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T Panda

Bio: T Panda is an academic researcher from Indian Institute of Technology Madras. The author has contributed to research in topic(s): Esterase. The author has an hindex of 1, co-authored 1 publication(s) receiving 5 citation(s).

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Topics: Esterase
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Journal ArticleDOI
Boyapati Gokul1, Je Hyuk Lee2, Ki Bang Song2, T Panda1  +2 moreInstitutions (2)
TL;DR: (R)-β-acetylmercaptoisobutyric acid is an important intermediate for the chemical synthesis of various antihypertensive and congestive heart failure drugs and can be converted asymmetrically from (R,S)-ester.

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Abstract: (R)-β-acetylmercaptoisobutyric acid (RAM), a chiral compound, is an important intermediate for the chemical synthesis of various antihypertensive and congestive heart failure drugs. Microorganisms capable of converting (R,S)-β-acetylmercaptoisobutyric acid ((R,S)-ester) to RAM were screened from soil microorganisms. A strain ofPseudomonas sp. 1001 screened from a soil sample was selected to be the best. Cells showed an activity of 540 U/mL from culture broth and the enzyme was thermostable up to 70°C. This strain could produce RAM asymmetrically from (R,S)-ester.

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5 citations


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Journal ArticleDOI
TL;DR: To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source.

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Abstract: L-Homophenylalanine (L-HPA) was asymmetrically synthesized from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two-step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K-1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314-fold higher than that of the wild-type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2-OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L-HPA at high concentrations of 2-OPBA. To avoid the substrate inhibition by 2-OPBA, intermittent addition of the solid-state substrate was attempted to obtain a high concentration of L-HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L-HPA at 840 mM of 2-OPBA resulted in >94% of conversion yield and >99% ee of L-HPA of optical purity. Due to the low solubility ( 99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction.

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62 citations


Journal ArticleDOI
TL;DR: Two enantioselective strains were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates and showed high esterase activity, but showed low lipase activity onp-nitrophenyl palmitate (pNPP).

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Abstract: About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (E app>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

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11 citations


Journal ArticleDOI
Chul Ho Kim1, J H Lee1, Joo Hyung Heo1, Oh Suk Kwon1  +2 moreInstitutions (1)
TL;DR: Aims: To screen and clone a novel enzyme with specific activity for the resolution of (R)‐β‐acetylmercaptoisobutyrate (RAM) from (R,S)‐α‐sub 2,3,4,5‐triene‐2,6,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27

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Abstract: Aims: To screen and clone a novel enzyme with specific activity for the resolution of (R)-β-acetylmercaptoisobutyrate (RAM) from (R,S)-β-acetylmercaptoisobutyrate [(R,S)-ester]. Methods and Results: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. Conclusion: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. Significance and Impact of the Study: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.

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9 citations


Journal ArticleDOI
Je Hyuk Lee1, Gokul Boyapati2, Ki Bang Song1, Sang Ki Rhee1  +1 moreInstitutions (2)
TL;DR: The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues.

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Abstract: The est A gene encoding the enzyme that catalyzes the production of ( R )-β-acetylmercaptoisobutyric acid from ( R,S )-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A+T and C+G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the est A gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70–100 amino acids upstream of the G-X-S-X-G consensus sequence.

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9 citations


Journal ArticleDOI
Sang-Yoon Lee1, Byung-Hyuk Min1, Seong-Won Song, Sun-Young Oh  +3 moreInstitutions (1)
TL;DR: Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxAcin butyl ester and could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantiOSElectivity.

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Abstract: Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxacin butyl ester. The initial activity of immobilized esterase was found to be significantly affected by the polyacrylamide gel composition. The optimum concentrations of monomer and crosslinker were determined to be 20% and 8.3%, respectively. The activity of immobilized esterase was 55.4% compared to a free enzyme. Enantiomeric excess was maintained at 60%, almost the same level as that of free enzyme. In addition, the immobilized esterase could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantioselectivity.

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4 citations


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Author's H-index: 1

No. of papers from the Author in previous years
YearPapers
20001

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