방선균에서 신규 항생물질의 탐색과 해당유전자의 클로닝을 위한 분자유전학적 접근

현창구,홍순광,서주원 한국산업미생물학회 1998 생물산업 Vol.11 No.3

(주)오리온제주용암수

현창구 한국식품저장유통학회 2020 식품저장과 가공산업 Vol.19 No.1

(재) 제주테크노파크 용암해수산업화지원센터

현창구 한국식품저장유통학회 2018 식품저장과 가공산업 Vol.17 No.2

(주)제주사랑농수산

현창구 한국식품저장유통학회 2019 식품저장과 가공산업 Vol.18 No.2

활성형 Vitamin D₃ 전환능을 소유한 방선균에서 P-450 Hydroxylase유전자의 클로닝 전략

서주원,현창구,김정미,김승영,홍순광 명지대학교 자연과학연구소 1998 자연과학논문집 Vol.17 No.-

비타민 D₃는 포유동물이나 새의 간에서 25-hydroxyvitamin D₃[25(OH)D₃]로 전환되는데, 이 hydroxylation은 P-450 hydroxylase에 의해 촉매된다고 알려져있다. 본 연구에서는 활성형 비타민 D₃전환능을 소유한 방선균에서P-450 hydroxylase효소를 신속하게 분리가능한 PCR방법을 보고하고자 한다. Primer는 방선균에서 보고된 P-450 hydroxylase들 사이의 아미노산 서열의비교를 통해 산소결합부위와 heme-ligand pocket 주의에서 제작되었고,5종의 방선균에서 효과적으로 PCR증폭산물을 획득할 수 있었다. 증폭산물을 획득할 수 있었다.증폭 산물들의 아미노산서열을 분석한 결과 기존에 밝혀진 많은P-450 hydroxylase유전자들과 상동성을 보이고 있었으며, 특히 Streptomyces griseolus에서 분리되어 sulfonylurea 등의 제초제의 분해능력을 가진 SuaC, SubC 효소 등과 높은 아미노산 상동성을 지니고 있었다. 이러한 PCR전략은 방선균에서 유용한 물질의 대사에 관여하는 P-450 hydroxylase효소의 클로닝에 도움을 줄 것이라 사료된다 In mammals and birds, vitamin D₃is converted to 25-hydroxyvitamin D₃[25(OH)D₃] and then to 1α25-dihydroxyvitamin D₃[25(OH)₂D₃]in the liver and kideny, respectively these hydroxylating reactions are known to be catalyzed by P-450 hydroxylases. In this paper we report a PCR method which can be used for the repid amplification of DNA fragments for _450 hydroxylase from actinomycetes which can convert to active from of vitamin D₃. Primers were designed based on several regions sharing strong similarities in amino acid sequence of P-450 hydroxylases from a variety of actinomycetes, primarily in the regions of an oxygen binding site and a heme ligand pocket. These primers were used to amplify DNA fragments from five different actinomycetes. The deduced amino acid sequences of the isolated fragments revealed significant similarities to known P-450 hydroxylase including the product of the suaC or subC genes from Streptomyces griseolus that is capable of metabolizing a number of sulfonylurea herbicides and to the product of the P450sca2 from S. carbophilus that produces a specific HMG-CoA reductase inhibitor. This method will be helpful for the researchers in cloning the genes for P-450 hydroxylase involved in the biosynthesis of useful compounds.

Streptomyces coelicolor에서 seeY 유전자의 클로닝과 염기서열 결정

김상숙,현창구,김영민,이주헌,정인권,김대명,서주원 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.6

단백질 분비기작에서 translocator의 역할을 하는 것으로 알려진 secY 유전자는 E.coli에서는 어느 정도의 연구가 이루어지고 있지만 산업적으로 유용한 그람양성 균주에서의 secY 유전자에 대한 연구는 거의 전무한 상태이다. 그러므로 현재 사용되는 항생제의 60% 정도를 생산하며 자연적인 단백질 분비체계를 갖고 있는 S. coelicolor에서의 secY 유전자의 분리는 유용 이종단백질 생산균주로서 Streptomyces를 이용하고자 하는 연구에 기폭제가 될 수 있을 것이다. 지금까지 분리된 secY 유전자 중 E. coli secY를 제외한 모든 secY 유전자는 모두 spectinomycin(spc)-operon 내의 L15과 adk 유전자 사이에 존재하며 높은 상동성을 보이고 있으므로 우리는 이 지역에서 4개의 primer를 제작하여 PCR 실험을 수행하였고 PCR 산물의 염기서열 결정 결과 아미노산 서열상에서 B. subtilis와 37.8%, M. luteus와 56% 정도의 동질성을 보였으며 secY 유전자의 양 옆으로 spc-operon 내의 rplO(ribosomal protein인 L15 유전자)와 adk-operon의 adk 유전자와 상동성을 보이는 지역을 확인할 수 있어 S. coelicolor로 부터 secY 유전자를 cloning 하였음을 알 수 있었다. 또한 hydrophobicity analysis결과 매우 유사한 양상이 나타나는 것으로 보아 S. coelicolor secY 유전자 역시 E. coli, B. subtilis, M. luteus와 같은 topology를 갖는 단백질임을 알 수 있었다. 그러나 S. coelicolor의 secY 유전자는 E. coli와 B. subtilis에서 L15와 translationally coupling되어 있는 것과는 대조적으로 자신의 promotor 지역을 가지고 있어 오히려 adk 유전자와 translationally coupling된 구조를 이루고 있는 것으로 보아 adk operon에 포함되어 존재하고 있다고 사료된다. SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane of Escherichia coli. In order to study the mechanism of protein secretion in Streptomyces, we have done cloning and sequencing of the Streptomyces coelicolor secY gene by using polymerase chain reaction method. The nucleotide sequence of the gene for SecY from S. coelicolor showed over 58% identity to that of M. luteus. The deduced amino acid sequences were highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments, and especially second, fifth, and tenth segments were particularly conserved, sharing greater than 75% identity with M. luteus SecY. We propose that the conserved membrane-spanning segments actively participate in protein export. In B. subtilis and E. coli, the secY gene is a part of the spc operon, is preceded by the gene coding for ribosomal protein L15, and is likely coupled transcriptionally and translationally to the upstream L15 gene. In the other hand, secY gene of S. coelicolor and M. luteus have its own promoter region, are coupled translationally with adk gene and presented in adk operon.

