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Tao W.,Dong - A University | Lee M.H.,Dong - A University | Wu J.,Wuhan Polytechnic University | Kim N.H.,Dong - A University | And 4 more authors.
Applied and Environmental Microbiology | Year: 2012

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic. © 2012, American Society for Microbiology. All Rights Reserved.

Tao W.,Dong - A University | Lee M.H.,Dong - A University | Yoon M.-Y.,Chemical Biotechnology Research Center | Kim J.-C.,Chemical Biotechnology Research Center | And 4 more authors.
Journal of Microbiology and Biotechnology | Year: 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 (≤C5) 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 C1, 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.

Hong J.-S.,Chemical Biotechnology Research Center | Kim Y.-W.,Chemical Biotechnology Research Center | Chung K.-W.,Chemical Biotechnology Research Center | Jeong S.-H.,Daegu University
Applied Chemistry for Engineering | Year: 2010

n-Paraffin and saturated fatty acid methyl esters in the diesel and bio-diesel fuel crystallize at low temperature. Many articles have addressed various solutions for the low temperature crystallization problem and one of them is the use of methacrylate copolymers. In this work, we synthesized a series of copolymers in the reaction condition of 70: 30 molar ratio of lauryl methacrylate (LMA) (or stearyl methacrylate (SMA)) and alkyl methacrylates. The structures of the copolymers were characterized by 1H-NMR and FT-IR spectroscopy, and the molecular weight of copolymers were obtained from Gel Permeation Chromatography (GPC) method. The concentrations of additives were 500~1000 ppm and 1000~10000 ppm in diesel fuels and bio-diesel fuel (BD5 and BD20), respectively. The addition of copolymers changes the many properties of fuel such as the pour point (PP), cloud point (CP) and cold filtering plugging point (CFPP). For example, the low temperature properties of the copolymers containing SMA (PSMAmR 2n) were excellently improved about 15, 7, and 10 °C for PP, CP and CFPP, respectively.

Yoon B.-T.,Chemical Biotechnology Research Center | Kim Y.-W.,Chemical Biotechnology Research Center | Chung K.-W.,Chemical Biotechnology Research Center | Kim J.-S.,Chemical Biotechnology Research Center
Applied Chemistry for Engineering | Year: 2011

Algae is an abundant and potential fermentation substrate. The enzymatic hydrolysis of algae was investigated by pre-treating an alkaline hydrogen peroxide with commercial cellulase and viscozyme. Algae used in this study was the Ulva pertusa. The evaluated response was the yield of released glucose after the enzymatic hydrolysis. Alkaline hydrogen peroxide containing mixtures of 1 wt% hydrogen peroxide and 1 ̃1.75 wt% sodium hydroxide was also used. The results show that the highest glucose conversion was obtained for Ulva pertusa using 5 wt% hydrogen peroxide at 60 °C for 3 h. The required amount of enzymes after the pre-treatment with alkaline hydrogen peroxide were reduced by far compared to that of untreated Ulva pertusa. Also, the amount of glucose that is released during the enzymatic hydrolysis was increased.

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