• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.315-325
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• 2016

A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40ºC and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an S-enantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.277-288
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• 2017

Rhizomucor miehei NRRL 5282 and Rhizopus oryzae NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified R. miehei and Rh. oryzae enzymes, respectively. p-Nitrophenyl palmitate (pNPP) hydrolysis was maximal at $40^{\circ}C$ and pH 7.0 for the R. miehei lipase, and at $30^{\circ}C$ and pH 5.2 for the Rh. oryzae enzyme. The enzymes showed almost equal affinity to pNPP, but the $V_{max}$ of the Rh. oryzae lipase was about 1.13 times higher than that determined for R. miehei using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM $Hg^{2+}$, $Zn^{2+}$, or $Mn^{2+}$, 10 mM N-bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of n-hexane, cyclohexane, n-heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the pNPP hydrolyzing activity of R. miehei lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed p-nitrophenyl (pNP) esters with C8-C16 acids, exhibiting maximum activity towards pNP-caprylate (R. miehei) and pNP-dodecanoate (Rh. oryzae). The purified lipases are promising candidates for various biotechnological applications.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.604-610
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• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1203-1210
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• 2011

Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (${\leq}C_5$) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3-acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at $C_1$, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

• The Plant Pathology Journal
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• v.24 no.2
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• pp.191-201
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• 2008

Bacillus sp. SB-007 was isolated from pea leaves harvested from the southwestern parts of South Korea through screening on a minimal medium containing 0.2% purified cutin for its ability to induce the cutinase production. However, no cutinase was produced when it was grown in a minimal medium containing 0.2% glucose. A proteomic approach was applied to separate and characterize these differentially secreted proteins. The expression level of 83 extracellular proteins of the cutinase-producing Bacillus sp. strain SB-007 incubated in a cutinase-induced medium increased significantly as compared with that cultured in a non cutinase-induced medium containing glucose. The extracellular proteome of Bacillus sp. SB-007 includes proteins from different functional classes, such as enzymes for the degradation of various macromolecules, proteins involved in energy metabolism, sporulation, transport/binding proteins and lipoproteins, stress inducible proteins, several cellular molecule biosynthetic pathways and catabolism, and some proteins with an as yet unknown function. In addition, the two protein spots showed little similarities with the known lipolytic enzymes in the database. These secreted proteome analysis results are expected to be useful in improving the Bacillus strains for the production of industrial cutinases.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.216-225
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• 2020

An esterase gene, estA1, was cloned from Alteromonas sp. 39-G1 isolated from the Beaufort Sea. The gene is composed of 1,140 nucleotides and codes for a 41,190 Da protein containing 379 amino acids. As a result of a BLAST search, the protein sequence of esterase EstA1 was found to be identical to Alteromonas sp. esterase (GenBank: PHS53692). As far as we know, no research on this enzyme has yet been conducted. Phylogenetic analysis showed that esterase EstA1 was a member of the bacterial lipolytic enzyme family IV (hormone sensitive lipases). Two deletion mutants (Δ20 and Δ54) of the esterase EstA1 were produced in Escherichia coli BL21 (DE3) cells with part of the N-terminal of the protein removed and His-tag attached to the C-terminal. These enzymes exhibited the highest activity toward p-nitrophenyl (pNP) acetate (C₂) and had little or no activity towards pNP-esters with acyl chains longer than C₆. Their optimum temperature and pH of the catalytic activity were 45℃ and pH 8.0, respectively. As the NaCl concentration increased, their enzyme activities continued to increase and the highest enzyme activities were measured in 5 M NaCl. These enzymes were found to be stable for up to 8 h in the concentration of 3-5 M NaCl. Moreover, they have been found to be stable for various metal ions, detergents and organic solvents. These salt-tolerant and chemical-resistant properties suggest that the enzyme esterase EstA1 is both academically and industrially useful.

• Journal of Applied Biological Chemistry
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• v.62 no.1
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• pp.73-79
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• 2019

A novel esterase gene (est7S) was cloned from an enriched metagenomic library derived from an oil-spill area. The gene encoded a protein of 505 amino acids, and the molecular mass of the Est7S was estimated to be 54,512 Da with no signal peptide. Est7S showed the highest identity of 40% to an esterase from a sludge metagenome compared to the characterized enzymes with their properties, although it showed 99% identity to a carboxylesterase in the genome sequence of Alcanivorax borkumensis SK2. Est7S had catalytic triad residues, Ser183, Glu312, and His420, and the GESAG motif in most family VII lipolytic enzymes. Est7S was purified from the crude extract of clone SM7 using Sephacryl S-200 HR and HiTrap Q column chromatographies. The purified Est7S was optimally active at $50^{\circ}C$ and pH 10.0. Est7S showed a high specific activity of 366.7 U/mg protein. It preferred short length esters, particularly p-nitrophenyl acetate, efficiently hydrolyzed R- and S-enantiomers of methyl-3-hydroxy-2-methylpropionate, and glyceryl tributyrate. These properties of Est7S may provide potential merits in biotechnological applications such as detergent and paper processing under alkaline conditions.

