천연 김 유래 porphyra-334에 관한 항노화 및 탈모방지 효능 연구

김수연 성균관대학교 일반대학원 2021 국내석사

Porphyra-334 is a kind of mycosporine-like amino acids(MAAs) which can protect marine organs and cells from UV-radiation by absorbing UVA. In this study, we investigated anti-aging and anti-hair loss functions of Porphyra-334, and estimated whether Porphyra-334 is suitable for using as cosmetics agents. We purchased the Porphyra yezoensis from a market. We obtained porphyra extract by heat extraction and Porphyra-334 was purified from the extract. We conducted MTT assays to measure the effect of porphyra-334 on the cell viability of 3 human cell lines: Detroit 551 cells derived from human skin fibroblast cells; human keratinocyte (HaCaT); human follicle dermal papilla (HFDP) cells, and found the optimal concentration of porphyra-334 without cytotoxicity. To estimate improvement in collagen synthesis after treatment with different concentration of porphyra-334, we determined pro-collagen expression level in response to porphyra-334 treatment. We used TGF-β1, known for promoting Collagen Type I, as a positive control. The application of porphyra-334 resulted in similar PIP contents to TGF-β1’s in Detroit 551 cell. To investigate the proliferation of keratinocyte in response to Porphyra-334, we measured the expression of Ki-67 protein, cell-proliferation marker, by immunohistochemistry. We treated Porphyra-334 on 3D cultured skin, using ascorbic acid normally used for promoting skin elasticity as a positive control. We found the expression of Ki-67 proteins on both samples. To estimate the potential of pophyra-334 as an anti-hair loss agent, We extracted total RNA after treatment of Porphyra-334 on HFDP cell. By RNA-seq, We found differentially expressed genes(DEGs) up or down-regulated by Porphyra-334. To estimate gene expression changes, we selected some genes associated with hair among the DEGs. The expression of some Wnt family genes, the hair growth cycle-associated genes was up-regulated after the treatment. We also found Fibroblast growth factor 5, hair growth inhibitor, was down-regulated in response to Porphyra-334 treatment. The expression of some genes encoding Keratin, main components of hair and epidermis, was up-regulated by Porphyra-334. We also found CDH3 encoding P-Cadherin which can promote Wnt pathway signaling, was also up-regulated. In this study, We confirmed Porphyra-334 help to promote the synthesis of collagens in fibroblast and cell-proliferation of keratinocyte. we also found Porphyra-334 can promote hair growth by up or down-regulating the gene expressions associated with hair growth and hair maintenance. Overall, we examined the potential of used of Porphyra-334 as cosmetics agents, especially for anti-aging and anti-hair loss. Porphyra-334는 UVA를 흡수하는 것으로 알려진 마이코스포린 유사 아미노산(Mycosporine-like amino acid, MAA)의 일종이다. 본 연구에서는 Porphyra-334의 항노화 및 탈모 방지 효능에 대해 알아보고, 화장품 소재로서 Porphyra-334의 활용 가능성에 대해 평가하였다. 시중에서 판매하는 방사무늬 김을 이용해 김 추출물을 생성한 후, 단일 물질인 Porphyra-334를 분리 정제하여 물질을 규명하였다. 3종류의 Human Cell: Detroit 551 cells(human skin fibroblast cells); human keratinocyte (HaCaT); human follicle dermal papilla (HFDP) cells에 Porphyra-334를 처리하여 세포 독성을 확인 한 뒤, 독성을 보이지 않은 농도에서 콜라겐 합성 및 단백질 발현 실험을 수행하였다. 콜라겐 합성에 대해 Procollagen Type I C-Peptide의 함량 변화 정도를 측정하여 콜라겐 합성능력을 평가하였다. 콜라겐 type I를 촉진하는 것으로 알려진 TGF-β1을 양성대조군으로 하여 실험을 진행한 결과, TGF-β1와 비슷한 수준에서 콜라겐 합성이 증가한 것으로 나타났다. 표피를 구성하며, 케라틴을 생성하는 keratinocyte가 Porphyra-334 처리 후, 증식에 영향을 미치는지 알아보기 위해 증식의 결과로 분비되는 물질이자 세포 증식의 maker로 활용되는 Ki-67 단백질을 면역반응염색을 이용하여 발현 여부를 확인하였다. 주름개선 및 피부탄력 향상에 도움을 주는 것으로 알려진 Ascorbic Acid을 양성대조군으로 하여 3D 배양피부모델에 도포하여 면역 염색을 실험한 결과, 두 시료 모두 Ki-67이 발현됨을 확인하였다. 탈모 방지 효능 평가를 위하여 Human Follicle Dermal Papilla cell에 Porphyra-334를 처리하고 total RNA 추출 후, RNA-sequencing을 통해 대조군 대비 발현양에 유의미한 변화가 있는 유전자를 확인하였다. 그 중, 두피 구조 및 모발 성장주기 관련된 유전자를 선별하여 발모에 어떠한 영향을 미치는 지 알아보았다. 모발 생성 주기와 밀접한 연관이 있는 유전자 중, Wnt family 유전자의 발현이 Porphyra-334 처리 후 up-regulation 되었고, hair growth inhibitor로 알려진 Fibroblast growth factor 5(FGF5)는 down-regulation되는 양상을 보였다. 모낭 구조 및 표피 구조 관련 유전자 변화에서는 모발 주요 성분인 케라틴 관련 유전자의 발현이 상향조절 되었으며, Wnt pathway를 조절하는 것으로 알려진 P-Cadherin을 암호화하는 CDH3 유전자도 up-regulation된 것을 확인하였다. 본 연구를 통해 Porphyra-334가 진피 내 콜라겐 합성 및 각질세포 증식을 촉진하며, 모발 성장주기 및 발모 관련 유전자 발현을 Up 혹은 Down regulation 하여 발모 및 탈모 효능이 있음을 확인하였으며, 이를 통해 천연 유래 단일물질로써 Porphyra-334가 향후 주름 개선 및 탈모 완화에 도움을 주는 화장품 소재로써 활용할 수 있는 가능성이 있는 물질임을 확인하였다.

