초고열성 고세균 Pyrococcus horikoshii 유래 샤페로닌의 ATPase 활성 특성

최성석,김세원,서용배,김군도,이혜영,김연희,전숭종,남수완 한국미생물·생명공학회 2019 한국미생물·생명공학회지 Vol.47 No.4

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. It is thought that the folding activity of group II chaperonin is strongly correlated with the ATP-dependent conformational change ability. In order to confirm the dependence of the reaction temperature and ATP concentration of PhCpn, the ATPase activities were measured under different reaction temperatures and ATP concentrations. The maximal ATPase activity of PhCpn was observed at 80℃ and 3 mM ATP concentration. As a result of ATPase activity according to the type of salt ions, the highest activity was observed at 300 mM LiCl among the univalent cations and 5 mM MgCl2 among the divalent cations, respectively. The values of Km and Vmax for ATP substrate were estimated as 2.17 mM and 833.3 μM/min, respectively. This results provide the enzymatic information of PhCpn when the prolonged and high activities of pharmaceutical and industrial proteins (or enzymes), by using chaperonin molecules, are required. Group II형 샤페로닌은 단백질의 캡슐화를 유도하기 위해열린 기질 결합 형태에서 닫힌 형태로 형태를 변화시키며, 이 때 ATP를 필요로 한다. 샤페로닌의 폴딩 유도는 ATP에의한 샤페로닌의 구조 변화와 관련이 있는 것으로 보여진다. 본 연구에서는 Pyrococcus horikoshii OT3의 group II형샤페로닌인 PhCpn의 ATPase 활성을 다양한 조건에서 측정하였다. PhCpn의 반응온도(37−85℃)와 ATP 농도(1.5− 10 mM) 의존성을 확인한 결과, 반응 온도는 80℃에서, ATP 농도는 3 mM에서 최적 활성을 보였다. 염의 종류에 따른ATPase의 활성을 분석한 결과, 1가 양이온은 300 mM LiCl, 2가 양이온은 5 mM MgCl2에서 최적 활성을 나타내었다. ATP 기질에 대한 Km 값은 2.17mM, Vmax 값은 833.3 μM/ min으로 계산되었다. 이러한 결과는 의약학용 및 바이오 산업용 단백질(효소)을 장기간 활성유지하는데 PhCpn을 이용할 경우에 귀중한 기초 자료를 제공할 것이다.

Multiplex PCR과 Real-Time PCR을 이용한 창난젓과 가이양젓 원료 검사법 개발

최성석,서용배,김종오,양지영,신지영,김군도,Choi, Seong Seok,Seo, Yong Bae,Kim, Jong-Oh,Yang, Ji-Young,Shin, Jiyoung,Kim, Gun-Do 한국식품위생안전성학회 2021 한국식품위생안전성학회지 Vol.36 No.4

본 연구에서 multiplex PCR과 real-time PCR을 이용하여 창난젓의 원료를 감별할 수 있는 새로운 판별법을 개발하였다. 명태와 가이양의 종 특이 프라이머를 디자인하고, 명태와 가이양의 genomic DNA를 template로 single PCR과 multiplex PCR을 실시하였다. PCR을 실시한 결과, single PCR에서 명태(297 bp)와 가이양(132 bp)에 해당하는 PCR 밴드를 확인하였으며 교차 반응이 일어나지 않는 것을 확인하였다. Multiplex PCR에서 명태와 가이양 사이에 교차반응 없이 증폭이 일어나는 것을 확인하였다. Real-time PCR 결과, 명태 종 판별 프라이머에서 명태의 Ct 평균값은 20.765±0.691, 가이양 시료에서 Ct 평균값은 35.719±1.828이었으며, 가이양 종 판별 프라이머에서 명태 시료의 Ct 평균값은 35.996±1.423, 가이양 시료의 Ct 평균값은 20.096±0.793으로 프라이머의 효율성, 특이성 및 교차 반응성에서 유의한 차이가 나타났다. 이러한 결과를 바탕으로 시중에서 판매되는 7개 제품을 multiplex PCR 및 real-time PCR로 확인하였으며, 모든 시료에서 유효한 결과를 확인하였다. 본 연구에서 제작된 명태와 가이양에 대한 종 특이적 프라이머는 가공된 젓갈 시료의 원료의 판별 가능하며, 이러한 결과는 식품안전관리에 기여할 수 있을 것으로 기대된다. In this study, multiplex PCR and real-time PCR were performed on Theragra chalcogramma (walleye pollock), Pangasianodon hypophthalmus (iridescent shark) and their processed foods, such as changnan-jeot and gaiyang-jeot (salted iridescent shark intestine). Species-specific primers for T. chalcogramma and P. hypophthalmus were designed, and genomic DNA was directly extracted from each sample to perform single PCR and multiplex PCR. As a result of PCR, in the case of single PCR, PCR bands of T. chalcogramma (297 bp) and P. hypophthalmus (132 bp) were identified, and in the case of multiplex PCR, it was confirmed that amplification occurred without cross-reaction between T. chalcogramma and P. hypophthalmus. As a result of checking the PCR sensitivity, the concentration of genomic DNA was detected up to 0.1 ng/µL in both single PCR and multiplex PCR. The real-time PCR results showed that the average Ct value of T. chalcogramma was 20.765±0.691, and the average Ct value of P. hypophthalmus sample was 35.719±1.828 in the T. chalcogramma species-specific primers. In the P. hypophthalmus species-specific primers, the average Ct value of the T. chalcogramma sample was 35.996±1.423, and the mean Ct value of the P. hypophthalmus sample was 20.096±0.793. These results demonstrated the significant differences in the efficiency, specificity and cross-reactivity of species-specific primers in real-time PCR. Based on these findings, 7 of changnan-jeot or gaiyang-jeot products were confirmed by multiplex PCR and real-time PCR, and valid results were confirmed in all samples.

