새로운 섬유소분해 균주 Trichoderma sp. C-4에서 분리한 Endoglucanase (F-I-III)에 대한 연구

설옥주,정대균,한인섭,정춘수,Sul Ok Ju,Chung Dae Kyun,Han In Seob,Jeong Choon Soo 한국미생물학회 2005 미생물학회지 Vol.41 No.1

One of the endoglucanases, F-I-III, was purified from the culture filtrate of T. sp. C-4 through procedures including chromatography on Sephacryl S-200, DEAE-Sepharose A-50, and Chromatofocusing on Mono-P (FPLC). The molecular weight of the enzyme was determined to be about 56,000 Da by SDS-PAGE, and pI of 4.9 by analytical isoelectric focusing. F-I-III showed the highest enzyme activity at $55^{\circ}C$, and the pH optimum of the enzyme was 5.0. There was no loss of activity when the enzyme was incubated at $50^{\circ}C$ for 24 hours. The specific activity of the enzyme F-I-III toward the CMC was 315.4 U/mg. The Km value for $PNPG_2$ of F-I-III was 2.69 mM. N-terminal sequence of F-I-III was analyzed to be QPGTSTPEVHPKKLTTYK. It showed $95\%$ of homology to that of EGI from T. reesei. The presence of some metal ions (1 mM) had only a little effect on CMCase activity. The treatment of the reducing agents resulted in the increase of endoglucanase activity. 국내에서 분리된 우수섬유소분해 균주인 Trichodema sp. C-4가 생성하는 endoglucanase 중하나를 $(NH_4)_2SO_4$ 침전, Sephacryl S-200 gel filtration, DEAE-Sepharose A-50 ion exchange, Mono-P chromatofocusing (EPLC)의 단계로 정제하고 이를 F-I-III라 명명하였다. 분리된 효소 F-I-III는 분자량 56,000Da, 둥전점 4.9로 측정된 단일 단백질이었다. F-I-III는 $55^{\circ}C$에서 가장 높은 활성을 보였으며, pH 5.0이 반응 최적 조건이었다. $50^{\circ}C$에서 24시간 동안 안정하였으며, pH 4-7의 범위에서 안정하였다. CMC에 대한 비활성은 315.4U/mg 이었으며, PNPG2에 대한 Km 값은 2.69 mM이었다. 이 효소는 같은 균주에서 분리한 다른 endoglucanase와 exoglucanase를 섞었을 때 결정형 섬유소인 Avicel분해에 대한 상승효과를 보였다. $Mg^{2+},\;CO^{2+},\;Fe^{2+},\;Ca^{2+},\;CS^+,\;Li^+$ 등의 이온은 1 mM의 농도에서 효소의 활성에 큰 영향을 미치지 않았고, 1 mM의 환원제 (cystein, EDIA, \beta-mercaptoethanol, dithiothreitol(DTT), L-ascorbic acid)들은 효소의 활성을 증가시켰다. E-I-III의 N-말단 서열을 분석하여 QPGTSTPEVHPKKLTTYK의 서열을 얻었다. 이는 Trichodema reesei의 endoglucanase인 EGI과 $95\%$의 유사도를나타내었다. 분리된 효소 F-I-III는 높은 비활성을 가지고 있어서 활용가치가 높을 것으로 사료되었다.

Trichoderma sp. C-4에서 분리한 endoglucanase(F-II-II)의 특성에 대한 연구

설옥주,최지영,손영준,신지원,한인섭,정대균,정춘수 한국미생물학회 2000 미생물학회지 Vol.36 No.1

Trichoderma sp. C-4의 배양액으로부터 한 종류의 endoglucanase(F-II-II)를 Sephacryl S-200 및 Sephacryl S-100 chromatography를 통하여 분리하였다. 분리된 효소는 SDS-PAGE및 isoelectric focusing을 통하여 단일 band로 나타났으며, 분자량이 26,000, 등전점이 8.0으로 나타났다. 이 효소의 반응 최적온도와 최적 pH는 각각 $50^{\circ}C$, 5.0 이었으며, $50^{\circ}C$에서 24시간 동안 안정하였다. 분리된 효소의 carboxymethylcellulose에 대한 specific activity는 776.2 U/mg protein으로 나타났다. 이 효소의 아미노산 조성을 조사하였다. 효소를 trypsin으로 가수분해한 후 분해산물에 대한 단백질서열 분석을 행하였다. 그 결과 이 효소의 서열은 현재까지 밝혀진 다른 단백질과 homology를 갖지 않음이 확인되었다. 이 효소는 그 specific activity가 대단히 높고 아직 보고되지 않은 novel protein일 가능성이 대단히 높기 때문에 좋은 유전자 자원이 될 것으로 사료되었다. One of endoglucanases(F-II-II) was purified from the culture filtrate of Trichoderma sp. C-4 through two step procedures including chromatography on Sephacryl S-200 and Sephacryl S-100. The molecular weight of the enzyme was determined to be about 26,000 by SDS-PAGE and the isoelectric point as 8.0 by analytical isoelectric focusing. The optimum temperature of the enzyme was $50^{\circ}C$ and the optimum pH was 5.0. No loss of activity was observed when the enzyme was preincubated at $50^{\circ}C$ for 24 hours. The specific activity of the enzyme toward carboxymethylcellulose (CMC) was estimated to be 776.2 U/mg. The internal amino acid sequence was analysed.