Biochemical features of red rot disease fungus pythium porphyrae isolated from porphyra cultivation farms in Korea and implication for disease prevention

박찬선 Mie University 2002 해외박사

Pythium porphyrae Takahashi et Sasaki is the causative organism of red rot disease in Porphyra (Rhodophyta). Although no work has been done on the morphological and physiological characteristics of Pythium sp. isolated from Korea, the overlap in geographical distribution and similarities in the disease symptoms in Korea and Japan would suggest that the same species of Pythium is involved. The present study was undertaken to explore the biological, biochemical and genetical characteristics of P. porphyrae isolates isolated from Asan, Mokpo, Wando, and Pusan in Korea by comparison with those from Fukuoka, Miyagi, and Aichi in Japan, and to develop an effective countermeasures for prevention and control of red rot disease by using the characteristics acquired by comparison among the isolates from Korea and Japan. The biological characteristics such as general mycological characteristics, responses to environmental conditions such as temperature, pH, salinity, and nutritional requirements for growth of P. porphyrae isolated from Wando in Korea were compared with those from Fukuoka, Miyagi, and Aichi in Japan. The number of hyphal swelling per visual field (31 mm2) of the isolate from Wando was significantly higher (p < 0.05) than those of the Japanese isolates. The other morphological characteristics such as the width of hyphae, diameter of hyphal swelling, oogonium diameter, oospore diameter, oospore cell membrane thickness, zoosporangium diameter and number of zoospore per zoosporangium of four isolates differed slightly. The four isolates grew at between 5 and 30 ℃ (growth maxima at 20 ℃), and at between pH 5 and 8.5 (growth maxima at between pH 7.5 and pH 8). The optimal seawater concentration for their growth of the isolates from Wando and Fukuoka was 80 %, while those from Miyagi and Aichi was 50 %. Growth responses of mycelium were high for the bovine serum and layer extract in the isolates from Wando and Fukuoka, and for the bovine serum and yeast extract in the isolates from Miyagi and Aichi. Growth responses for the carbon and nitrogen source of mycelium were the highest for the starch and DL-methionine in the isolate from Wando, for the D-glucose and L-asparatate in the isolates from Fukuoka and Aichi, and for the D-glucose and DL-methionine in the isolate from Miyagi. It was apparent that nutritional requirements of the four isolates differed slightly suggesting that the isolate from Wando was P. porphyrae. Biochemical characteristics of the isolate from Wando were surveyed by comparison with those from Aichi, Miyagi, and Fukuoka by means of electrophoretic patterns of soluble proteins and isozymes. SDS- and native polyacrylamide gel electrophoretic patterns of soluble proteins revealed slight differences among the four isolates. The dissimilarity (DS) in the isozyme banding patterns was highest (0.51) between the isolate of Wando and Miyagi or Fukuoka, and lowest (0.19) between the Miyagi and Aichi. These results indicate that the four isolates are well resemble each other in biochemical characters. Genetic variation of the P. porphyrae isolated from Asan, Mokpo, Pusan, and Wando in Korea, and Aichi, Fukuoka, and Miyagi in Japan was investigated by random amplified polymorphic DNA (RAPD) cluster analysis. RAPD-PCR using arbitrary primers consisting of 10 nucleotide sequences amplified DNA fragments in size range from 0.8 kbp to 5.1 kbp. The amount of polymorphic DNA bands varied from 1 to 6 by each primer. The total 67 RAPD markers were generated from 38 isolates and 33 of them indicated polymorphisms. The dissimilarity coefficients calculated from RAPD banding patterns for the 38 isolates ranged from 0.0010 to 0.6983. The dendrogram generated by unweighted pair-group method using arithmetic averages (UPGMA) indicated that the 38 isolates were classified into three clusters (Groups 1, 2, and 3). The first cluster (Group 1) consisting of 2 isolates from Miyagi was separated from all other isolates by a genetic distance of 0.6983. Groups 2 and 3 containing was branched on genetic distance of 0.3957. These two clusters subdivided into 4 and 3 subclusters, respectively, which were apparently associated with geographic origins of the isolates. Interestingly, the isolates from Asan were close to Japanese isolates rather than Korean isolates on genetic diversity. In addition, the genetic distances (0.0010 - 0.2419) of intra-isolates from Japan were higher than those (0.0010 - 0.1277) from Korea. It is suggest that the genetic diversity of the isolates in Japan associates with geographic distance of isolates which was more widely dispersed. Genetical characteristics of the isolate from Wando were determined by comparison with those from Aichi, Miyagi, and Fukuoka by means of sequences analysis of the internal transcribed spacer (ITS) of the rDNA. The result supports that P. porphyrae isolated from Wando is the same species with those isolated from Fukuoka, Miyagi, and Aichi. The species-specific PCR primers [PP-1 (5'-TGTGTTCTGTGCTCCTCTCG-3') and PP-2 (5'-CCCAAATTGGTGTTGCCTCC-3')] based on the ITS region of the rDNA of P. porphyrae (IFO 30800) and P. marinum (CBS 312.93) were developed to detect zoospores in seawater before the onset of infection in Porphyra cultivation farms. Specificity of these primers were tested by PCR using total DNA prepared from six fungal pathogens [P. aphanidermatum (IFO 7030), P. irregulare (IFO 30346), P. spinosum (IFO 7031) and P. ultimum (IFO 32210)] including two isolates, P. porphyrae and P. marinum as template. PCR using P. porphyrae and P. marinum as templates successfully produced DNA fragment whose molecular size was about 700 bp, corresponding to the expected one in combination of PP-1 and PP-2 primers. Only one zoospore of P. porphyrae was able to detect by PCR using PP-1 and PP-2 primers. These primers developed in this study will be useful to rapid and accurate detection of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried P. yezoensis sheets was also achieved by PCR using the primers PP-1 and PP-2. Digestion of the amplified product by EcoRI produced two fragments of 493 by and 214 bp. Quantities of the amplified product varied according to the time samples (early, middle, and late) which were harvested after the occurrence of red rot disease in Porphyra cultivation farms. This method will be useful for quality management and determination of quality ranking in the Porphyra processing industry. The quantitative analysis of P. porphyrae zoospores was performed by competitive PCR using PP-1 and PP-2 primers and an internal standard plasmid (pPPISC) containing a modified DNA fragment whose. Both ends were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments of approximately 700 and 400 by from the target DNA of P. porphyrae zoospores and from the pPPISC, respectively. To perform quantitative PCR, known quantities of pPPISC were added to reaction mixtures containing the experimental DNAs extracted from zoospores. After a co-amplification reaction, the two different sized PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The number of zoospores was estimated by comparing the fluorescence intensities of the PCR products. The result shows that competitive PCR using P. porphyrae specific primers and competitor pPPISC are useful tools for the quantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms. The red rot disease-resistant isolates, Porphyra yezoensis-AP2 and -AP3, were isolated from single alive cells obtained from an infected lesion of the blades of P. yezoensis. The resistance degree of these isolates for red rot disease was evaluated with environmental conditions such as temperature (10, 15, and 20℃), salinity (20, 26, and 32 %), and pH (7.5, 8.0, and 8.5) that favor infection of Porphyra blades by P. porphyrae. P. yezoensis and P. suborbiculata with low and high partial resistance to red rot disease used as controls. Disease infection occurred in a various environmental conditions regardless of resistance degree of resistant isolates. The disease incidence, severity and expansion in their isolates increased with increasing of temperature and decreasing of salinity and pH. The resistance degree of resistant isolates for red rot disease in a various environmental conditions was very higher than that of P. yezoensis, but not than that of P. suborbiculata, and the resistance degree did not influenced in relation to environmental conditions and in alternative of their generations. The resistant isolates obtained in the present study are evaluated as relatively partial resistant to P. porphyrae, and as having potential and actual value as a means for controlling of red rot disease. To determine the biochemical characterizations of P. yezoensis-AP3 isolate accompanies the resistant reaction of partly to the red rot disease, the contents of pigments, free amino acids, total protein, carbohydrate and sulfate of P. yezoensis-AP3 (resistant) were compared with those of P. yezoensis. The contents of chlorophyll a, phycoerythrin, and phycocyanin were higher in P. yezoensis-AP3 than in P. yezoensis. The analysis of total protein of P. yezoensis-AP3 and P. yezoensis by two-dimensional electrophoresis shown a distinct spots of 135 and 128, respectively. About 89.6 % of the spots of these were common in both P. yezoensis-AP3 and P. yezoensis. The content of total free amino acids in the P. yezoensis-AP3 was less than that of the P. yezoensis. However, L-taurine, L-glutamic acid, L-citrulline, and L-arginine contents were higher in the P. yezoensis-AP3 than those of P. yezoensis. The contents of porphyran and 3,6-anhydro-L-galactose for the P. yezoensis-AP3 and P. yezoensis thallus were 11.9 ± 0.5 %, 2.3 ± 0.1 % and 9.5 ± 0.4 %, 1.6 ± 0.2 %, respectively. The concentrations of galactose and 3,6-anhydro-L-galactose by 4M trifluroacetic acid hydrolysis of the porphyran of P. yezoensis-AP3 and P. yezoensis were 91.8 ± 0.9 %, 8.2 ± 0.2 % and 93.9 ± 1.0 %, 6.1 ± 0.3 %, respectively. The sulfate contents of the porphyran of P. yezoenssis-AP3 and P. yezoensis were 7.7 ± 0.3 % and 10.8 ± 0.5 %, respectively. These results suggest that the biochemical characteristics of P. yezoensis-AP3 differed from those of P. yezoensis, and this difference would be related with the resistant reaction of partly to the red rot disease. This study is the first report of the Korean Pythium isolates for biological, biochemical, and genetical characteristics. Although have a little biological, biochemical, and intraspecies-genetic variation between Pythium isolates isolated from Korea and Japan, the results presented in this study suggest that isolates from Korea seem to be the same P. porphyrae species as Japanese isolates because the rDNA sequences of the ITS region were identical, and that the new approach of countermeasures for prevention and control of red rot disease in Porphyra cultivation is effective.