Molecular Cloning and Overexpression of Phytoene Desaturase (CrtI) from Paracoccus haeundaensis

최성석,서용배,임한규,남수완,김군도 한국미생물·생명공학회 2018 한국미생물·생명공학회지 Vol.46 No.2

Among the carotenoid biosynthesis genes, crtI gene encodes the phytoene desaturase (CrtI) enzyme, and phytoene desaturase convert phytoene to lycopene. Phytoene desaturase is involved in the dehydrogenation reaction, in which four single bonds in the phytoene are introduced into a double bond, eliminating eight hydrogen atoms in the process. Phytoene desaturase is one of the key regulating enzyme in carotenoid biosynthetic pathway of various carotenoid biosynthetic organisms. The crtI gene in genomic DNA of Paracoccus haeundaensis was amplified and cloned into a T-vector to analyze the nucleotide sequence. As a result, the crtI gene coding for phytoene desaturase from P. haeundaensis consists of 1,503 base pairs encoding 501 amino acids residues. An expression plasmid containing the crtI gene was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 55 kDa, equivalent to the molecular weight of phytoene desaturase. The expressed protein in cell lysate showed enzymatic activity similar to phytoene desaturase. Phytoene and lycopene were analyzed by HPLC and measured at wavelength of 280 nm and 470 nm, respectively. The Km values for phytoene and NADPH were 11.1 μM and 129.3 μM, respectively.

원환풀내에서 Quencher Device에 의한 고온수 분출로 일어나는 혼합유동에 관한 연구

최성석,김종보,Choi, Seong-Seok,Kim, Jong-Bo 대한설비공학회 1985 설비저널 Vol.14 No.1

One of the problems with the Boiling Water Reactor involves the flow and thermal mixings in the suppression water pool high pressure steam discharge into the pool in case of emergency core relief. Varioos heat sensitive devices and pumps for the reactor core cooling are installed in the middle of the suppression pool. Especially the pumps utilize pool water in order to cool the reactor core in emergency cases. In this case, the water temperature for the reactor cool ins should be below a certain temperature specified by the reactor design. In the present investigation, in other to determine the optimum locations of these pumping devices, numerical solutions have been obtained for the model to determine the f low mixing characteristics. Experimental investigations have also been carried out for the flow mixing and for the thermal mixing in the pool during the discharge. Considering that the discharge steam through the Quenching Device becomes hot water immediately in the water pool, the steam- equivalent hot water has been utilized. Examining these characteristices, it becomes possible to deform me the best locations for RCIC, LPCI , HPCI pumps in the suppression water pool for the emermency reactor core cooling.

