Cloning, Expression, and Characterization of a Novel GH-16 β-Agarase from Agarivorans sp. JA-1

Myong Je Jeon(전명제),A-Ram Kim(김아람),Dong-Geun Lee(이동근),Sang-Hyeon Lee(이상현) 한국생명과학회 2012 생명과학회지 Vol.22 No.11

이전 연구에서 저자들이 Glycoside hydrolase family 50 (GH-50)과 GH-118 β-agarase들의 발현과 특성을 보고한 Agarivorans sp. JA-1 균주로부터 신규의 GH-16 β-agarase를 보고하고자 한다. 본 유전자는 1,362 염기쌍으로 구성되어 있으며, 453 아미노산 잔기로 구성된 49,830 Da의 단백질을 암호화한다. 본 효소는 Pseudoalteromonas sp. CY24 유래의 GH-16 β-agarase와 98%의 염기서열 상동성과 99%의 아미노산서열 상동성을 나타냈다. 신호서열을 제외한 429 아미노산으로 구성된 성숙단백질에 해당하는 유전자를 E. coli BL21 (DE3) 세포에서 재조합 발현 시킨 후, 친화성 크로마토그래피로 효소를 정제하였다. 정제된 효소는 40℃와 pH 5.0에서 67.6 U/mg의 최적 활성을 보였다. 아가로스를 기질로 한 효소분해산물의 박막크로마토그래피 분석결과, neoagarohexaose와 neoagarotetraose가 주산물로 생산되는 것을 알 수 있었다. 본 효소는 기능성 한천올리고당의 산업적 생산에 활용 가능할 것으로 기대된다. Authors report the glycoside hydrolase (GH) family 16 β-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 β-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 β-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at 40°C and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

Whitening Effect of Hizikia fusiformis Ethanol Extract and Its Fractions

Myong Je Jeon(전명제),Mihyang Kim(김미향),Hye-Ji Jang(장혜지),Seung-Woo Lee(이승우),Jae-Hoon Kim(김재훈),Hyung-Suk Kim(김형석),Sang-Hyeon Lee(이상현) 한국생명과학회 2012 생명과학회지 Vol.22 No.7

멜라닌 생합성은 tyrosinase의 효소적 산화반응에 의해 일어난다. 톳(Hizikia fusiformis)의 미백효과를 검증하기 위하여 메탄올, 헥산, 부탄올 및 물을 용매로 이용한 분획과정을 통해 톳 에탄올 추출물로부터 분획물을 제조하였다. 에탄올 추출물과 분획물들을 대상으로 B16F10 흑색종 세포에서 tyrosinase 활성 저해효과 및 멜라닌 생합성 억제효과를 평가하였다. 생쥐의 에탄올 추출물 및 수층 분획물은 미백효과를 나타냈지만, 세포독성은 나타내지 않았다. 에탄올 추출물은 시료들 중에서 가장 높은 미백활성을 보였다. 에탄올 추출물은 100 μg/ml의 농도에서 arbutin 10 μg/ml의 활성보다 높은 저해활성을 보였으나, kojic acid 10 μg/ml의 활성보다는 낮은 저해 활성을 보였다. 또한, 메탄올, 헥산, 부탄올 및 수층 분획물은 100 μg/ml의 농도에서 arbutin 10 μg/ml의 활성과 유사한 저해활성을 보였다. 항산화 활성은 L-ascorbic acid를 양성 대조군으로 하여 수치를 비교하여 나타냈다. 에탄올 추출물 및 수층 분획물의 DPPH 라디칼 소거활성은 다른 시료들보다 비교적 높게 나타났다. 또한, 에탄올 추출물및 수층 분획물은 500 μg/ml의 농도에서 LPS에 의해 유도된 iNOS 발현을 각각 82와 80% 감소시켰다. 이러한 결과들로 톳의 에탄올 추출물과 수층 분획물은 미백효과 및 피부 보호효과를 가지는 화장품 소재로 이용 가능할 것으로 기대된다. Melanin synthesis is catalyzed by tyrosinase. To investigate the whitening effect of Hizikia fusiformis, fractions from ethanol extract of H. fusiformis were prepared by a systematic fractionation procedure with solvents such as methanol, hexane, butanol, and H2O. The ethanol extract and its fractions were then subjected to evaluate the inhibitory effects on the tyrosinase activity and melanin synthesis in murine B16F10 melanoma cells. The ethanol extract and aqueous fraction exhibited a whitening effect with no cytotoxicity. The ethanol extract showed the highest whitening effect among the samples. The inhibitory effect of 100 μg/ml of ethanol extract was higher than that of 10 μg/ml of arbutin, but it was lower than that of 10 μg/ml of kojic acid. Furthermore, the inhibitory effects of 100 μg/ml of methanol, hexane, butanol, and aqueous fractions were similar to those of 10 μg/ml of arbutin. The antioxidant activities were examined by comparing the results with that of ascorbic acid as a positive control. The ethanol extract and aqueous fraction showed relatively higher DPPH radical-scavenging activities compared with the other samples. Furthermore, 500 μg/ml of ethanol extract and aqueous fraction diminished LPS-induced iNOS expression to 82 and 80%, respectively. These results suggest that ethanol extract and aqueous fraction of H. fusiformis could be used as cosmetic ingredients for whitening and skin protection effects.