Sustainable production of shinorine and porphyra-334 by metabolically engineered Saccharomyces cerevisiae

정채림 전남대학교 2023 국내석사

Random Amplified polymorphic DNA Variation of Porphyra dentata in the Southern West Coast of Korea (Bangiales, Rhodophyta) : 한국 남서해안산 잇바디돌김(Porphyra dentata)의 종내 RAPD 변이

崔成劑 여수대학교 1999 국내석사

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique was used to analyze DNA level variation in 12 isolates of Porphyra dentata from the southern west coast of Korea. Of thirty-eight arbitrary primers tested, three single-primers (kc-13, kc-15 and kc-24) and two double-mixed-primers (kc-29 kc-30 and kc-37 kc-38) were able to generate reproducible amplication products. The main band of amplication patterns was shared between populations of P. dentata in question and a population of P. pseudolinearis used as an outgroup, the band sharing indices (BSI) between these two species, however, were much lower and ranged from 0.22 to 0.30, and the genetic distance indices (DI) were much higher and varied from 1.204 to 1.561. The level of BSI was relatively high among nine populations (BSI = 0.48∼1.00) of P. dentata except for among Gatmyeong, Byeongpung and Shinwol populations (BSI = 0.42), and DI was relatively low among nine populations (DI = 0.0733∼0.734) compared with among Galmyeong, Byeongpung and Shinwol populations (DI = 0.868). These results suggest that the RAPD-PCR method provides evidence for identification and estimation of genetic differentiation among populations within a species of the genus Porphyra.

