• Toxicological Research
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• 제18권3호
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• pp.249-257
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• 2002

Cytochrome P450 3As such as 3A4 and 3A5 metabolize a wide range of pharmaceutical compounds. The vectors for the expression of fusion protein containing an N-terminal human P450 3A4 or P450 3A5 sequences and a C-terminal rat NADPH-cytochrome P450 reductase moiety were constructed. These plasmids were used to express the fusion protein in Escherichia coli DH5$\alpha$ cells. High levels of expression were achieved (100~200 nmol/liter) and the expressed fusion protein in E. coli membranes were catalytically active for nifedipine oxidation, a typical enzymatic activity of P450 3A4. The NADPH-P450 reductase activities of these fusion protein were also determined by measuring reduction of cytochrome c. To fine a specific Inhibitor of P450 3A4 from naturally occurring chemicals, a series of isothiocyanate compounds were evaluated for the inhibitory activity of P450 using the fusion proteins in E. coli membranes. Of the five isothiocyanates (phenethyl isothiocyanate, phenyl isothiocyanate, benzol isothiocyanate, benzoyl isothiocyanate and cyclohexyl isothiocyanate) tested, benzoyl isothiocyanate showed a strong inhibition of P450 3A4 with an $IC_{50}$value of 2.8 $\mu\textrm{M}$. Our results indicate that the self-sufficient fusion protein will be very useful tool to study the drug metabolism and benzyl isothiocyanate may be valuable for characterizing the enzymatic properties of P450 3A4.

• Mycobiology
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• 제35권4호
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• pp.219-225
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• 2007

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and $28^{\circ}C$. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH ($7{\sim}10$) and temperature ($30^{\circ}C{\sim}70^{\circ}C$) profiles with the optimal for keratinase activity at pH 8 and $45^{\circ}C$. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

• Journal of Applied Biological Chemistry
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• 제43권3호
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• pp.125-130
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• 2000

An antiviral protein (CAP30) with ribosome-inactivating activity was purified from the leaves of Chenopodium album L. through ammonium sulfate precipitation and column chromatography using S-Sepharose, Blue-Sepharose, FPLC Suprose12 HR, and FPLC Mono-S. The molecular wight of CAP30 was estimated to be 30kD. CAP30 was thermostable, maintaing its activity even after incubation at $70^{\circ}C$ for 30 min, and was stable in the pH range of 6 to 9. In a cell-free in vitro translation system using rabbit reticulocyte lysate, protein synthesis was inhibited by the addition of CAP30 with an $IC_{50}$ of 2.26pM. The comparison of N-terminal amino acid sequences of this protein with known ribosome-inactivating proteins (RIPs) revealed that it had some sequence homology with PAP-S and PAP-R from pokeweed (Phytolacca americana)and dodecandrin from P. dodecandra, but had no sequence homology with RIPs from other plants belonging to different orders. The mosaic symptoms on tobacco leaves caused by cucumber mosaic virus infection was completely inhibited by 100 ng/ml of the pure CAP30 protein.

• Journal of Microbiology and Biotechnology
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• 제24권11호
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• pp.1525-1535
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• 2014

The xynC gene, which encodes high molecular weight xylanase from Paenibacillus sp. DG-22, was cloned and expressed in Escherichia coli, and its nucleotide sequence was determined. The xynC gene comprised a 4,419bp open reading frame encoding 1,472 amino acid residues, including a 27 amino acid signal sequence. Sequence analysis indicated that XynC is a multidomain enzyme composed of two family 4_9 carbohydrate-binding modules (CBMs), a catalytic domain of family 10 glycosyl hydrolases, a family 9 CBM, and three S-layer homologous domains. Recombinant XynC was purified to homogeneity by heat treatment, followed by Avicel affinity chromatography. SDS-PAGE and zymogram analysis of the purified enzyme identified three active truncated xylanase species. Protein sequencing of these truncated proteins showed that all had identical N-terminal sequences. In the protein characterization, recombinant XynC exhibited optimal activity at pH 6.5 and $65^{\circ}C$ and remained stable at neutral to alkaline pH (pH 6.0-10.0). The xylanase activity of recombinant XynC was strongly inhibited by 1 mM $Cu^{2+}$ and $Hg^{2+}$, whereas it was noticeably enhanced by 10 mM dithiothreitol. The enzyme exhibited strong activity towards xylans, including beechwood xylan and arabinoxylan, whereas it showed no cellulase activity. The hydrolyzed product patterns of birchwood xylan and xylooligosaccharides by thin-layer chromatography confirmed XynC as an endoxylanase.

