• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2001년도 추계학술대회
• /
• pp.16-25
• /
• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

• 한국식품영양과학회지
• /
• 제33권7호
• /
• pp.1169-1174
• /
• 2004

본 연구에서는 감마선 및 전자선종이 ovalbumin(OVA)의 구조 변화에 미치는 영향을 비교 평가하였다. 표준항원 OVA을 3,5,7 및 10 kGy의 흡수선량으로 감마선 및 전자선 조사하였으며 그에 따른 구조적인 변화는 SDS-PAGE, GPC-HPLC 및 단클론 항체를 사용한 Ci-ELISA법으로 측정하였다. Native OVA분자는 감마선 및 전자선 조사에 의해 소편화 및 응집화 되었으며, 조사 선량이 증가할수록 OVA의 분자량이 감소하였다. 또한 OVA의 면역화학적 인 구조는 방사선 조사에 의해 항원-항체간의 결합능이 감소하는 것으로 나타났으나 감마선 및 전자선종에 따른 차이는 없었다. 이상의 결과들은 식품 알레르겐의 제거 및 면역원의 개발에 대한 전자선 조사의 기초자료로 이용될 수 있을 것으로 사료된다.

• 한국동물학회지
• /
• 제28권4호
• /
• pp.257-278
• /
• 1985

Phenyalanine, tryptophan, tyrosine 과 같은 방향족 아미노산을 계배 초기에 투여하였을 때 somite 형성에 미치는 영향을 광학 및 전자현미경을 사용하여 형태적으로 추구한 결과 아미노산을 투여한 계배에서는 불완전한 체절 분절 현상이 일어나고 신경계에도 감쇠 영향을 미치며 somite의 발생이 불완전하고 그 크기도 다양하였다. 또한 체절 세포는 chromatin이 응축되고 미토콘드리아의 일부는 파괴되었고 핵이 변형된 경우도 있었다. 부란 24시간 후 아미노산을 투여하고 15일간 부란한 계배의 경우 단백질이나 핵산은 대조군에 비해 크게 저하되지는 않았으나 lactate dehydrogenase, succinate dehydrogenase, malate dehydrogenase 및 glucose 6-phosphate dehydrogenase와 같은 기초대사에 중요한 구실을 하는 효소활성은 크게 저하되었다. 이와 같은 실험결과로부터 초기계배에 아미노산을 투여하면 아마도 yolk granule의 분해가 지연되며 결과적으로 세포내의 아미노산 균형이 파괴되어 정상 대사가 이루어지지 못하여 비정상적인 체절형성의 현상이 나타나게 되는 것으로 생각된다.

• Restorative Dentistry and Endodontics
• /
• 제24권1호
• /
• pp.200-211
• /
• 1999

The periapical response to injury is a complex interaction of inflammatory, immune, neural, vascular and synthetic activity. TGF-${\beta}$ is a potent modulator of proliferation and differentiation in various tissue, seems to lead to an increase in extracellular matrix. MMP are a family of proteolytic enzyme that mediate the degradation of extracellular matric macromolecules, but little is known about theirs possible role in periapical tissue. The purpose of this study is to investigate the differential expression of TGF-${\beta}$ and MMP-1 in tooth follicle, periapical abscess, granuloma and cyst. The expression of TGF-${\beta}$ and MMP-1 in Periapical tissue was evaluated by immunohistochemical staining and Western blot analysis. Correlationship among the periapical lesions were stastically analyzed. The degree of MMP-1 expression in periapical abscess was higher than in any other periapical lesion, and stastically significant. TGF-${\beta}$ expression is the prominent in granuloma than other periapical lesion, which was stastically significant. The increased expression of MMP and TGF-${\beta}$ was not co-related with inflammatory cell infiltration degree of the periapical cyst. The expression degree of MMP and TGF-${\beta}$ was not co-related with periapical abscess and cyst, but expression of MMP and TGF-${\beta}$ showed strong positive co-relationship with periapical granuloma, which was stastically significant. TGF-${\beta}$ expression by Western blot analysis was prominent in granuloma and cyst, and similar to the results by imunohistochemistry. MMP-1 expression is less than TGF-${\beta}$, but there is not extreme difference between periapical lesion. These results suggest that TGF-${\beta}$ and MMP may be involved in tissue remodeling and has an important role in progress or mediation of periapical lesions.

• Journal of Microbiology and Biotechnology
• /
• 제20권4호
• /
• pp.712-717
• /
• 2010

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole-cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a hemeand diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. The surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of a heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins into practically useful whole-cell biocatalysts for extensive biotechnological applications, including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and biochip development.

