• Title, Summary, Keyword: Chloramphenicol acetyltransferase

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Selective overproduction of chloramphenicol acetyltransferase in the T7 expression system (T7 발현체계에서 chloramphenicol acetyltransferase의 선택적 과잉생산)

  • 김한복;강창원
    • Korean Journal of Microbiology
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    • v.27 no.4
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    • pp.317-322
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    • 1989
  • A gene can be selectively overexpressed in E. coli by utilizing the phage T7 RNA polymerase's stringent recognition and active transcription of the T7 promoter. The T7 expression system was constructed such that the T7 RNA polymerase gene is under the control of lacUV5 promoter in one plasmid, and that the target gene, the promoterless chloramphenicol acetyltransferase (CAT) gene with E. coli ribosome binding site is under the control of T7 promoter in the other plasmid. Only the E. coli cells containing both plasmids show high resistance to chloramphenicol. When the copy number of the runaway plasmid containing the polymerase gene was varied by a temperature shift, amounts of the CAT protein synthesized upon induction was correspondingly changed as shown in SDS gel electrophoresis.

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Effect of Hexane Extract of Acori graminei Rhizoma on Chloramphenicol Acetyltransferase of Staphylococcus aureus SA2 (석창포 헥산 추출물이 Staphylococcus aureus SA2의 Chloramphenicol Acetyltransferase 에 미치는 영향)

  • 문경호;권주열;박민수;김혜경;이정규
    • YAKHAK HOEJI
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    • v.48 no.1
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    • pp.30-33
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    • 2004
  • One subfraction from the hexane fraction of Acri graminei Rhizoma, the E4 fraction which is mainly consisted of acorenone, showed a potential inhibitory activity against chloramphenicol acetyltransferase (CAT) of S. aureus SA2 that is a multidrug-resistant strain to 10 usual antibiotics. The combination therapy of this fraction with chloramphenicol resulted in reduction of the minimal inhibitory concentration from 128 $\mu\textrm{g}$/ml to 8 $\mu\textrm{g}$/ml. The E4 fraction also revealed to prevent the induction of CAT from this strain.

Characterization of Two Metagenome-Derived Esterases That Reactivate Chloramphenicol by Counteracting Chloramphenicol Acetyltransferase

  • Tao, Weixin;Lee, Myung-Hwan;Yoon, Mi-Young;Kim, Jin-Cheol;Malhotra, Shweta;Wu, Jing;Hwang, Eul-Chul;Lee, Seon-Woo
    • 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.

Purification and Characterization of Chloramphenicol Acetyltransferase from Morganella morganii

  • El-Gamal, Basiouny;Temsah, Samiha;Olama, Zakia;Mohamed, Amany;El-Sayed, Mohamed
    • BMB Reports
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    • v.34 no.5
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    • pp.415-420
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    • 2001
  • Chloramphenicol acetyltransferase (CAT) was purified to homogeneity from Morganella morganii starting with ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50, and G-100 Sephadex gel filtration. The enzyme was purified 133.3 fold and showed a final specific activity of 60 units/mg protein with a yield of 37%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it as a heterotetramer that consists of four subunits with close molecular weights (19.5, 19, 18, and 17.5 kDa). The molecular weight of the native enzyme was calculated to be 78 kDa, as determined by gel filtration, which approximated to that of the four subunits (74 kDa). The enzyme showed a maximum activity at pH 7.8 when incubated at $35^{\circ}C$. A Lineweaver-Burk analysis gave a Km of 5.0 uM and Vmax of 153.8 U/ml. The amino acid composition of the purified enzyme was also determined.

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Nucleotide Sequence and Inducibility Analysis of Chloramphenicol Acetyltransferase Gene from Staphylococcus aureus R-plasmid pSBK203 (Staphylococcus aureus에서 분리된 R-plasmid pSBK203상의 chloramphenicol acetyltransferase 인자의 염기서열 및 유발성 분석)

  • 권동현;변우현
    • Korean Journal of Microbiology
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    • v.27 no.3
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    • pp.194-200
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    • 1989
  • The nucleotide sequence of inducible chloramphenicol acetyl-transferase(CAT) gene isolated from a small plasmid pSBK203 of Staphylococcus aureus was determined. The base sequence shows that structural gene of pSBK203-CAT encodes a protein of 213 amino acids and has a leader region which encodes a short polypeptide of 9 amino-acids in its upstream. vertical bar /sup 35/S vertical bar-Methionine labelled CAT gene product in minicell showed almost same mobility with pC194-CAT of which molecular weight is 24Kdal on polyacrylamide gel electrophoresis. Predicted amino acid sequence of pSBK203-CAT has revealed a high degree of homology with the CATs of pC194 and pC221 than those of cat-86, Tn9 and proteus mirabilis PM13.

