단백질분해효소를 생산하는 Pseudoxanthomonas sp. WD12와 WD32의 분리

조운동,이제관,임채성,박아름,오용식,노동현,Cho, Woon-Dong,Lee, Je-Kwan,Lim, Chae-Sung,Park, A-Rum,Oh, Yong-Sik,Roh, Dong-Hyun 한국미생물학회 2010 미생물학회지 Vol.46 No.1

생리 및 상업적 응용분야에서 중요한 위치를 차지하는 단백질 분해효소는 펩타이드 결합의 가수분해를 촉매한다. 단백질 분해효소를 생산하는 신규 균주를 분리하기 위하여 부패한 나무로부터 균을 분리하여 1% skim milk가 포함된 LB 배지에서 투명환의 생성 능력이 높은 두 균주 WD12와 WD32를 선발하였다. 선발균주의 동정을 위하여 16S rRNA gene의 염기서열을 결정한 후 GenBank 상에 등록된 서열들과 상동성을 조사한 결과, WD12는 Pseudoxanthomonas mexicana와 97.8%, WD32는 99.8%의 상동성을 보여주었다. 계통적 유연관계를 조사한 결과 이들 균들은 P. mexicana와 P. japonensis와 cluster를 형성하였다. WD12와 WD32는 그람 음성 간균으로 catalase와 oxidase 활성을 보였다. WD12의 경우 P. mexicanna와 달리 malate를 동화할 수 있었으며, D-mannose를 동화할 수 없었다. 두 균주의 최적 단백질 분해효소를 생산하는 온도는 $35-37^{\circ}C$로 나타났으며, 최대활성은 WD12가 656 unit/ml, WD32가 267 unit/ml을 나타내었다. Proteases catalyze hydrolytic cleavage of a peptide bond between amino acids and occupy pivotal positions in application in physiological and commercial fields. During the screening for novel bacteria producing extracellular protease, two bacterial strains, WD12 and WD32, were isolated from rotten trees and they made clear zone on LB plates supplemented with 1% skim milk. The similarities of 16S rRNA gene sequence of either WD12 or WD32 to GenBank database showed the highest to Pseuoxanthomonas mexicana as 97.8 and 99.8%, respectively. Phylogenetic analysis showed that both isolated was located within the cluster comprising P. mexicana and P. japonesis. WD12 and WD32 were catalase- and oxidase-positive, Gram-negative rod strains. In case of WD12, it could assimilate malate, but could not assimilate D-mannose, which were different characteristics from P. mexicana. Both Pseuoxanthomonas sp. WD12 and WD32 optimally produced extracellular protease at $35-37^{\circ}C$, and maximal activity showed as 656 unit/ml and 267 unit/ml, respectively.

Pseudoxanthomonas sp. WD12와 WD32의 세포외 단백질분해효소 특성

조운동 ( Woon-dong Cho ),오지성 ( Ji-sung Oh ),노동현 ( Dong-hyun Roh ) 한국응용생명화학회(구 한국농화학회) 2016 Journal of Applied Biological Chemistry (J. Appl. Vol.59 No.4

단백질 분해효소는 단백질 내 아미노산의 펩티드 결합을 가수분해하며, 산업분야에서 가장 많이 사용되는 효소이다. 이전의 논문에서 부패한 나무로부터 Pseudoxanthomonas sp. WD12와 WD32를 분리하였다. 분비된 단백질 분해효소의 특성을 조사한 결과 WD12는 pH 9.0, 50 ℃, WD32의 경우 pH 8.0, 45 ℃에서 최적 활성을 나타내었다. 최적조건하에서 최고의 활성은 WD12는 768 unit/mL로 WD12의 활성이 WD32보다 2.6배 높았다. 금속 이온에 대한 분비효소 영향을 조사한 결과 두 균주 모두 KCl, NaCl, CaCl₂, MnSO₄를 첨가했을 때 활성이 증가하였으며, AgNO₃, CuSO₄, FeCl₃, AlCl₃를 첨가했을 때는 감소하였고, 최고의 활성은 MnSO₄의 첨가했을 때 두 균주 모두 활성증가를 보인 반면 FeCl₃, CuSO₄는 활성저해를 보였다. 신종으로 생각되는 WD12 균주의 효소는 알칼리성 조건의 산업 환경에서 이용가능 하다고 생각된다. Proteolytic enzymes perform hydrolysis of the peptide bonds in the protein and most commonly use in the industry. Pseudoxanthomonas sp. WD12 and WD32 were previously isolated as protease producers from a rotten wood sample. Here, we report the secreted proteolytic enzymes. The optimum enzyme reaction temperature for the secreted crude enzyme from the strain WD12 and WD32 were 50 ℃ at pH 9.0 and 45 ℃ at pH 8.0, respectively. The enzyme activities of both strains were increased by addition of KCl, NaCl, CaCl₂ or MnSO4, and decreased by addition of AgNO3, CuSO₄, FeCl_₄ or AlCl₃. Secreted enzymes of both strains were most strongly inhibited by addition of FeCl₃ or CuSO₄. Taken together these results, WD12 could be a candidate strain of industrial alkaline protease production.

