Ji-Youn Kim

Bio: Ji-Youn Kim is an academic researcher from Ajou University. The author has contributed to research in topics: Esterase & Escherichia coli. The author has an hindex of 4, co-authored 4 publications receiving 128 citations.

Screening, production and properties of a stereospecific esterase from Pseudomonas sp. S34 with high selectivity to (S)-ketoprofen ethyl ester

Abstract: To isolate novel strains expressing an esterase that hydrolyzed the rac-ketoprofen ethyl ester to (S)-ketoprofen in the stereospecific manner, we screened broad ecological niches and soil samples in which the activity was expected to be found. Thousands of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R, S)-ketoprofen ethyl ester. Twenty-eight strains were screened primarily and compared with respect to the potential to ketoprofen ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain S34 was isolated as a best producer and finally identified as a Pseudomonas sp. S34. We first formulated the optimal medium for the high level production of the enzyme, and as a preliminary experiment for enzymatic resolution, we characterized the fractionated enzyme. The enzyme with ketoprofen ethyl ester-hydrolyzing activity to (S)-ketoprofen showed a high degree of enantioselectivity (>94%) and was mainly found in cell extracts, whereas no distinct activity was detected in culture broth. The optimum pH and temperature of the enzyme were 9.5 and 35 °C, respectively. The activity of the enzyme was markedly increased (four-fold) by addition of a non-ionic detergent Triton X-100 and, resultantly, a high activity toward ketoprofen ethyl ester (52 U/mg) was found. The small-scale conversion of (R, S)-ketoprofen ethyl ester to (S)-ketoprofen using the partially purified enzymes was completed in 28 h, with optical purity of 99% and yield of 47%.

A new isolate Bacillus stearothermophilus JY144 expressing a novel esterase with high enantioselectivity to (R)-ketoprofen ethyl ester: strain selection and gene cloning

Abstract: In order to obtain novel strains that hydrolyzed the rac-ketoprofen ethyl ester to an enantiomer of ketoprofen, we enriched strains from broad ecological niches in which the thermostable enzymes were ubiquitously found The isolated strains were first analyzed to detect the ester-hydrolyzing activity by using a selective plate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester Twenty-six strains were screened primarily and further compared for the ethyl ester-hydrolyzing activity in terms of conversion yield and enantioselectivity Consequently, a strain JY144 was isolated as a novel strain that produced a (R)-stereospecific esterase with a high stability and systematically identified as a Bacillus stearothermophilus JY144 The enzyme was indeed stable at a broad range of pH (60–95) and temperature, up to 65 °C The optimal temperature and pH for enzymatic conversion were 50 °C and 90, respectively Since the wild-type strain showed a poor cell growth and enzyme activity, we further attempted the cloning of esterase gene into Escherichia coli, with a simple and rapid strategy for selecting the positive clones, and determined its primary structure The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 274 kDa The primary structure showed a relatively high homology (38–52%) to probable esterases unidentified to date, and thus presumed as a novel family enzyme In whole cell conversions, the recombinant enzyme had a superior potential than the parent strain, and it could completely convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with an optical purity of 99%

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)‐ketoprofen ethyl ester

Abstract: A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed. The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression. The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure. The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use. The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures. Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.

Production and applications of esterases

Abstract: Esterase plays a major role in the degradation of natural materials and industrial pollutants, viz, cereal wastes, plastics, and other toxic chemicals It is useful in the synthesis of optically pure compounds, perfumes, and antioxidants The potential applications of esterase with reference to agriculture, food, and pharmaceutical industries, are discussed in this review Promising applications in this avenue can be supported by appropriate production strategies

Lipase and esterase: to what extent can this classification be applied accurately?

Abstract: Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes - four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.

Lipase And Esterase - To What Extent Can This Classification Be Applied Accurately? [lipases E Esterases: Como Definir E Classificar?]

Abstract: Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes – four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.