro 3 hydroxybutanoate with high space time yield
Applied Microbiology and Biotechnology, 2014Co-Authors: Zhongyu You, Zhiqiang Liu, Yuguo ZhengAbstract:A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-Chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-Chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg(-1). The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K m and V max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min(-1) mg(-1), respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous-organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3% and e.e. of 99% was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP(+) to (S)-CHBE were 26.5 mmol L(-1) h(-1) g(-1) DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.
Hou Cao - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a novel nadh dependent carbonyl reductase from pichia stipitis involved in biosynthesis of optically pure ethyl s 4 Chloro 3 hydroxybutanoate
Bioresource Technology, 2011Co-Authors: Hou Cao, Ming Yan, Yan Wang, Jian Xiong, Pingkai Ouyang, Yueyuan Zhang, Guanglou Zang, Hanjie YingAbstract:A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-Chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the Km of PsCRII for ethyl 4-Chloro-3-oxobutanoate (COBE) was 3.3 mM, and the corresponding Vmax was 224 μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study.
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biosynthesis of s 4 Chloro 3 hydroxybutanoate ethyl using escherichia coli co expressing a novel nadh dependent carbonyl reductase and a glucose dehydrogenase
Bioresource Technology, 2010Co-Authors: Hou Cao, Ming Yan, Yong Chen, Yan Wang, Jian Xiong, Pingkai Ouyang, Hanjie YingAbstract:Abstract A novel NADH-dependent carbonyl reductase (PsCR II) gene with an open reading frame of 855 bp encoding 285 amino acids was cloned from Pichia stipitis . Analysis of the amino acid sequence of PsCR II revealed less than 55% identity to known reductases that produce ( S )-4-Chloro-3-hydroxybutanoates ethyl [( S )-CHBE]. When NADH was provided as an electron donor, Escherichia coli with pET-22b-PsCRII exhibited an activity of 15 U/mg protein using 4-Chloro-3-oxobutanoate ethyl (COBE) as a substrate. This activity was the highest ever reported for reductases, with the exception of PsCR I, which in our previous analysis required NADPH for catalysis. Biocatalysis of COBE to ( S )-CHBE was investigated using E. coli with a polycistronic plasmid pET-BP II co-expressing PsCR II and a glucose dehydrogenase in a water/butyl acetate system for 24 h. The transformants gave a molar yield of 91%, and an optical purity of the ( S )-isomer of higher than 99% enantiomeric excess.
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construction and co expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl s 4 Chloro 3 hydroxybutanoate
Bioresource Technology, 2010Co-Authors: Hou Cao, Ming Yan, Yong Chen, Jian Xiong, Pingkai Ouyang, Fei Cao, Yueyuan Zhang, Hanjie YingAbstract:Abstract Biocatalysis of ethyl 4-Chloro-3-oxobutanoate (COBE) to ethyl ( S )-4-Chloro-3-hydroxybutanoate [( S )-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium . An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine–Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to ( S )-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles ( S )-CHBE formed per mole NADP + , was 12,600, which was more than 10-fold higher than in aqueous systems.
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a new member of the short chain dehydrogenases reductases superfamily purification characterization and substrate specificity of a recombinant carbonyl reductase from pichia stipitis
Bioresource Technology, 2009Co-Authors: Ming Yan, Hou Cao, Yong Chen, Hanjie Ying, Jian Xiong, Zhong Yao, Jianxin Bai, Pingkai OuyangAbstract:Abstract A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl ( S )-4-Chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K m value of PsCR for ethyl 4-Chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.
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a novel carbonyl reductase from pichia stipitis for the production of ethyl s 4 Chloro 3 hydroxybutanoate
Biotechnology Letters, 2009Co-Authors: Ming Yan, Hou Cao, Yong Chen, Hanjie YingAbstract:An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-Chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-Chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.
S A Shevelev - One of the best experts on this subject based on the ideXlab platform.
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efficient procedure for high yield synthesis of 4 substituted 3 5 dinitropyrazoles using 4 Chloro 3 5 dinitropyrazole
ChemInform, 2012Co-Authors: I L Dalinger, Irina A Vatsadze, T K Shkineva, G P Popova, S A ShevelevAbstract:The reactions of readily prepared 4-Chloro-3,5-dinitropyrazole (II) and its N-methyl analogue with O,S, and N-nucleophiles are studied.
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efficient procedure for high yield synthesis of 4 substituted 3 5 dinitropyrazoles using 4 Chloro 3 5 dinitropyrazole
Synthesis, 2012Co-Authors: I L Dalinger, Irina A Vatsadze, T K Shkineva, G P Popova, S A ShevelevAbstract:The transformations of readily available 4-Chloro-3,5-dinitropyrazole and its N-methylated derivative under the action of anionic S-/O-nucleophiles and neutral N-nucleophiles were studied. Independent of the substrate’s charge (anionic, R = H, or neutral, R = Me), the nucleophilic substitution proceeds exclusively at position 4 replacing the chlorine nucleofuge. As a result of this study, the effective synthetic method for the preparation of 4-substituted 3,5-dinitropyrazoles via the nucleophilic substitution in 4-Chloro-3,5-dinitropyrazole was elaborated.