전통막걸리에서 분리한 효모균주를 이용한 막걸리 발효과정 중의 물성 및 미생물 군집의 변화

전명제(Myong Je Jeon),장민경(Min Kyung Jang),이솔지(Sol Jee Lee),박성환(Sung Hwan Park),김미향(Mihyang Kim),손재학(Jae Hak Sohn),이한승(Han-Seung Lee),이동근(Dong-Geun Lee),이상현(Sang-Hyeon Lee) 한국생명과학회 2013 생명과학회지 Vol.23 No.6

전통막걸리들에서 순수분리한 효모균주 5종을 이용하여 각각 막걸리를 제조하면서 발효 시일에 따른 막걸리의 물성 변화와 PCR-DGGE를 이용하여 미생물 군집구조 변화를 조사하였다. 막걸리의 pH는 0일째 pH 6 에서 발효 2일째 pH 3 정도의 큰 감소를 보였고 이후 큰 변화가 없었다. 발효일별로는 차이를 보였지만(ANOVA, p<0.001) 균주별로는 차이가 없었다(p=0.60). 산도는 0.19~1.04% 범위였으며 발효 2일째부터 증가하였고 발효일자별(p<0.001) 및 균주별(p=0.006) 모두 차이가 있었다. C 균주의 산도 증가가 가장 컸으며 S 균주의 산도 증가가 가장 적었다. 아미노태 질소는 S 균주가 8일째 0.442%로 높은 값을 보였지만 다른 균주들은 모두 0.150% 이하를 나타내었다. 발효일자별(p=0.4558) 및 균주별(p=0.3513) 모두 큰 차이를 보이지 않았다. 당도는 빵효모균주인 C가 발효 4일 째부터 다른 균주들에 비해 높았고 발효일자별(p<0.0001) 및 균주별(p=0.007) 모두 유의미한 차이를 보였다. 알코올 함량은 모든 효모균주에서 발효 2일째 10%의 급격한 증가를 보였으며 그 이후에 큰 차이가 없었다. 발효일자별(p<0.0001)로는 차이를 보였지만 균주별로는 차이는 없는 것으로 나타났다(p=0.1464). DGGE 밴드로 검출된 우점세균은 산성물질을 생산하며 기능성을 보이는 Lactobacillus fermentum와 Pediococcus pentosaceus였고, 우점진균은 Saccharomyces cerevisiae였는데 이는 효모균주 첨가에 의한 결과로 판단된다. Property changes and bacterial characterizations by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were investigated during the fermentation of Makgeollies by 5 isolated yeast strains. Changes of pH were large between day 0 (pH 6) and day 2 (pH 3) and showed less variation after then. ANOVA analyses revealed that pHs were statistically different with fermentation times (p<0.001), while strains (p=0.60) did not. Acidities were changed from 0.19 to 1.04% and showed rather high increase from day 2, and fermentation times (p<0.001) and strains (p=0.006) represented statistical differences. All strains showed less than 0.150% at amino-type nitrogen contents except S strain showed 0.442% at day 8, and there were no statistical differences with fermentation times (p=0.4558) and strains (p=0.3513). Saccharinities of C strain were higher from day 4, and fermentation times (p<0.0001) and strains (p=0.007) showed statistical differences. Large variation of alcohol concentrations (%) were observed between day 0 (0%) and day 2 (10%) and showed less variation after day 2, and there was no statistical difference with strains. Dominant prokaryotes were Lactobacillus fermentum and Pediococcus pentosaceus, which producing acids and functional materials. Dominant eukaryote was Saccharomyces cerevisiae, which might be resulted from addition of yeasts.

전통막걸리로부터 분리된 효모균주를 이용해 제조된 막걸리의 물성 분석

전명제(Myong Je Jeon),김미향(Mihyang Kim),이동근(Dong-Gun Lee),황현정(Hyun-Jung Hwang),강민숙(Min Suk Kang),김보경(Bo Kyung Kim),이승우(Seung Woo Lee),장혜지(HyeJi Jang),이상현(Sang-Hyeon Lee) 한국생물공학회 2012 KSBB Journal Vol.27 No.1

Five yeast strains were isolated from traditional Makgeollies, Makgeollies were made by isolated yeasts after cultivation, and then property changes of Makgeollies were analyzed according to yeasts, storage temperatures and storage periods. Average pHs were shown to be 3.22~3.88 and statistically changed according to yeasts used, storage temperatures and storage periods. Total acidities were statistically changed according to storage periods. Amino-type nitrogen contents were in the ranges of 0.009~0.245% and statistically changed according to storage temperatures especially at 18 and 25℃ for 15 days. Average alcohol concentrations were in the ranges of 7.5~18.5% and reduced until 10 days and increased for 15 days according to yeasts used and storage periods. Consequently, Makgeollies, made by isolated yeast strains originated from traditional Makgeollies, revealed that alcohol concentrations and amino-type nitrogen contents were changed but pHs and total acidities were not dramatically changed according to yeasts used. It suggests that development of various Makgeollies would be possible using isolated yeast strains in this study, and optimal storage condition of ready-made Makgeollies to maintain its original property turned out to be at 4℃ for 5 days. Especially, Makgeolli made by F strain showed the best quality on its property, therefore Makgeolli which maintains its property stably until 10 days when stored at 4℃ could be made using this strain.