Antioxidant and anti-aging effects of Porphyra-334 in human skin primary cells and human artificial skin tissue models

박소은 을지대학교 대학원 2026 국내석사

Antioxidant and anti-aging effects of Porphyra-334 in human skin primary cells and human artificial skin tissue models Ultraviolet radiation (UV) causes damage on skin barrier and induces skin aging. Porphyra-334 (PPR-334) is one of the spices of mycosorprine-like amino acids (MAAs). MAAs containing PPR-334 are known as biological UV protection ingredients. This study developed a large-scale purification process to extract PPR-334 from Saccharomyces cerevisiae, confirmed the previously identified efficacy of PPR-334 in primary cells and human artificial skin tissue models on UV irradiation, and recognized its independent efficacy. We observed antioxidant effect of PPR-334 using 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid, ABTS). PPR-334 scavenged reactivity oxygen species (ROS) and increased catalase (CAT) gene expression in human epidermal keratinocyte cells (HEKa). In both HEKa cells exposed by UVA/UVB and normal human dermal fibroblast cells (NHDF), PPR-334 suppressed gene expression of matrix metalloproteinase-1 (MMP-1). NHDF cells treated with PPR-334 showed collagen expression increased and proliferation, while advanced glycation end-products (AGEs) production was decreased. It was confirmed that the efficacy in vitro was also reproduced on human artificial skin tissue models. Above all, the mechanism of antioxidant efficacy of PPR-334 in HEKa cells through nuclear factor erythroid 2–related factor 2 (NRF2) and Caspase-9 signals was identified. It was determined that the proliferation efficacy of PPR-334 was due to factors related to the cell cycle. These results demonstrate the anti-aging efficacy of PPR-334 independent of UV irradiation, while enhancing the UV-blocking and antioxidant effects. Thus, we suggest the potential of PPR-334 as a sunscreen agent as well as dual- or multi- functional material.

한국산 양식 김에 발병하는 붉은갯병 원인균의 생활사와 감염 기작

김제윤 공주대학교 대학원 2015 국내석사

The red algal genus Pyropia is the most valuable seaweed worldwide, with a global industry of US $ 1.8 billion. Pythium porphyrae, the agent of red rot disease is responsible for devastating outbreaks in seaweed farms during the initial stage of cultivation causing serious economic loss to the farmers, but little is known about its life cycle. During sexual reproduction, the oogonium containing a single haploid nucleus is produced. Antheridium develops in the vicinity of oogonium and migrate its nucleus to oogonial cell via a fertilization tube to fertilize an oocyte. During asexual reproduction, zoosporangia containing flagellate motile zoospores are produced within 1-2 days after the change of medium. After the release, the asexual zoospores germinate and develop coenocytic hyphae upon arrival on the suitable Pyropia surface. The development of zoosporangia was significantly affected by the presence of its host alga, Pyropia yezoensis. The number of zoosporangia was three times bigger when P. yezoensis was present. The results of membrane filtering and heat-denaturation experiment showed that the causative agent inducing the development of zoosporangium in P. porphyrae was a 10-30 kDa protein released from Pyropia blades. 홍조류 김은 우리나라 연안에서 대표적으로 양식되는 해조류 중에 하나로 해외로의 수출도 증가하는 추세이다. 하지만 이러한 김의 양식은 해마다 지속적인 피해를 입고 있다. 김 양식장에서 심각한 손실을 초래하는 붉은 갯병은 강한 전파력을 가지고 있는 균류성 질병으로 Pythium porphyrae에 의해서 발생한다고 알려져 있지만 그 생활사에 대해서 아직 많은 것이 연구되지 않았다. 기본적으로 붉은 갯병균은 기본적으로 주어진 조건에 따라 유성생식과 무성생식의 다른 생식유형을 보인다. 숙주의 유무에 관계없이 일어나는 유성생식은 수배우체인 Antheridium과 암배우체인 Oogonium의 수정을 통해서 일어나게 된다. 숙주의 존재에 의해서 촉발된다고 생각되는 무성생식은 유주자낭의 형성과 유주자의 방출을 통해 숙주에 대한 효과적인 감염을 보인다. 김 양식장에서 서로 다른 김 엽체로의 붉은 갯병의 전파는 이 유주자를 통해서 일어나게 되며 매우 빠르게 일어나게 된다. 실험실 상에서 이러한 유주자의 방출과 유주자낭의 형성은 숙주인 김의 유무에 따라서 차이를 보였다. 따라서 김에서 분비되는 어떠한 요인이 붉은 갯병균의 유주자낭 형성을 유도한다고 생각되었다. 유주자낭의 형성을 유도하는 물질을 특정하기 위하여 김을 배양했던 물을 크기, 온도, 농축 정도에 따라 측정하였으며 나아가 이 물질을 정제하고 알아내기 위하여 실험을 수행하였다. 농축된 배양물은 10-30 kDa에서 유주자낭의 형성이 촉진되었으며 65 ℃의 열처리에 의해 그 활성이 감소하고 0 ℃조건에서 활성이 유지되는 것으로 보아 10-30 kDa 사이의 단백질이라고 생각되었다. 김 배양물을 농축하여 SDS-PAGE 상에서 확인하였을 때 30 kDa 이하의 단백질이 있었으며 이를 분석하였다. 분석 결과 해당 단백질은 박테리아 유래의 아미노산 바인딩 단백질인 것으로 나타났으며 김의 metagenome data 상에서도 발견되어 김과 밀접한 관련이 있는 박테리아로부터 분비되었다고 생각하였다.