• Journal of Marine Life Science
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• v.8 no.1
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• pp.56-67
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• 2023

Marine macroalgae are important in coastal ecosystems and interact with marine microorganisms. In this study, we isolated fungi from seven types of marine macroalgae including Cladophora sp., Gloiopeltis furcate, Gracilariopsis chorda, Hydroclathrus clathratus, Prionitis crispata, Sargassum micracanthum, and Ulva lactuca collected in Korea. Morphological and phylogenetic analyses identified the isolates as four Aspergillus spp. (A. fumigatus, A. sydowii, A. tamarii, and A. terreus), three Penicillium spp. (P. crustosum, P. jejuense, and P. rubens), and Cladosporium tenuissimum. Among them, A. fumigatus TOP-U2, A. tamarii SH-Sw5, and A. terreus GJ-Gf2 strains showed the activities of all enzymes examined (amylase, chitinase, lipase, and protease). Based on the enzymatic index (EI) values in solid media, A. terreus GJ-Gf2 and C. tenuissimum UL-Pr1 exhibited the highest amylase and lipase activities, respectively. Chitinolytic activity was only observed in A. terreus GJ-Gf2, A. tamarii SH-Sw5, and A. fumigatus TOP-U2. Penicillium crustosum UL-Cl2 and C. tenuissimum UL-Pr1 showed the highest protease activities. To the best of our knowledge, this is the first report of lipolytic and proteolytic activities in a marine-derived C. tenuissimum strain. Overall, the fungal strains isolated from the marine macroalgae in this study actively produced industrially important enzymes.

• Journal of Animal Science and Technology
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• v.55 no.4
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• pp.303-314
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• 2013

Knowledge regarding lipid catabolism has been of great interest in the field of animal sciences. In the livestock industry, excess fat accretion in meat is costly to the producer and undesirable to the consumer. However, intramuscular fat (marbling) is desirable to enhance carcass and product quality. The manipulation of lipid content to meet the goals of animal production requires an understanding of the detailed mechanisms of lipid catabolism to help meticulously design nutritional, pharmacological, and physiological approaches to regulate fat accretion. The concept of a basic system of lipases and their co-regulators has been identified. The major lipases cleave triacylglycerol (TAG) stored in lipid droplets in a sequential manner. In adipose tissue, adipose triglyceride lipase (ATGL) performs the first and rate-limiting step of TAG breakdown through hydrolysis at the sn-1 position of TAG to release a non-esterified fatty acid (NEFA) and diacylglycerol (DAG). Subsequently, cleavage of DAG occurs via the rate-limiting enzyme hormone-sensitive lipase (HSL) for DAG catabolism, which is followed by monoglyceride lipase (MGL) for monoacylglycerol (MAG) hydrolysis. Recent identification of the co-activator (Comparative Gene Identification-58) and inhibitor [G(0)/G(1) Switch Gene 2] of ATGL have helped elucidate this important initial step of TAG breakdown, while also generating more questions. Additionally, the roles of these lipolysis-related enzymes in muscle, liver and skin tissue have also been found to be of great importance for the investigation of systemic lipolytic regulation.

• Molecular & Cellular Toxicology
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• v.1 no.3
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• pp.189-195
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• 2005

Industrial development has increase consumption of crude oil and environmental pollution. A large number of microbial lipolytic enzymes have been identified and characterized to date. To development for a new lipase with catalytic activity in degradation of crude oil as a microbial enzyme, Acinetobactor sp. B2 was isolated from soil samples that were contaminated with oil in Daejon area. Acinetobactor sp. B2 showed high resistance up to 10 mg/mL unit to heavy metals such as Ba, Li, Al, Cr, Pb and Mn. Optimal growth condition of Acinetobactor sp. B2 was confirmed $30^{\circ}C$. Lipase was purified from the supernatant by Acinetobactor sp. B2. Its molecular mass was determined to the 60 kDa and the optimal activity was shown at $40^{\circ}C$ and pH 10. The activation energies for the hydrolysis of p-nitrophenyl palmitate were determined to be 2.7 kcal/mol in the temperature range 4 to $37^{\circ}C$. The enzyme was unstable at temperatures higher than $60^{\circ}C$. The Michaelis constant $(K_{m})\;and\;V_{max}$ for p-nitrophenyl palmitate were $21.8{\mu}M\;and\;270.3{\mu}M\;min^{-1}mg\;of\;protein^{-1}$, respectively. The enzyme was strongly inhibited by $Cd{2+},\;Co^{2+},\;Fe^{2+},\;Hg^{2+},\;EDTA$, 2-Mercaptoethalol. From these results, we suggested that lipase purified from Acinetobactor sp. B2 should be able to be used as a new enzyme for degradation of crude oil, one of the environmental contaminants.