• 제목/요약/키워드: hybrid enzyme

검색결과 70건 처리시간 0.022초

Magnaporthe oryzae Effector AVR-Pii Helps to Establish Compatibility by Inhibition of the Rice NADP-Malic Enzyme Resulting in Disruption of Oxidative Burst and Host Innate Immunity

  • Singh, Raksha;Dangol, Sarmina;Chen, Yafei;Choi, Jihyun;Cho, Yoon-Seong;Lee, Jea-Eun;Choi, Mi-Ok;Jwa, Nam-Soo
    • Molecules and Cells
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    • 제39권5호
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    • pp.426-438
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    • 2016
  • Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

Saccharomycopsis속과 Saccharomyces속의 잡종형성 균주에서 생산하는 ${\alpha}$-amylase의 특성 (Characterization of ${\alpha}$-amylase Producing Hybrid Constructed between Saccharomycopsis and Saccharomyces)

  • 양영기;문명님;임채영;이영하;김정호
    • 미생물학회지
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    • 제35권4호
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    • pp.315-321
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    • 1999
  • 핵전이 방법을 이용하여 효모에서 전분분해능이 향상된 새로운 우수 균주를 개발하고자 하였다. Saccharomycopsis fiburigera KCTC 7393과 Saccharomyces cerevisiae KCTC 7049에서 핵을 분리한 후 영양요구 돌연변이주인 S. cerevisiae의 안으로 전달시켜 잡종(MN-16)을 형성하여 전분 분해능이 증가된 잡종을 선별하였다. 세포 배양액으로 조효소 용액을 만든 후 ammonium sulfate 침전, DEE-Sephacel column chromatography, Sephacryl S-200 column chromatography 등의 정제과정을 통해 9.7%의 회수율로 약 10.6배 정제 된 효소를 얻을 수 있었다. 정제된 효소는 SDS-PAGE 전기영동을 통해 단일 band를 보여 주었으며, SDS-PAGE 와 Sephacryl S-200 column chromatography를 통해서 53kDa으로 나타났다. 정제효소의 최적 활성 온도는 40${\circ}C$이고, 안정성은 40~45${\circ}C$로 나타났다. 최적 활성 pH는 5.5이었고, pH 5.0~7.0 정도에서 pH안정성이 80%정도 유지되었다. 가용성 전분에 대한 $K_{m}$ 값은 2.5㎎/㎖이었다. 또한, 정제 효소의 금속 이온의 효과로 $Ca^{2+}, Co^{2+}, EDTA, Mg^{2+}, Mn^{2+}, Zn^{2+}$ 첨가시 활성이 촉진되었고, $Ca^{2+}$의 경우 가장 높은 반면 $Cu^{2+}, Fe^{2+}, Ni^{2+}$의 경우는 오히려 활성이 감소되었다.

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구기자 품종과 교배종의 부위별 심혈관관련 질환 예방 기능성 및 항산화 활성비교 (Cardiovascular Biofunctional Activity and Antioxidant Activity of Gugija (Lycium chinensis Mill) Species and Its Hybrids)

  • 박원종;이봉춘;이주찬;이은나;송정은;이대형;이종수
    • 한국약용작물학회지
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    • 제15권6호
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    • pp.391-397
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    • 2007
  • 새로운 고부가가치의 구기자 품종을 개발하기 위하여 구기자 표준 품종들과 이들의 교배종들의 순과 잎, 열매와 뿌리등의 물 추출물과 메탄올 추출물을 제조한 후 심혈관 질환 예방생리기능성과 노화억제 항산화 활성을 조사하여 비교하였다. 구기자 표준품종들의 생리기능성 가운데, 항산화 활성은 청운품종의 구기순의 메탄올 추출물이 93%로 가장 높았고 항고혈압성 안지오텐신 전환효소 저해활성은 청양7호의 지골피의 물 추출물에서 84.1%로 제일 높았다. 구기자 교배종의 생리 기능성중 항산화 활성은 명안A-2의 지골피의 메탄올 추출물이 93.1%로 제일 높았고 ACE저해활성은 DO148-72 (A11) 교배종 열매의 메탄올 추출물이 96.9%로 제일 높았다. 그러나 혈전용해활성과 HMG-CoA reductase 저해활성은 표준품종과 교배종 모두에서 대체로 미약하거나 없었다. 이상의 결과를 종합하였을 때 청운 표준품종의 구기순과 명안A-2 교배종의 지골피가 우수한 항산화 자원으로, DO148-72 (A11)교배종의 구기열매가 훌륭한 항고혈압성 ACE저해제 자원으로 각각 기능성 제품생산에 유용하게 활용될것으로 사료된다.

Mass-Spectral Identification of an Extracellular Protease from Bacillus subtilis KCCM 10257, a Producer of Antibacterial Peptide Subtilein

  • SONG HYUK-HWAN;GIL MI-JUNG;LEE CHAN
    • Journal of Microbiology and Biotechnology
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    • 제15권5호
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    • pp.1054-1059
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    • 2005
  • An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

핵전이에 의한 Aspergillus oryzae var. Oryzae와 Aspergillus nidulans 514의 잡종으로부터 생산된 Xylanase의 특성 (Characterization of Xylanase from an Hybird between Aspergillus oryzae var. oryzae and Aspergillus Nidulans 514 by Nuclear Transfer)

  • 양영기;문명님;박형남;임채영
    • 한국미생물·생명공학회지
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    • 제24권1호
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    • pp.50-58
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    • 1996
  • Interspecific hybrids between Aspergillus oryzae var oryzae and A. nidulans 514 were obtained by nuclear transfer technique. Several autotrophic mutants isolated from conidiospores of the two strains were mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were $3{\times}10^{-5}{\sim}1{\times}10^{-5}$. From observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrids showed 1.1~1.4 fold higher xylanase activities than parental strains did. The xylanase of Aspergiilus sp. TAVD514-3 was purified and some of it's enzymatc characteristics were investigated. The enzyme was purified about 85 fold with an overall yield of 17% from the culture medium by ammonium sulfate fractionation, Sephadex G-75 gel permeation chromatography, and CM-sephadex A-50 ion exchange chromatography. The purified enzyme functions optimally at pH 9.0 and 80$^{\circ}C$. The enzymatic activity was increased by the presence of $Mg^{2+}$ and $Mn^2$ ions.