• 식물병연구
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• 제29권4호
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• pp.445-451
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• 2023

Zamioculcas zamiifolia is a popular indoor ornamental plant in Korea. In August 2021, a severe outbreak of soft rot disease affected Z. zamiifolia in Emseong, Chungcheongbuk-do, Korea. Infected plants displayed wilting, water-soaked lesions, stem collapse, and green-brown discoloration. The bacterial strain KNUB-05-21 was isolated from infected stems and identified as Pectobacterium aroidearum using 16S rRNA nucleotide sequencing and multilocus sequence analysis based on partial sequences of dnaX, leuS, and recA genes. Confirmation of its affiliation with P. aroidearum was also obtained through biochemical and morphological characterization. To confirm the pathogenicity of strain KNUB-05-21, its suspension was injected into Z. zamiifolia stems. Within a week, soft rot developed on the stems, exhibiting symptoms similar to those observed in field-infected plants. The reisolated strain was identical to those of P. aroidearum. Before this study, P. aroidearum was not reported as a causative pathogen of Z. zamiifolia soft rot in Korea.

• 한국미생물·생명공학회지
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• 제34권3호
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• pp.278-287
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• 2006

토양으로부터 분리한 P. polymyxa 균주가 생산하는 CFTase의 각 repeat region의 결손 변이체를 제작하였으며 그에 따른 야생형 효소와 변이 효소의 활성변화 및 효소특성을 비교 검토하였다. 야생형 CFTase를 CFT148로, N-말단의 R1과 R3를 제거한 결손변이체 CFTase를 CFT108로, C-말단의 R4를 제거한 결손변이체는 CFT130, 모든 R 영역을 제거한 것은 CFT88로 명명하였다. 각각 정제된 재조합 단백질은 148 kDa, 108 kDa, 130 kDa, 그리고 88 kDa의 크기를 나타내었다. Inulin을 기질로 각 재조합 단백질의 활성을 검토한 결과 CFT108은 CF생성, CF분해 반응을 모두 가지고 있었으며 CFT130, CFT88의 경우에는 CF생성 활성을 나타내지 않았으며 endo-inulinae와 동일한 inulin 분해활성을 나타내었다. 따라서 N-말단의 repeat region인 R1과, R3의 역할은 균체 외로의 분비를 담당하며 C-말단의 R4영역은 CFTase의 중요 활성인 cyclization반응을 담당하고 있는 것으로 확인되었다. Repeat region의 모두 제거한 결손 변이체는 기질 inulin을 F2-F4 사이의 중합도를 가진fructooligosaccharide를 생산하는 endo-inulinase의 활성을 가지고 있었다. CFTase는 다기능 효소로 여러 domain으로 구성되어 있으며 각 domain의 결손에도 단백질의 활성을 유지하였으며, 특히 결손 변이체 CFT108은 고효율의 CF 생산이 가능하여 산업적으로 유용하게 사용될 수 있는 효소이다.