Improved Mycosporine-like Amino Acid Production by Reducing By-products in Engineered Saccharomyces cerevisiae with Optimizing Fermentation Conditions

박병렬 전남대학교 2024 국내석사

Mycosporine-like amino acids (MAAs) is used as sunscreen materials because of having UV- absorbing characteristics. The currently, commercially utilized MAAs production method is extraction from nature substance such as red algae Porphyra umbilicalis, but this extraction method has a problem in that extraction yield is low and the process is complex. To resolve this problem, MAAs production methods from microorganism have been researched. In recent study, MAAs have been produced by engineered Saccharomyces cerevisiae, but production levels are feasible for industrial applications. In this study, industrial polyploid S. erevisiae (JHS200) is selected to host strain for high production levels of MAAs. To construct MAAs production strain, especially, shinorine, MAAs biosynthetic pathway is introduced and TAL1 gene was deleted to accumulate sedoheptulose 7-phosphate (S7P) by Cas9-mediated genome editing system in polyploid S. cerevisiae. The engineered polyploid S. cerevisiae (JdT-M) produced 6.14 mg/g of shinorine contents. To improve the shinorine production instead of ethanol production, the PDC1, PDC6 genes that converts pyruvate to acetaldehyde were deleted and ADH1 gene that converts acetaldehyde to ethanol was deleted in JdTdA1P16-M strain. In addition, NADH oxidase (noxE) was integrated into ADH1 chromosome site of polyploid S. cerevisiae to avoid any cell growth problem caused by cofactor imbalance. The resulting strain (JdTdA1P16-M), shinorine contents was 1.22- fold higher than JdT-M and ethanol yields was 0.79.-fold less than JdT-M strain. For efficient MAAs production, various resources such as carbon source (lignocellulosic hydrolysate) and nitrogen base (CSL, YP medium) were used in fermentation using engineered S. cerevisiae strains. In batch fermentation using lignocellulosic hydrolysate as carbon source, the engineered S. cerevisiae (DXdT-M89-pS, DXdT-M89-aP) produced 293.3 mg/L shinorine and 185.3 mg/L porphyra- 334, respectively. The optimized composition of CSL medium (35 g/L CSL, 5 g/L MgSO4, 0.075 g/L CuSO4 and 0.5 g/L urea) was used in fed-batch fermentation and the DXdT-M89- pS strain produced 1.7 g/L shinorine. Also, fed-batch fermentation using YP medium was performed to obtain high cell density. The DXdT-M89-aP strain was cultured at 250 of OD600 and produced 2.2 g/L of porphyra-334. Thus, the results were expected that the engineered yeast strains could be feasible to industrial application of MMAs production with environmental and economic benefits. Keywords: Mycosporine-like amino acids (MAAs), shinorine, porphyra-334, polyploid Saccharomyces cerevisiae, CSL, lignocellulosic hydrolysate, fed-batch fermentation

Engineering of organic/inorganic nanomaterial-embedded DNA thin films for applications on physical devices