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Secretion of Bacillus subtilis Cytidine Deaminase by the Aid of Signal Sequences in Escherichia coli

  • Yoon, Soo-Ryun;Kim, Sung-Il;Lee, Se-Young;Song, Bang-Ho
    • Journal of Microbiology and Biotechnology
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    • 제1권1호
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    • pp.22-30
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    • 1991
  • In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtilis cdd gene in E. coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the $cdd^{+}$ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the $NH_2-terminal$ of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave $cdd^{+}$ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.

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세포사멸 조절 단백질인 IEX-1의 새로운 결합단백질로서의 CATHEPSIN B의 발견 (Identification of CATHEPSIN B as a Novel Binding Protein of the Cell Death Regulating Protein IEX-1)

  • 유상미;이강석;배지현
    • 한국발생생물학회지:발생과생식
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    • 제16권2호
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    • pp.129-135
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    • 2012
  • 선행연구에서 본 연구자는 IEX-1 단백질이 난소 암세포에서 세포사멸(apoptosis)을 유도하는 기능을 수행함을 확인하였으나, 세포사멸과 세포생존의 여러 단계에서 어떠한 신호전달 체계로 IEX-1이 작용하는지는 정확히 알지 못하고 있다. 따라서 IEX-1 단백질과 결합하는 새로운 단백질을 찾기 위해 yeast two-hybrid system을 이용하였다. 그 결과 IEX-1이 여러 다양한 인간 암 세포에서 세포사멸을 유도하는 CATHEPSIN B 단백질과 결합함을 밝혔다. 본 연구에서는 리소좀 프로테아제의 일원인 CATHEPSIN B 단백질과 IEX-1 단백질의 결합을 면역침강법과 western blot 분석으로 확인하였다. 그러므로 이 결과들을 통해서 IEX-1과 CATHEPSIN B는 세포 내에서 서로의 기능에 영향을 미칠 것으로 예상되며, 세포사멸을 유도하는 IEX-1 단백질이 lysosome-mediated apoptotic pathway에 관여할 가능성을 시사한다.

Mapping, Tissue Distribution and Polymorphism Study of the Porcine SOCS2 and SOCS3 Genes

  • Li, X.Y.;Liu, B.;Fan, B.;Yu, M.;Zhu, M.J.;Xiong, T.A.;Li, K.
    • Asian-Australasian Journal of Animal Sciences
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    • 제19권2호
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    • pp.165-170
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    • 2006
  • Using the somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel, porcine SOCS2 gene was mapped at SSC5 (1/2) q21-q24 and closely linked with SW1383 marker (47 cR in distance), while SOCS3 gene was assigned to SSC12p11-(2/3p13) and closely linked with SW2490 (43 cR). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the expression of these two genes in the different tissues and the results showed that both SOCS2 and SOCS3 genes were widely expressed in tissues investigated (heart, liver, spleen, lung, kidney skeletal muscle, fat and brain), although some tissues showed lower gene expression. Moreover, SOCS2 and SOCS3 genes had different expression levels at different stages, in different tissues and in different breeds. A G/A substitution, which can be recognized by restriction enzyme of Cfr421, was observed in 5' untranslated region (5'-UTR) of SOCS2 gene. The allele frequencies was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) method and it showed that the allele frequency among Dahuabai, Erhualian, Yushan, Qingping, Large white and Landrace tested were different. Association analysis in a cross experimental populations revealed no significant association between the SOCS2 gene polymorphism and the economic traits investigated. The full-length coding regions (CDs) of porcine SOCS3 gene was obtained by RT-PCR.

RTP1, a Rat Homologue of Adenovirus ElA-associated Protein BS69, Interacts with DNA Topoisomerase II

  • Oh, Misook;Rha, Geun-Bae;Yoon, Jeong-Ho;Sunwoo, Yang-Il;Hong, Seung-Hwan;Park, Sang-Dai
    • Animal cells and systems
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    • 제6권3호
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    • pp.277-282
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    • 2002
  • Topoisomearse II is an essential enzyme in all organisms with several independent roles in DNA metabolism. Recently, it has been demonstrated that the C-terminal region of topoisomerases II is associated with hetero-logous protein-protein interactions in human and yeast. In this study, we identified that RTP1, a rat homologue of EIA binding protein BS69, is another topoisomerae II interacting protein by yeast two-hybrid screening. RTP1 has an E1A-binding domain and a MYND motif, which are known to be required for transcriptional regulation by binding to other proteins and interaction with the leucine zipper motif of topoisomerase II. The physical interaction between RTP1 and topoisomerase ll$\alpha$ was examined by GST pull-down assay in vitro. The expression level of RTP1 peaks in S phase as that of topoisomerase ll$\alpha$. These results suggest that the interaction between topoisomerase ll$\alpha$ and RTP1 might play an important role in regulating the transcription of genes involved in DNA metabolism in higher eukaryotes.