초임계 이산화탄소를 이용한 피트모스 추출물의 정제 및 생리활성 검증

김미향(Mihyang Kim),전명제(Myong Je Jeon),박미라(Mira Park),이승우(Seung Woo Lee),황현정(Hyun-Jung Hwang),장해지(HyeJi Jang),강민숙(Min Suk Kang),김보경(Bo Kyung Kim),최승태(Seung Tae Choi),박미연(Mi Yeon Park),이상현(Sang-Hyeon Lee) 한국생물공학회 2011 KSBB Journal Vol.26 No.5

In this study, the canadian peat moss extract was purified by a supercritical CO₂ using three different conditions and assessed its biological activities. Peat moss was extracted by acid-alkaline extraction method (sample 1) and purified by a supercritical CO₂ at 40℃ under pressure of 100 bar (sample 2), 120 bar (sample 3) or 150 bar (sample 4). We evaluated the antioxidant activities of the samples by 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging, Fe<SUP>2+</SUP>/ ascorbate (FTC) and 2-thiobarbituric acid (TBA) methods. The antioxidant activities were examined by comparing the results with that of ascorbic acid as a positive control. Sample 3 showed relatively higher DPPH radical-scavenging activities than other samples. The antioxidant activity by FIC method exhibited similar results as the DPPH radicalscavenging activities. On the other hand, sample 2 showed higher antioxidant activity measured by TBA method of all. The whitening effects of the samples were examined using mushroom tyrosinase and B16F10 melanoma cells. Sample 3 exhibited overall significant whitening effects, however, other samples showed relatively lower effects. These results suggest that the peat moss extract purified by a supercritical CO₂ could be used as a cosmetic ingredient for the anti-aging and whitening effects.

In vitro 검출 시스템을 이용한 한약재 추출물들의 에스트로겐 유사활성 검증

박성환(Seong Hwan Park),전명제(Myong Je Jeon),장민경(Min Kyung Jang),이솔지(Sol Jee Lee),김보경(Bo Kyung Kim),전명정(Myeong Jeong Jeon),김서연(Seo Yeon Kim),김미향(Mihyang Kim),이동근(Dong Geun Lee),이태호(Tae Ho Lee),남재섭(Jae Sub 한의병리학회 2013 동의생리병리학회지 Vol.27 No.6

해양성 Flammeovirga sp. mbrc-1 균주의 분리 및 한천분해기능의 특성조사

장혜지(Hye Ji Jang),이동근(Dong-Geun Lee),이승우(Seungwoo Lee),전명제(Myong Je Jeon),천원주(Won Ju Chun),권개경(Kae Kyoung Kwon),이희순(Hee-Soon Lee),이상현(Sang-Hyeon Lee) 한국생물공학회 2011 KSBB Journal Vol.26 No.6

A novel agar-degrading bacterium mbrc-1 was isolated from seashore of Kyungpo at Gangwon province and cultured in marine broth 2216 medium. Isolated bacterium mbrc-1 was named as Flammeovirga sp. mbrc-1 based on the 16S rDNA sequence. Its agarase showed maximum activity of 923 units/L at pH 7.0 and 45℃ and sustained 90% remaining activity after exposed to 45℃ for 2 hours. The enzyme hydrolyzed agarose to yield neoagarohexaose (18.5%), neoagarotetraose (38%) and neoagarobiose (43.5%), indicating that the enzyme is β-agarase. Thus, isolated bacterium and its β-agarase would be useful for the industrial production of neoagarotetraose and neoagarobiose.

iNOS 발현 검출을 위한 in vitro 시스템의 확립 및 적송잎 추출물에 의한 저해효과 검증

김남영(Nam-Young Kim),장혜지(HyeJi Jang),이동근(Dong-Geun Lee),장민경(Min-Kyung Jang),이승우(Seung Woo Lee),전명제(Myong Je Jeon),김미향(Mihyang Kim),김성구(Sung Gu Kim),이상현(Sang-Hyeon Lee) 한국생물공학회 2011 KSBB Journal Vol.26 No.2

This study was aimed to verify suppressive effect of pine-needle extract on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression. In order to evaluate suppressive effect on iNOS expression, RAW 264.7 cells were stably transfected using an iNOS promoterluciferase reporter plasmid yielding RAW 264.7/pGL2-NeomiNOS_ pro11 cells. Established in vitro detection system revealed to diminish LPS-induced iNOS expression by 0.1~500 μg/mL of saponin at the concentration-dependant manner. Pine needle extract also diminished LPS-induced iNOS expression to 92 and 88% at 500 and 50 μg/mL, respectively. These results suggest that the in vitro detection system developed here could be useful for the verification of suppressive materials on iNOS expression and pine needle extract could be used for the development of functional foods.