블록체인을 이용한 드론 무인 택배 배송 시스템

최성석(Seong Seok Choi),강응선(Eung Seon Kang),피승재(Seung Jae Pee),송재근(Jae Keun Song),장주욱(Ju Wook Jang) 대한전기학회 2018 정보 및 제어 심포지엄 논문집 Vol.2018 No.10

박테리아에 의한 카로티노이드 생산; 카로티노이드 생산성 및 활용 가능성

최성석(Seong Seok Choi),김군도(Gun-Do Kim) 한국생명과학회 2022 생명과학회지 Vol.32 No.5

카로티노이드는 자연계에 존재하는 붉은색, 주황색, 황색의 지용성 색소이며, provitamin A, 항산화, 항염증, 항암 등 다양한 기능성을 가지는 생리활성물질로 알려져 있다. 생리 활성과 색소 활용성 때문에 카로티노이드는 식품, 화장품, 양식 산업 등에 널리 이용되고 있다. 현재 산업적으로 활용되는 카로티노이드는 대부분 생산 단가가 저렴한 합성 색소를 이용하고 있지만, 안전성과 생리활성효과 때문에 천연 카로티노이드가 각광받고 있다. 하지만 식물과 동물의 카로티노이드 생산은 경제적인 원인으로 생산에 제한이 있다. 박테리아에서 생산되는 카로티노이드는 화합 합성으로 생산되는 카로티노이드를 대체하기 위한 좋은 장점을 가지고 있다. 박테리아에서 생산되는 카로티노이드는 낮은 생산성 때문에 산업적 활용에 제한이 있으며, 지속적으로 박테리아로부터 카로티노이드의 생산성을 증가시키기 위한 연구가 이루어져 왔다. 박테리아로부터 카로티노이드 생산량을 증가시키기 위해 진행된 연구로는 박테리아의 카로티노이드 생합성 경로와 각 효소들의 활성, 돌연변이를 이용한 균주 개발, 유전공학을 통한 카로티노이드 생산성 증가 균주구축, 스트레스 유도를 통한 카로티노이드 축적, 발효 배지의 조성과 배양 조건, 다른 균주와의 공배양 등을 포함하고 있다. 이 논문의 목적은 박테리아로부터 카로티노이드의 생산성을 증가시키기 위해 진행된 연구들을 검토하는 것이다. Carotenoids are red, orange, and yellow fat-soluble pigments that exist in nature, and are known as physiologically active substances with various functions, such as provitamin A, antioxidant, anti-inflammatory, and anticancer. Because of their physiological activity and color availability, carotenoids are widely used in the food, cosmetics, and aquaculture industries. Currently, most carotenoids used industrially use chemical synthesis because of their low production cost, but natural carotenoids are in the spotlight because of their safety and physiologically active effects. However, the production of carotenoids in plants and animals is limited for economic reasons. Carotenoids produced by bacteria have a good advantage in replacing carotenoids produced by chemical synthesis. Since carotenoids produced from bacteria have limited industrial applications due to low productivity, studies are continuously being conducted to increase the production of carotenoids by bacteria. Studies conducted to increase carotenoid production from bacteria include the activity of enzymes in the bacterial carotenoid biosynthesis pathway, the development of mutant strains using physical and chemical mutagens, increasing carotenoid productivity in strain construction through genetic engineering, carotenoid accumulation through stress induction, fermentation medium composition, culture conditions, co-culture with other strains, etc. The aim of this article was to review studies conducted to increase the productivity of carotenoids from bacteria.

한국산과 중국산 새꼬막(Scapharca subcrenata)의 원산지 판별을 위한 SNP 마커의 개발 및 검증

최성석(Seong Seok Choi),유승현(Seung Hyun Yoo),서용배(Yong Bae Seo),김종오(Jong Oh Kim),권익정(Ik Jung Kwon),배소희(So Hee Bae),김군도(Gun Do Kim) 한국생명과학회 2023 생명과학회지 Vol.33 No.12