Prathamesh, Chopade Sungkyunkwan university 2019 국내박사

The thought that Deoxyribonucleic acid (DNA) can be applied in modern-day electronics has risen interest amongst researchers. DNA has bases known as nucleotides viz., Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) joined together by hydrogen bonds (stabilized by π-stacking) and are held together by a phosphodiester linkage forming the backbone. This arrangement yields a double helix that can interact with functional nanomaterials (i.e., metal ions, nanoparticles, naturally occurring metabolites, conducting polymers, organic compounds, carbon nanotubes, drug molecules, proteins) chemically as well as physically. Subsequently, this opens up new opportunities to develop biomaterials with various functionalities. Reproducibility, low-yield, incompatibility with physical devices are some of the pressing concerns that bionanotechnology faces in current times thus hampering the use of biomolecules with physical devices. As a result, efforts in resolving such problems have given rise to interdisciplinary research interests merging biology with physics, chemistry. The use of salmon DNA (extracted from salmon fish) can be thought of overcoming such hurdles with ease in process-ability for developing novel biomaterials. Considering these drawbacks, we base our efforts to develop functionalized DNA materials that can be used for various applications. We have used two types of DNA viz., aqueous DNA (artificial double-crossover (DX) nanostructures and salmon DNA) and non-aqueous DNA (CDNA) in our studies. Divalent metal ions (M2+), Mycosporine-like amino, conducting polymers and metal nanoparticles are the types of functional materials used to modify aqueous DNA, while organic light-emitting molecule (OLEM) are the choice of compounds used to modify non-aqueous DNA and its properties. In the first part, Porphyra, a type of Mycosporine-like amino acids (extracted from algae) an UV-absorbing compound was mixed with DNA and its current-voltage (Silicon nanowire/nanoribbon field effect transistors) and electrochemical (cyclic voltammetry) properties under the influence UV light illumination was evaluated. Next, DNA was mixed with a conducting polymer (PEDOT: PSS) and its energy-storage capability/dielectric properties were studied by measuring the capacitance of a metal-insulator-metal device. The interaction between PEDOT:PSS and DNA was observed by UV-Vis and X-ray photoelectron spectroscopy. Next, a method by mixing OLEMs with CDNA (i.e. Cetyltrimethylammonium chloride modified DNA) in a mixed solvent (Chlorobenzene: Butanol) and developed red, green, blue, violet, yellow, orange and white light emitting nanolayers was developed and nanolayers were characterized to understand the parameters influencing the emission process. Further in the second part, the effect of divalent ions (M2+, Cu2+, Ni2+ and Co2+) doping with DNA and its nanostructures was studied. In order to study the structural stability, M2+ doped-2D DX-DNA lattices were constructed with double and triple ion combinations. Here, we measured the optimum concentration of M2+ after which DX-DNA lattices changed to amorphous followed by doping M2+ with DNA for investigating their optical (UV-Vis, PL) and electrical (Current-Voltage) properties. Finally, we mixed plasmonic nanoparticles (PNP, i.e., Au, Ag and Cu) with DNA in order to develop multilayer and multiple-mixed DNA thin films. The stacking order and layer dependent effect on physical processes was investigated by measuring the sample combinations for UV-Vis. absorption, PL, and Raman spectroscopy. The interaction between PNPs and DNA was studied by XPS and XRD measurements while the energy storage capability was estimated by calculating the dielectric constant from the measured capacitance. Subsequently, we provide an insight into using DNA with functional nanomaterials in order for applications in physical devices, sensors, energy storing and optical devices and hope our efforts offer a way in using biomaterials in various fields of nanotechnology.

Effects of Gelidium amansii Extract on Lipid Metabolism and Mitochondrial Dysfunction in High-Fat Diet-Induced Hepatic Steatosis

백서영 세종대학교 대학원 2025 국내석사

Non-alcoholic fatty liver disease (NAFLD), a representative metabolic liver disorder induced by obesity, is characterized by excessive hepatic lipid accumulation, elevated oxidative stress, and impaired mitochondrial function. These metabolic abnormalities are accompanied by increased lipogenesis and suppressed lipolysis in adipose tissue and Liver tissue, leading to disrupted lipid homeostasis, inflammation, and hepatic fibrosis. Gelidium amansii, a red algae traditionally used as a raw material for agar, has recently attracted attention as a marine-derived functional ingredient due to its diverse bioactive components. This study aimed to evaluate the anti-obesity and anti-steatosis effects of Gelidium amansii extract (GAE) and elucidate its underlying molecular mechanisms using high-fat diet (HFD)-induced obese mice and cellular models. GAE administration significantly reduced body weight gain and white adipose tissue hypertrophy, decreased hepatic triglyceride accumulation, and normalized serum AST and ALT levels without nephrotoxicity. Histological analyses revealed that GAE alleviated hepatic lipid droplet accumulation, inflammatory cell infiltration, and hepatocellular ballooning. GAE downregulated SREBP-1c and FAS, while upregulating PPARα and CPT1A, indicating inhibition of lipogenesis and enhancement of fatty acid β-oxidation. Furthermore, GAE reduced lipid peroxidation markers (4- HNE and MDA), increased antioxidant enzyme SOD1, and decreased IL-6 and TNF-α expression, demonstrating its antioxidative and anti-inflammatory effects. In addition, GAE restored mitochondrial biogenesis through activation of the LKB1–AMPK– SIRT1–PGC-1α–NRF1–TFAM pathway and enhanced mitophagy via the PINK1/Parkin/LC3B axis, improving mitochondrial quality control and mitochondrial homeostasis. It also reactivated PI3K/AKT signaling and inhibited the TGFβ1/Smad2/3 pathway, thereby suppressing COL1A1 expression and collagen deposition. Overall, GAE effectively ameliorates obesity-induced NAFLD by regulating lipid metabolism, attenuating oxidative stress and inflammation, restoring mitochondrial function, and inhibiting hepatic fibrosis. LC–MS/MS analysis identified Porphyra-334, a mycosporine-like amino acid (MAA), as a major active compound contributing to these protective effects, highlighting the potential of GAE as a marine-derived functional ingredient for the prevention and management of metabolic liver diseases.