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Solubilities and Activities of Chloramphenicol Acetyltransferase and $\beta$-Lactamase Overproduced by the T7 Expression System in Escherichia coli (대장균에서의 T7 발현체계에 의하여 과잉생산된 클로람페니콜 아세틸전이효소와 베타-락타메이즈의 수용성과 활성)

  • Kim, Han-Bok
    • Korean Journal of Microbiology
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    • v.31 no.4
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    • pp.274-278
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    • 1993
  • Overproduced proteins in many cases result in forming insoluble inclusion bodies, and their formation might be due to high concentration of protein. To investigate how proteins become insoluble, chloramphenicol acetyltransferase (CAT) and .betha.-lactamase were overproduced, and their solubilities and activities were determined. CAT was accumulated from 9 to 45% of total cellular protein in a fully soluble form without inclusion body formation. CAT specific activity was shown to be proportional to the amount of the protein produced. Moderately produced .betha.-lactamase by the phase T7 expression system at 30.deg.C comprised only mature forms in a soluble form. However, overproduced .betha.-lactamase at 37.deg.C became insoluble. Most precursor forms of .betha.-lactamase in the cytoplasm were insoluble, whereas majority of the mature forms in the periplasm space were soluble. Also, chaperone GroE proteins which assist proper protein folding and translocation did not increase .betha.-lactamase solubility significantly under the experimental condition. It seems that the formation of inclusion bodies in the cell is related to the nature of protein itself rather than just to high concentration of protein.

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Effect of Hexane Extract of Acori graminei Rhizoma on the Growth of Chloramphenicol Resistant Bacteria

  • Moon, Kyung-Ho;Kwon, Joo-Yeoul;Kim, Hye-Kyung;Seo, Bong-Soo;Lee, Chung-Kyu
    • Natural Product Sciences
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    • v.9 no.3
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    • pp.183-185
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    • 2003
  • The combination of hexane extract (E4) of rhizome of Acorus gramineus with chloramphenicol (Cm) was applied to Gram negative Cm resistant microbials to find the possibility of clinical use and to clarify the relationship of the activity of chloramphenicol acetyltransferase (CAT). The combination of $1,000\;{\mu}g/ml$ of E4 and $8\;{\mu}g/ml$ of Cm entirely ceased the growth of S. aureus SA2, a gram positive resistant strain to 10 antibiotics. But in Gram negative strains which possess CAT activity, some showed considerably strong resistances to Cm and some did weakly.

Synthetic Regulatory Elements of the Nopaline Synthase Promoter in Higher Plants (고등 식물에서 Nopaline Synthase Promoter의 합성 조절 요소)

  • Kim, Young-Hee
    • Korean Journal of Plant Tissue Culture
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    • v.22 no.4
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    • pp.201-205
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    • 1995
  • The synthetic oligomers called nos right palindrome (RP) element and left palindrome (LP) element were inserted into nos.minimal promoter nos 5'-101 deletion mutant The activity of nos promoter was measured by studying the expression pattern of gene fusion between nos promoter and reporter genes such as chloramphenicol acetyltransferase and $\beta$-glucuconidase. Analysis of transgenic tobacco plane carrying transgene showed that the activity of nos minimal promoter activity was recovered by insertion of synthetic nos RP element. Nos RP element insertion of nos minimal promoter was induced by auxin, dithiothreitol, salicylic acid and methyl jasmonate.

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Screening and Isolation of Antibiotics Resistance Inhibitors from Herb Materials. V.- Resistance Inhibition by Acorenone from Acorus gramineus Solander

  • Kim, Hye-Kyung;Moon, Kyung-Ho;Lee, Chung-Kyu
    • Natural Product Sciences
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    • v.6 no.1
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    • pp.36-39
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    • 2000
  • Acorenone, a diterpene isolated from Acorus gramineus, showed strong resistance inhibitory activity against multi-drug resistant microorganisms such as Staphylococcus aureus SA2, which has resistance to 10 usual antibiotics including chloramphenicol (Cm). At the level of $5\;{\mu}g/ml$ when combined with $50\;{\mu}g/ml$ of Cm. Bacterial resistance to Cm is due to the presence in resistant bacteria of an enzyme, chloramphenicol acetyltransferase (CAT), which catalyses the acetyl-CoA dependent acetylation of the antibiotic at C-3 hydroxyl group. To elucidate the mechanism of resistant inhibitory effect, the acorenone which had the strongest resistant inhibitory activity, was investigated on the CAT assay. As the result, the combination of Cm and acorenone showed the strongest inhibitory activity on CAT as noncompetitive and dose dependent manner.

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Screening of Promoter Sequences from Lactic Acid Bacteria Using a Promoter-Selection Vector (Promoter-Selection Vector를 사용한 유산균 Promoter의 탐색)

  • 우승희;김갑석
    • KSBB Journal
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    • v.11 no.4
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    • pp.504-509
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    • 1996
  • Promoters which are useful for constructing expression vectors for lactic acid bacteria were obtained from the chromosomal DNA of Lactococcus lactis ssp. lactis MG1363. pBV5030, a promoter-selection vector, replicates in L. lactis and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene (cat-86). After examining E. coli transformants which grew on LB media containing chloramphenicol (Cm, 20$\mu\textrm{g}$/mL) , many MG1363 derived DNA fragments which encompass promoter sequences were identified. Some recombinant E. coli cells can grow at the Cm concentration of 1,000$\mu\textrm{g}$/mL. When plasmids from those highly resistant E. coli cells were purified and introduced into L. lactis ssp. lactis MG1614 cells by electroporation, lactococcal transformants showing Cm resistance were obtained. So far, five plasmids with different promoter inserts were introduced into L. lactis MGl614 cells. The maximum level of Cm resistance in L. lactis MG1614 transformants was quite low (20$\mu\textrm{g}$/mL) when compared with that observed in recombinant E. coli cells harboring the same plasmids.

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