• 약학회지
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• 제48권1호
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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.

• 미생물학회지
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• 제27권4호
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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.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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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.

• BMB Reports
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• 제34권5호
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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.

• 미생물학회지
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• 제27권3호
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• pp.194-200
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• 1989

S. aureus에서 분리된 plasmid pSBK203 상의 CAT 유전자 염기서열을 결정하였으며 유발성 발현현상이 확인되었다. 염기서열 결과에 의해 예측된 단백질의 아미노산 서열 분석결고 pC221-CAT 와는 78%의 가장 높은 상동성을 나타냈으며 pC194-CAT와는 55%, 그람음성균 유래의 CAT 중 하나인 Tn9-CATdhkss 38%의 상동성을 각각 보여주고 있었다.

• 미생물학회지
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• 제31권4호
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• pp.274-278
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• 1993

단백질이 어떻게 비수용성이 되는지를 알기위해, 클로람페니콜 아세틸전이효소와 베타-락타메이즈를 과잉생산하여 그들의 수용성과 활성을 측정하였다. 클로람페니콜 아세틸전이효소는 총단백질의 9에서 45%를 차지하였으며, inclusion body 형성없이 완전히 수용성이었으며, 효소활성은 만들어진 양과 비례하였다. 또한 30℃에서 T7 발현체계에 의해 생성된 베타-락타메이즈는 수용성의 숙성체였으나, 37℃에서는 비수용성이 되었다. 세포질에 있는 대부분의 베타-락타메이즈는 비수용성이었고. 페리플라즘 공간에서는 대부분이 수용성이었다. 단백질의 올바른 폴딩을 도와주는 chaperone의 일종인 GroEL 단백질은 본 실험조선에서는 베타-락타베이즈의 수용성을 별로 높이지는 못했다. 세포 내에서 inclusion body의 형성은 단백질의 높은 종도보다는 각각 단밸질 자체의 특성과 관련된 듯하다.

• Natural Product Sciences
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• 제9권3호
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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.

• 식물조직배양학회지
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• 제22권4호
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• pp.201-205
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• 1995

Nopaline synthase promoter의 upstream element region을 본딴 nos-RP 요소라고 불리워진 합성 oligomer는 nos wild type promotor의 5' end에서부터 - 101까지를 절단한 promoter의 upstream에 삽입하였다. Nos promoter의 활성은 nos promoter와 연결되어 있는 reporter gene인 Chlorarnphenicol과 $\beta$-glucuronidase유전 인자들이 발현되는 현상을 연구함으로써 측정하였다. 형질 전환된 유전인자를 가지고 있는 담배 식물에 대한 분석은 nos minimal promoter의 활성이 합성 nos-RP 요소가 삽입됨으로써 회복될 수 있음을 보여 주었다. 또한 Nos minimal promoter의 upstream에 nos-RP 요소의 삽입은 여러 가지 환경적인 요소들인 auxin, dithiothreitol, salicylic acid 그리고 methyl jasmonate에 의해서 활성이 증가됨을 보여 주었다.

• Natural Product Sciences
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• 제6권1호
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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.

• KSBB Journal
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• 제11권4호
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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.