Paenibacillus sp. DG-22로부터 열에 안정한 β-xylosidase를 암호화하는 유전자의 클로닝, 염기서열결정 및 발현

이태형,이용억,Lee, Tae-Hyeong,Lee, Yong-Eok Korean Society of Life Science 2007 생명과학회지 Vol.17 No.9

세균인 Paenibacillus sp. DG-22의 유전체 DNA library가 제조되었으며, ${\beta}-xylosidase-$양성 클론이 형광기질인 $4-methylumbelliferyl-{\beta}-D-xylopyranoside$ $({\beta}MUX)$를 사용하여 확인되었다. 이 클론으로부터 재조합 플라스미드가 분리되었고 삽입된 4.3-kb 크기 DNA의 염기서열이 결정되었다. ${beta}-xylosidase$ 유전자는 분자량이 78.710 dal-ton이고 pI가 5.0인 701개의 아미노산을 암호화하는 2,106 염기쌍의 열린해독틀(ORF)로 구성되어있었다. xylA 유전자산물의 추론된 아미노산 서열은 과(family) 52에 속하는 클리코실 가수분해효소로 분류된 ${beta}-xylosidase$들과 상당한 유사성을 가지고 있었다. 이 xylA 유전자에 6개의 히스티딘-꼬리표를 붙이기 위해 pQE60 발현벡터에 다시 클로닝하였다. 재조합 ${beta}-xylosidase$ $(XylA-H_6)$가 열처리와 고정화금속친화성 크로마토그래피(IMAC)에 의해 순수하게 정제되었다. $XylA-H_6$ 효소의 최적 pH와 온도는 각각 pH 5.5-6.0과 $60^{\circ}C$이었다. A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.

스테비아 추출물 발효액에서 분리된 유효 미생물들의 동정 및 항미생물 활성

이태형,박수상,이용억,Lee, Tae-Hyeong,Park, Su-Sang,Lee, Yong-Eok 한국생명과학회 2006 생명과학회지 Vol.16 No.6

스테비아(Stevia rebaudiana Bertoni)는 남미가 원산지인 국화과의 감미식물이다. 스테비아추출물 발효액으로부터 세균 23균주와 효모 10균주를 분리하여 일반적인 분류학적 방법과 분자유전학적 방법으로 동정하였다. 스테비아추출물 발효액에서 분리된 균주들은 5속 10종의 세균과 1종의 효모균에 속하는 것으로 나타났다.16S와 18S rDNA 염기서열 분석에 근거하여 계통수를 작성하였다. 분리균들의 항미생물 활성을 여러 세균과 식물병원성 진균들에 대해 조사하였다. 분리균들 중에서는 Lactobacillus paracasei SB13이 광범위한 세균들에 대해서 강한 항균활성을 나타내었다. 이들 결과는 토양개량을 위한 친환경적 미생물 제제를 개발하는데 도움을 줄 것이다. Stevia rebaudiana Bertoni is a sweet herb of the Asteraceae family originally derived from South America. Twenty three bacterial strains and ten yeast strains were isolated from fermented Stevia extract and identified by general taxonomic methods and molecular genetic method. Isolated strains from fermented Stevia extract include ten species of bacteria which belong to five genus and one species of yeast. Based on 16S and 18S rDNA sequence analysis, phylogenetic trees were constructed. Antimicrobial activity of the isolated strains was examined against various bacteria and plant-pathogenic fungi. Among them, Lactobacillus paracasei SB13 showed strong antibacterial activity towards a wide range of bacteria. These results may be useful to develop environmentally friendly microbial agent for soil improvement.

Purification and Characterization of β-Xylosidase from Paenibacillus sp. DG-22

Tae Hyeong Lee(이태형),Pyung Ok Lim(임평옥),Yong-Eok Lee(이용억) 한국생명과학회 2007 생명과학회지 Vol.17 No.10