Comparative Depigmentation Effects of Resveratrol and Its Two Methyl Analogues in a-Melanocyte Stimulating Hormone-triggered B16/F10 Murine Melanoma Cells

윤훈석,현창구,이남호,박성수,신동범 한국식품영양과학회 2016 Preventive Nutrition and Food Science Vol.21 No.2

Previous research showed that resveratrol (trans-3,4’,5-trihydroxystilbene) and pinostilbene (trans-3-methoxy-4’,5-dihydroxystilbene) were able to inhibit tyrosinase directly; however, anti-melanogenic effects of pterostilbene (trans-3,5-dimethoxy-4’-hydroxystilbene) and resveratrol trimethyl ether (RTE) have not been compared. To investigate the hypopigmentation effects of pterostilbene and RTE, melanin contents and intracellular tyrosinase activity were determined by western blot analysis. Firstly, pterostilbene showed the inhibitory effects on a-melanocyte stimulating hormone (MSH)-induced melanin synthesis stronger than RTE, resveratrol, and arbutin. Pterostilbene inhibited melanin biosynthesis in a dose-dependent manner in a-MSH-stimulated B16/F10 murine melanoma cells. Specifically, melanin content and intracellular tyrosinase activity were inhibited by 63% and 58%, respectively, in response to treatment with 10 mM of pterostilbene. The results of western blot analysis indicated that pterostilbene induced downregulation of tyrosinase protein expression and suppression of a-MSH-stimulated melan-A protein expression stronger than RTE or resveratrol. Based on these results, our study suggests that pterostilbene can induce hypopigmentation effects more effectively than resveratrol and RTE, and it functions via downregulation of protein expression associated with hyprepigmentation in a-MSH-triggered B16/F10 murine melanoma cells.

Differential Effects of Methoxylated p-Coumaric Acids on Melanoma in B16/F10 Cells

윤훈석,이남호,현창구,신동범 한국식품영양과학회 2015 Preventive Nutrition and Food Science Vol.20 No.1

As an approach to search for chemopreventive agents, we tested p-coumaric acid, 3-methoxy-p-coumaric acid (ferulic acid), and 3,5-dimethoxy-p-coumaric acid (sinapic acid) in B16/F10 melanoma cells. Intracellular melanin contents were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cytotoxicity of the compounds were examined by lactate dehydrogenase (LDH) release. p-Coumaric acid showed inhibitory effect on melanogenesis, but ferulic acid increased melanin content, and sinapic acid had almost no effect on melanogenesis. Treatment with ferulic acid resulted in a 2 to 3 fold elevation in the production of melanin. Correlatively, cell viability decreased in a dose-dependent manner when treated with ferulic acid. However, ferulic acid did not affect the LDH release from the cells. Treatment with sinapic acid resulted in a 50∼60% elevation in the release of LDH when treated with a 200 mg/mL concentration and showed neither cytostasis nor increase of melanin synthesis in a dose-dependent manner. Taken together, p-coumaric acid inhibits melanogenesis, ferulic acid induces melanogenesis, and sinapic acid exerts cytotoxic effects in B16/F10 murine melanoma cells. The results indicate that the addition of methoxy groups to p-coumaric acid shows the melanogenic or cytotoxic effects in melanoma cells compared to the original compound. Therefore, this study suggests the possibility that methoxylated p-coumaric acid, ferulic acid can be used as a chemopreventive agent.

LPS로 자극된 RAW 264.7 세포에서 암대극 추출물의 항염증 효과

양은진,김민선,김승영,현창구 한국생물공학회 2019 KSBB Journal Vol.34 No.2

The Euphorbia jolkini (J11) is traditional medicinalplant, has been used for treatment diabetes, flu, toothache,etc. Recently studies shown that J11 extract has been evaluatedto anti-oxidant, anti-bacterial, and anti-melanogenesis. However, Anti-inflammatory effect of J11 extract has notbeen reported yet. In this study, we studied the effects of J11extract on anti-inflammatory effect in lipopolysaccharide(LPS)-stimulated RAW 264.7 cells. We also studied the effectof J11 extract on expression of protein in RAW 264.7 cells. The cells were treated with J11 extract, ranging between 25and 100 μg/mL. The results indicated that J11 extract dramaticallydecreased nitric oxide (NO) and prostaglandin 2 (PGE2)production in the cells without any cytotoxicity. In addition,J11 extract strongly inhibited expression of pro-inflammatorycytokines such as interleukin (IL)-6 and interleukin (IL)-1β. Moreover, western blot result showed that the J11 extractinhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase(COX-2) expression in a does-dependent manner. Therefore, this study suggest that J11 extract may be appliedfor potential source as a natural anti-inflammatory agent.

New Dibenzofurans from the Branches of Distylium racemosum Sieb. et Zucc

Ryeo Kyeong Ko,이선주,현창구,이남호 대한화학회 2009 Bulletin of the Korean Chemical Society Vol.30 No.6