Lipopolysaccharide (LPS)-Induced Autophagy Is Responsible for Enhanced Osteoclastogenesis

설옥주,박현정,손호정,최혜선 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.11

We hypothesized that inflammation affects number and activity of osteoclasts (OCs) via enhancing autophagy. Lipopolysaccharide (LPS) induced autophagy, osteoclastogenesis, and cytoplasmic reactive oxygen species (ROS) in bone marrow-derived macrophages that were pre-stimulated with receptor activator of nuclear factor-κB ligand. An autophagy inhibitor, 3-methyladenine (3-MA) decreased LPS-induced OC formation and bone resorption, indicating that autophagy is responsible for increasing number and activity of OCs upon LPS stimulus. Knockdown of autophagy-related protein 7 attenuated the effect of LPS on OC-specific genes, supporting a role of LPS as an autophagy inducer in OC. Removal of ROS decreased LPS-induced OC formation as well as autophagy. However, 3-MA did not affect LPS-induced ROS levels, suggesting that ROS act upstream of phosphatidylinositol-4,5-bisphosphate 3-kinase in LPS-induced autophagy. Our results suggest the possible use of autophagy inhibitors targeting OCs to reduce inflammatory bone loss.

Cellulose 분해효소를 분비하는 Trichoderma sp. C-4

손영준,설옥주,정대균,한인섭,최윤재,정춘수 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.4

분해중인 볏짚으로부터 cellulase를 분비하는 곰팡이 C-4를 분리하였다. 분리된 균주의 형태적 배양적 특성을 조사한 결과 Trichoderma sp.로 분류되어 Trichoderma sp. C-4라고 명명하였다. 균을 Mandels 배지에서 6일동안 28℃에서 진탕배양한 후 배양액에 분비된 효소의 활성을 조사한 결과 CMCase 활성은 8.2 U/㎖(28.1 U/㎎), Avicelase활성은 0.75 U/㎖(2.58 U/㎎), β-glucosidase활성은 1.67 U/㎖(5.68 U/㎎)로 나타났다. 균배양 및 효소유도의 최적조건은 28℃, pH 6.2였다. 조효소는 50℃, pH 5.0에서 안정하였다. 1mM의 CsCl, LiCl, MgCl_2, CoCl_2에 대하여 CMCase활성이 영향받지 않았다. Trypsin과 chymotrypsin(2% w/w)에 의하여 10분 동안 30%의 활성이 상실되엇으나 그 후 60분 동안 더 이상의 활성 감소는 없었다. Rumen fluid에 대하여 24시간 동안 비교적 안정하였고, rumen fluid에 존재하는 효소와 CMC 및 Avicel에 대하여 각각 130% 및 118%의 상승효과를 나타내었다. During the screening of cellulase producing microorganisms, a fungal strain C-4 was selected from etiolated leaves. Based on taxonomic studies, the fungus could be classified as a strain of Trichoderma sp. When the strain C-4 was cultured in Mandel's media at 28℃ for 6 days, the enzyme activities detected in broth were as follows: 8.2 U/㎖ (28.1 U/㎎) of CMCase activity, 0.75 U/㎖ (2.58 U/㎎) of Avicelase activity, 1.67 U/㎖ (5.68 U/㎎) of β-glucosidase activity. The optimum pH for enzyme induction was 6.2. The crude enzyme retained 100% of its original CMCase activity at 50℃ for 1 hr (pH 5.0), and at 4℃ for 24 hrs (pH 5.0). There was no effect on the CMCase activity in the presence of 1 mM of CsCl, LiCl, MgCl_2 and FeCl_2, respectively. When the crude enzyme was treated with trypsin and chymotrypsin (2% w/w) for 10 minutes, the remaining CMCase activity was 70%, but there was no further loss of activity for 60 minutes treatment at 30℃. The crude enzyme showed the synergism with rumen fluid for the hydrolysis of Avicel and CMC by 118% and 130%, respectively.

Platinum nanoparticles reduce ovariectomy-induced bone loss by decreasing osteoclastogenesis

김운기,김진천,박현정,설옥주,Mi-Hyun Lee,김지순,최혜선 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.7

Platinum nanoparticles (PtNP) exhibit remarkable antioxidant activity. There is growing evidence concerning a positive relationship between oxidative stress and bone loss, suggesting that PtNP could protect against bone loss by modulating oxidative stress. Intragastric administration of PtNP reduced ovariectomy (OVX)-induced bone loss with a decreased level of activity and number of osteoclast (OC) in vivo. PtNP inhibited OC formation by impairing the receptor activator of nuclear factor-κB ligand (RANKL) signaling. This impairment was due to a decreased activation of nuclear factor-κB and a reduced level of nuclear factor in activated T-cells, cytoplasmic 1 (NFAT2). PtNP lowered RANKL-induced long lasting reactive oxygen species as well as intracellular concentrations of Ca2+oscillation. Our data clearly highlight the potential of PtNP for the amelioration of bone loss after estrogen deficiency by attenuated OC formation.

Increased Fat Due to Estrogen Deficiency Induces Bone Loss by Elevating Monocyte Chemoattractant Protein-1 (MCP-1) Production

Youn-Young Kim,Song-Hee Kim,Sora Oh,설옥주,Hye-Young Lee,Hyun-Ju Kim,김신윤,최혜선 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.3

Ovariectomy (OVX)-induced estrogen withdrawal resulted in both bone loss and an increase in fat. We observed ele-vated osteoclast (OC) formation by bone marrow-derived macrophages treated with medium conditioned by fats from OVX mice, but not from sham-operated mice. Fats from OVX mice expressed and secreted higher levels of monocyte chemoattractant protein-1 (MCP-1) than those from sham-operated mice. Increased fat resulting from estrogen deficiency is thus responsible for bone loss due to enhanced OC formation, which is, at least partly, a con-sequence of elevated MCP-1 production.