Micrococcus sp. HJ19에서 체외분비 단백질 분해효소의 생산조건과 효소특성

차인태,오용식,조운동,임채성,이제관,이오석,노동현,Cha, In-Tae,Oh, Yong-Sik,Cho, Woon-Dong,Lim, Chae-Sung,Lee, Je-Kwan,Lee, Oh-Seuk,Roh, Dong-Hyun 한국미생물학회 2009 미생물학회지 Vol.45 No.1

단백질 분해효소는 아미노산 간에 존재하는 공유결합인 펩티드 결합을 절단하는 효소이며, 세계적으로 가장 많이 판매되는 효소이다. 해양 심층수로부터 분리된 Micrococcus sp. HJ19의 체외분비 단백질 분해효소를 생산하는 최적의 배지조건을 조사하기 위하여 기본배지로 변형 STY 배지(1% tryptone, 0.15% yeast extract, 0.01% NaCl, 여과살균한 바닷물)를 사용하였다. 탄소원으로 포도당보다 갈락토오스를 사용하였을 때 1.3배로 높은 활성을 보여주었으며, 질소원으로는 casamino acid를 사용하였을 때 가장 낮은 활성을 보여준 반면, 맥아추출물을 사용하였을 때 가장 높은 활성을 나타내었다. 생산된 체외단백질 분해효소는 $35^{\circ}C$에서 최적의 활성을 나타내었으며, 최적 pH는 8.5로 판명되었다. Proteases are degradative enzymes which hydrolyze a peptide bond between amino acids and they are abundantly applied to commercial field. In order to investigate optimal medium compositions of carbon and nitrogen source for enzyme production, modified STY medium containing 0.15% yeast extract were used as basal medium. When galactose was used as carbon source, enzyme activity showed 1.3 higher than that of glucose. For nitrogen source addition of casamino acids to basal medium in place of tryptone showed lowest activity, whereas addition of malt extract showed maximal activity. The optimum temperature and pH of extracellular protease were found to be $35^{\circ}C$ an pH 8.5.

Characterization of Two Novel mAbs Recognizing Different Epitopes on CD43

김솟을,홍정원,조운동,문유리,윤상순,김민영,홍권표,이용문,이재혁,함영준,나형철,김승렬,송형근 대한면역학회 2014 Immune Network Vol.14 No.3

JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.

Characterization of Monoclonal Antibodies against Human Leukocyte Common Antigen (CD45)

신향미,이기형,송형근,조운동,이건국,이선화,이경미,지길용,윤상순,구지혜,이호창 대한면역학회 2011 Immune Network Vol.11 No.2

Background: The leukocyte common antigen (CD45) is a transmembrane-type protein tyrosine phosphatase that has five isoforms. Methods: We generated seven murine mAbs against human CD45 by injecting cells from different origins, such as human thymocytes, PBMCs, and leukemic cell lines. By using various immunological methods including flow cytometry, immunohistochemistry, and immunoprecipitation, we evaluated the reactivity of those mAbs to CD45 of thymus as well as tonsil lysates. Furthermore, we transiently transfected COS-7 cells with each of gene constructs that express five human CD45 isoforms respectively, and examined the specificities of the mAbs against the transfected isoforms. Results: In case of thymocytes, lymphocytes, and monocytes, all the seven mAbs demonstrated positive reactivities whereas none was reactive to erythrocytes and platelets. The majority of immune cells in formalin-fixed paraffin-embedded thymus and tonsil tissues displayed strong membranous immunoreactivity, and the main antigen was detected near 220 kDa in all cases. Among the mAbs, four mAbs (AP4, DN11, SHL-1, and P6) recognized a region commonly present in all the five isoforms. One mAb, YG27, recognized four isoforms (ABC, AB, BC, and O). Two mAbs, P1 and P14, recognized the isoforms that contain exon A encoded regions (ABC and AB). Conclusion: In this study, we confirmed that AP4, DN11, SHL-1, YG27 and P6, are mAbs reactive with the CD45 antigen whereas P1 and P14 are reactive with the CD45RA antigen.