• BMB Reports
• /
• 제48권2호
• /
• pp.91-96
• /
• 2015

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of over-expressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

• Natural Product Sciences
• /
• 제10권6호
• /
• pp.315-320
• /
• 2004

Alpha-viniferin was previously isolated as a cyclooxygenase (COX)-2 inhibitor from Carex humilis (Cyperaceae) and is an oligomeric stilbene compound with benzofuran (BF) moieties in its chemical structure. In the present study, a chemically synthetic BF compound, named as 3,3-dimethyl-2,3,4,6,7,8,9,10,11,12,13,14,15,16,17,18-hexadecahydro-1H-benzo[b] cyclopentadeca[d]furan-1-one, was discovered to inhibit bacterial lipo polysaccharide (LPS)-induced prostaglandin $E_2$ $(PGE_2)$ production in macrophages RAW 264.7. The BF compound exhibited a selectively preferred inhibitory effect on COX-2 activity over COX-1 activity. Furthermore, BF compound inhibited LPS-induced COX-2 expression at transcription level. As a down-regulatory mechanism of COX-2 expression shown by BF compound, suppression of nuclear factor $(NF)-{\kappa}B$ activation has been demonstrated. BF compound inhibited LPS-induced $NF-{\kappa}B$ transcriptional activity and nuclear translocation of $NF-{\kappa}B$ p65, in parallel, but did not affect LPS-induced degradation of inhibitory ${\kappa}B{\alpha}$ protein $(I{\kappa}B{\alpha})$. Taken together, anti-inflammatory effect of BF compound on $PGE_2$ production was ascribed by its down-regulatory action on LPS-induced COX-2 synthesis in addition to inhibitory action on enzyme activity of COX-2.

• Applied Biological Chemistry
• /
• 제35권6호
• /
• pp.507-512
• /
• 1992

대두(Glycine max)의 ${\beta}$-1,3-glucanase를 분리동정하고 benzyladenine(BA)이 이 효소의 세포내 함량과 활동도에 미치는 영향을 조사하였다. 또 세포벽의 callose함량과 protoplast의 세포벽재생에 미치는 BA의 영향을 조사하여, cytokinin이 식물의 세포벽재생을 촉진하는 기능이 있음을 확인하고 세포벽재생에 있어서 cytokinin의 작용기구를 검토하였다. 대두 ${\beta}-1,3-glucanase$는 21 kD의 polypeptide로 동정되었는데 이 polypeptide의 세포내함량과 효소활성은 BA처리에 의하여 저하되었다. 그리고 callus세포벽의 callose함량과 protoplast의 세포벽재생율이 BA처리에 의하여 증가되었다. 이 결과들은 cytokinin이 세포의 ${\beta}-1,3-glucanase$수준을 저하시켜 callose분해를 억제함으로써 세포벽 재생을 촉진할 수 있음을 보여주었다.

• Applied Biological Chemistry
• /
• 제14권2호
• /
• pp.131-135
• /
• 1971

1. Aspergillus oryzae의 균체에는 L-tyrosine-${\alpha}$-ketoglutaric acid transaminase와 p-hydroxyphenlypyruvic acid oxidase가 존재해 있다. 2. L-Tyrosine 산화효소는 액침배양한 Aspergillus oryzae의 acetone powder, cell free extract 및 배양액에도 존재하며 L-tyrosine은 ${\alpha}$-ketoglutaric acid의 첨가로 더욱 빨리 전환되었다. 3. ${\alpha}$-Ketoglutaric acid와 pyridoxal phosphate는 transamination의 amino기의 수용체로 생각되었다. 4. 이들 효소계는 L-tyrosine와 p-hydroxyphenlypyruvic acid를 homogentisic acid로 산화시켰다. 5. Ascorbic acid는 p-hydroxyphenlypyruvic가 homogentisic acid로 산화되는데 특별한 역할을 하는 것 같다. 6. L-Tyrosine-${\alpha}$-ketoglutaric acid transaminase와 p-hydroxyphenlypyruvic acid oxidase의 최적 pH는 각 각 pH $6.0{\sim}6.5$와 pH 7.5이었다.

• Food Science and Biotechnology
• /
• 제16권5호
• /
• pp.728-733
• /
• 2007

Bamboo grass, Sasa quelpaertensis, is a native plant to Jeju Island, Korea. The leaves of Sasa plants are widely used in traditional Korean medicine to treat inflammation-related diseases. We investigated the effect of hot water extract from Sasa quelpaertensis leaves (HWE-SQ) on nitric oxide (NO) production and nuclear $factor-{\kappa}B\;(NF-{\kappa}B)$ activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. HWE-SQ inhibited LPS-induced NO production and inducible NO synthase (iNOS) protein expression in a dose-dependent manner. Reporter gene assays indicated that HWE-SQ decreases LPS-induced $NF-{\kappa}B$ transcriptional activation. However, HWE-SQ did not affect the phosphorylation and degradation of inhibitory ${\kappa}B{\alpha}\;(1{\kappa}B{\alpha})$. HWE-SQ also directly inhibited iNOS enzyme activity in a dose-dependent manner. These results suggest that HWE-SQ suppresses NO synthesis in macrophages by attenuating $NF-{\kappa}B-mediated$ iNOS protein expression and inhibiting iNOS enzymatic activity, thereby implicating a mechanism by which HWE-SQ is able to ameliorate inflammation-related diseases by limiting excessive or prolonged NO production in pathological events.