• 미생물학회지
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• 제55권2호
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• pp.103-111
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• 2019

DNA 메틸화는 유전체의 무결성의 유지 및 유전자 발현 조절과 같은 박테리아의 다양한 과정에 관여한다. Alphaproteobacteria 종에서 보존된 DNA 메틸 전이 효소인 CcrM은 S-아데노실 메티오닌을 공동 기질로 사용하여 $N^6$-아데닌 또는 $N^4$-시토신의 메틸 전이 효소 활성을 갖는다. Celeribacter marinus IMCC 12053는 해양 환경에서 분리된 알파프로테오박테리아로서 GpC 시토신의 외향고리 아민의 메틸기를 대체하여 $N^4$-메틸 시토신을 생산한다. 단일 분자 실시간 서열 분석법(SMRT)을 사용하여, C. marinus IMCC12053의 메틸화 패턴을 Gibbs Motif Sampler 프로그램을 사용하여 확인하였다. 5'-GANTC-3'의 $N^6$-메틸 아데노신과 5'-GpC-3'의 $N^4$-메틸 시토신을 확인하였다. 발현된 DNA 메틸전이 효소는 계통 발생 분석법을 사용하여 선택하여 pQE30 벡터에 클로닝 후 $dam^-/dcm^-$ 대장균을 사용하여 클로닝된 DNA 메틸라아제의 메틸화 활성을 확인하였다. 메틸화 효소를 코딩하는 게놈 DNA 및 플라스미드를 추출하고 메틸화에 민감한 제한 효소로 절단하여 메틸화 활성을 확인하였다. 염색체와 메틸라아제를 코드하는 플라스미드를 메틸화시켰을 때에 제한 효소 사이트가 보호되는 것으로 관찰되었다. 본 연구에서는 분자 생물학 및 후성유전학을 위한 새로운 유형의 GpC 메틸화 효소의 잠재적 활용을 위한 외향고리 DNA 메틸라제의 특성을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.531-535
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• 1998

The gene encoding alginate lyase was isolated from a library constructed with the vector, pUC19, and expressed in Escherichia coli. The nucleotide sequence of the cloned alginate lyase gene (ALY) from Pseudomonas sp. W7 was determined. The nucleotide sequence revealed a 1,035 bp open reading frame (ORF), encoding 345 amino acid residues with a calculated molecular mass of 37,478 Da. The N-terminal amino acid sequences (15 residues) of purified alginate lyase corresponded to that of the deduced amino acid sequence.

• Journal of Microbiology and Biotechnology
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• 제11권3호
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• pp.534-538
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• 2001

The xynA gene encoding an alikali-tolerant endo-1,4-${\beta}$-xylanase (XYN) was cloned from the alkalophilic Bacillus pumilus A-30. The nucleotide sequence of a 974-bp DNA fragment containing the xynA was determined. An ORF of 684 nucleotides that encoded a protein of 228 amino aicds was detected. Asparagine-71 of XYN from B. Pumilus A-30 showed to be highly conservative in alkaline xylanases of family G/11, upon comparing the amino acid sequences of 17 family G/11 xylanases. Site-directed mutation of N71D of the xynA gene resulted in a decrease of 12.4% in the specific acitivity and a significant decline in the enzyme activity in the alkaline pH range.

• Bulletin of the Korean Chemical Society
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• 제1권3호
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• pp.101-104
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• 1980

A sequential copolypeptide with repeating unit sequences, in which a glycyl residue is flanked progressively by two ${\gamma}$-benzyl-L-glutamyl residues, has been synthesized by poymerizing p-nitrophenyl ester of ${\gamma}$-benzyl-L-glutamyl-${\gamma}$ -benzyl-L-gutamyl-glycine. Polymers obtained were characterized by viscosity and infra-red spectral data. The highest molecular weight obtained was 21,000. Molecular conformation in solid state was found to be a mixed form of and |${\beta}$-structure. Polymers obtained were insoluble in the most of the organic solvents except in a strong acid like dichloroacetic acid, but in binary mixtures of solvents such as dichloroacetic acid-ethylene dichloride or dichloroacetic acid-chloroform, they were soluble within certain ranges of solvent compositions.