본 연구에서는 한국산과 중국산 새꼬막(Scapharca subcrenata) 사이의 원산지 판별을 위하여 quantitative real-time PCR (qPCR) 분석을 기반으로 하는 Single-nucleotide polymorphism (SNP) 마커의 primer set를 개발 및 검증하였다. 총 180개의 새꼬막 sample을 genotyping by sequencing으로 분석하여 원산지 판별에 유용할 것이라 판단되는 7개의 후보 MS 마커와 15개의 후보 SNP 마커를 선정하였다. 후보 SNP 마커는 PCR과 sanger sequencing, SYBR green-based qPCR을 통해 원산지별 분리 여부를 확인하였다. 이 중 Insertion 1, SNP 21 마커가 qPCR 증폭 양상에서 집단이 확연히 분리되었으며 예상과 실제 증폭 형태가 일치하였다. 추가적으로 새꼬막을 무작위로 섞어서 진행한 blind test에서 Insertion 1은 새꼬막 100개에 대하여 74%의 정확도, 52%의 민감도, 96%의 특이도를 보였고, SNP 21은 새꼬막 137개에 대하여 86%의 정확도, 79%의 민감도, 93%의 특이도를 보였다. 따라서 개발된 두 개의 SNP 마커는 독립적 또는 복합적으로 사용하면 새꼬막 원산지 판별의 진위 여부를 검증하는 데 유용할 것으로 기대된다. In this study, we analyzed SNPs that appear between Korean and Chinese Scapharca subcrenata using the nucleotide sequence data of S. subcrenata analyzed by genotyping by sequencing (GBS). To distinguish the country of origin for S. subcrenata in Korean and Chinese, we developed a primer set as single nucleotide polymorphism (SNP) markers for quantitative real-time PCR (qPCR) analysis and validated by sequencing SNPs. A total of 180 samples of S. subcrenata were analyzed by genotyping by sequencing, and 15 candidate SNPs were selected. SNP marker selection for country of origin were identified through real-time qPCR. Insertion 1 and SNP 21 markers showed the most distinct separation between the sequence types as well as the country of origin through qPCR, with the observed amplification patterns matching the expected outcomes.. Additionally, in a blind test conducted by mixing samples of S. subcrenata at random, Insertion 1 showed 74% accuracy, 52% sensitivity, and 96% specificity, and SNP 21 showed 86% accuracy, 79% sensitivity, and 93% specificity. Therefore, the two SNP markers developed are expected to be useful in verifying the authenticity of the country of origin of S. subcrenata when used independently or in combination.

산업공학에서의 VE 중심 교육활동 사례

박재일,최성석 대한산업공학회 2014 ie 매거진 Vol.21 No.1

Enhanced Production of Astaxanthin by Metabolically Engineered Non-mevalonate Pathway in Escherichia coli

정태혁,조연수,최성석,김군도,임한규 한국미생물·생명공학회 2018 한국미생물·생명공학회지 Vol.46 No.2

Astaxanthin is one of the major carotenoids used in pigment has a great economical value in pharmaceutical markets, feeding, nutraceutical and food industries. This study was to increase the production of astaxanthin by co-expression with transformed Escherichia coli using six genes involved in the non-mevalonate pathway. Involved in the non-mevalonate biosynthetic pathway of the strain Kocuria gwangalliensis were cloned dxs, ispC, ispD, ispE, ispF, ispG, ispH and idi genes in order to increase astaxanthin production from the transformed E. coli. And co-expression with the genes to compared the amount of astaxanthin production. This engineered E. coli, containing both the non-mevalonate pathway gene and the astaxanthin biosynthesis gene cluster, produced astaxanthin at 1,100 μg/g DCW (dry cell weight), resulting in approximately three times the production of astaxanthin.

오징어류 종 판별을 위한 다중 유전자 검사법 개발 및 검증

김현수,서용배,최성석,김진희,신지영,양지영,김군도 한국식품위생안전성학회 2015 한국식품위생안전성학회지 Vol.30 No.1

In this study, single PCR and multiplex PCR tests were examined for identification of four types of squid species (giant squid, cuttlefish, octopus, beka squid) purchased from fish market as well as aquatic processed products in Busan. To design the specific primers against each species, the nucleotide sequences of the mitochondrial 16s rRNA gene of Architeuthis dux, Todarodes pacificus, Enteroctopus dofleini, Enteroctopus megalocyathus, Uroteuthis chinensis, Uroteuthis duvauceli, Uroteuthis edulis groups were analyzed for the identification of each species registered in the GeneBank (www.ncbi.nlm.nih.gov) and have been used for comparative analysis. In order to obtain the size variation of amplified fragments on multiplex PCR, we designed KOJ-F, OJ-F, OCT-F, HAN-F, ALLR primers for each species. The optimal PCR conditions and primers were selected for four types of squid species to determine target base sequences in its PCR products. In the case of single PCR, giant squid was only amplified by KOJ-F/ALLR primer; cuttlefish was only amplified by OJ-F/ALLR primer; octopus was only amplified by OCT-F/ALLR primer; and beka squid was only amplified by HAN-F/ALLR primer. For multiplex PCR, the mixture of four kinds of genomic DNA (giant squid, cuttlefish, octopus, beka squid) been prepared as a template and used together with the mixture of KOJ-F/OJ-F/OCT-F/HAN-F/ALLR primers in the reaction. By the multiplex PCR, it is confirmed that four samples are correspond to multiple simultaneous amplicon. Finally, we validated the established methods of multiplex PCR in the aquatic processed products. Although the mitochondrial 16s rRNA primers used in this study was useful as a marker for detection of each species among them, the study indicated that the established multiplex PCR method can be more useful tool for monitoring the processed products.