Seaweed Biotechnology: Isolation of Useful Compounds from Seaweed and Strain Improvement : 해조 생물공학: 해조류로부터 유용물질의 분리 및 품종개량

LUYEN QUOC HAI 부경대학교 대학원 2008 국내박사

Seaweed biotechnology is a multidisciplinary subject to produce food, pharmaceuticals, chemicals, and environmental remediation materials from seaweed resources. For centuries, seaweed has been of botanical, industrial and pharmaceutical interest. Compounds extracted from the seaweed are important in pharmaceutical industries. In particular, our survey effort in this area have led to two main themes in studies: (1) screening, isolation and biological analysis of biologically active substances, such as microalgal growth enhancer, antifouling agent and anti-inflammatory (2) tissue culture and mutant selection to overcome supply issues of useful substances. These studies are highly interdisciplinary in nature and draw on diverse methodologies in marine biotechnology. Firstly, in an attempt to identify the microalgal growth enhancer, we isolated the compound levoglucosan from the green seaweed Monostroma nitidum, which enhances cell growth in several microalgae in various culture media. Yield of the compound from seaweed powder was 5 × 10-3% (w/w). At 10 mM concentration, levoglucosan enhanced cell growth and the specific growth rate of all feed microalgal species tested (Chaetoceros gracilis, Chlorella ellipsoidea, Dunaliella salina, Isochrysis galbana, Nannochloris oculata, Navicula incerta, Pavlova lutheri, Tetraselmis suecica) in most culture media by approximately 150%. Cellular fatty acid profiles and cell size differed marginally between cultures with and without levoglucosan. Secondly, during our studies on allelopathic compounds as environmentally friendly antifouling agents led to the isolation of a novel fatty acid, heptadeca-5,8,11-trienoic acid (HDTA: C17:3). HDTA, an odd-numbered carbon atom fatty acid, showed more than 50% lysis at a concentration of 5 μg/mL against the spores of three chlorophyte species, eight rhodophytes, two phaeophytes, and the cells of three phytoplanktons. Lysis activity increased as the number of double bonds and carbon atoms in the fatty acid increased. HDTA showed ten-fold stronger activity with LC50 of 3.1 μg/mL than α-linolenic acid (C18:3). Thirdly, we summarize our study on the anti-inflammatory effects of C. fragile involving isolation and structure determination of active substances. The main active compound was isolated by acetonitrile extraction, fractionating by polarity, silica gel column chromatography, and reverse-phase HPLC to give pure compounds CF-2, CF-6 and CF-7. The structure of compound CF-2 was identified by 1D and 2D of 1H and 13C NMR spectroscopy and GC-MS data and suggested as eicosa-5,8,11,14,17- pentaenoic acid (EPA) (C20:5 ω-3). The structure of compound CF-6 and CF-7 were also studied by the NMR and MS experiments and its structures will be constructed. Purified compounds of EPA, CF-6 and CF-7 were tested for anti-inflammatory activities against the PMA-induced mouse ear inflammation symptoms of edema and erythema. The inhibitory effects of different concentrations of EPA, CF-6 and CF-7 topically applied to mouse ears were dose-dependent. The EPA concentrations producing IC50 were 230 and 462 μg per ear for edema and erythema, respectively. Finally, a strain improvement program was initiated based on mutagenesis with the goal of increasing the content of tryptophan and lysine (essential amino acids) of Porphyra. Three rounds of ultraviolet radiation and selected mutagenic agents (DL-5-methyltryptophan and aminoethyl-L-cysteine) were conducted using monospores of Porphyra as the parent strain. When analyzing the IC100 of selected monospores of the first stage, value of 8 mM and 116 mM, which is higher than original stage, were obtained for DL-5-methyltryptophan and aminoethyl-L-cysteine, respectively. When analyzing the IC100 of selected monospores of the final stage, value of 9.3 mM and 135 mM were obtained for DL-5-methyltryptophan and aminoethyl-L-cysteine, respectively. Our results appear promising that it is possible to increase the volumetric productivity of both essential amino acids using this method