Paenibacillus sp. DG-22로부터 세포내 효소인 β-xylosidase가 이온교환, 소수성 상호작용, 겔여과 크래마토그래피에 의해 순수하게 정제되었다. 이 효소의 분자량은 겔여과에 의해서는 156,000으로, SDS-PAGE에 의해서는 80,000으로 측정되었는데 이것은 이 효소가 동일한 두 소단위로 구성되어 있음을 나타낸다. 정제된 효소는 65℃와 pH 5.5에서 최대 활성을 나타내었다. 이 효소는 60℃에서 60분 까지 초기 활성의 80%를 유지하였고 65℃에서 25분의 반감기를 가지고 있었다. 이 효소는 기질로서 pNPX에 매우 특이적이었고 다른 p-nitrophenyl 글리코시드들과 자일란에는 활성을 나타내지 않았다. pNPX에 대한 Km과 Vmax는 각각 0.53 mM과 3.18 U/㎎ 단백질이었다. 이 β-xylosidase는 Ag?, Fe<SUP>2+</SUP>, Hg<SUP>2+</SUP> 및 Zn<SUP>2+</SUP>에 의해 강하게 억제되었으며 DTT에 의해서 약간 활성화되었다. 자일로바이오스, 자일로트라이오스 및 자일로테트라오스로부터의 가수분해 산물은 자일로오스이었다. An intracellular β-xylosidase from Paenibacillus sp. DG-22 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel-filtration chromatography. The molecular weight of the enzyme was measured to be 156,000 by gel filtration and 80,000 by SDS-PAGE, indicating that the enzyme consisted of two identical subunits. The purified enzyme exhibited maximum activity at 65℃ and pH 5.5. It retained 80% of its initial activity up to 60 min at 60℃ and had a half-life of 25 min at 65℃. The enzyme was highly specific for pNPX as the substrate. It showed little or no activity against other p-nitrophenyl glycosides and xylans. The Km and Vmax for pNPX was 0.53 mM and 3.18 U/㎎ protein, respectively. The β-xylosidase was strongly inhibited by Ag?, Fe<SUP>2+</SUP>, Hg<SUP>2+</SUP> and Zn<SUP>2+</SUP> and slightly activated by DTT. The hydrolysis product from xylobiose, xylotriose, and xylotetraose was xylose.

Paenibacillus sp. DG-22에서의 β-xylosidase 생합성 조절

이태형,임평옥,이용억,Lee, Tae-Hyeong,Lim, Pyung-Ok,Lee, Yong-Eok 한국생명과학회 2007 생명과학회지 Vol.17 No.3

효소생산을 최적화하기 위해서 Paenibacillus sp. DG-22에서의 ${\beta}-xylosidase$ 생합성 조절을 연구하였다. Paenibacillus sp. DG-22의 ${\beta}-xylosidase$는 배양액에 존재하는 탄소원에 의해 조절되는 것으로 관찰되었다. ${\beta}-Xylosidase$의 합성은 xylan과 methyl ${\beta}-D-xylopyranoside$ (${\beta}MeXyl$)에 의해 유도되었으나 쉽게 대사되는 단당류에 의해서는 약간 억제되었다. ${\beta}MeXyl$가 ${\beta}-xylosidase$의 유도를 위한 최적의 기질임을 확인하였고 가장 효과적인 유도는 10 mg/ml의 농도에서 얻어졌다. ${\beta}-Xylosidase$의 생산은 세포의 생장과 연관된 양상을 나타내었으며, 대수기 말에 최대양이 형성되었다. Glucose와 xylose가 존재하면 ${\beta}-xylosidase$의 활성 수준이 감소하는 것으로 보아 이 효소의 생합성은 catabolite repression을 받는것으로 보인다. SDS-PAGE와 활성염색 기술을 이용하여 ${\beta}Mexyl$가 이 효소의 생합성을 유도하며 약 80 kDa 크기의 하나의 ${\beta}-xylosidase$가 존재함을 알 수 있었다. Regulation of ${\beta}-xylosidase$ synthesis in Paenibacillus sp. DC-22 was studied to optimize the enzyme production. ${\beta}-Xylosidase$ synthesis of the Paenibacillus sp. DG-22 was observed to be regulated by carbon sources present in culture media. The synthesis of ${\beta}-xylosidase$ was induced by xylan and methyl ${\beta}-D-xylopyranoside$ (${\beta}MeXyl$) but slightly repressed by readily metabolizable monosaccharides. ${\beta}MeXyl$ was found to be the best substrate for the induction of ${\beta}$-xylosidase and the most effective induction was obtained at a concentration of 10 mg/ml. ${\beta}-Xylosidase$ production showed a cell growth associated profile with the maximum amount formed during the late exponential phase of growth. The presence of glucose and xylose decreased the level of ${\beta}-xylosidase$ activity indicating that its production was subjected to a form of carbon catabolite repression. SDS-PAGE and zymogram techniques demonstrated the induction by ${\beta}MeXyl$ and revealed the presence of one ${\beta}-xylosidase$ of approximately 80 kDa.

원가지수를 이용한 고속철도 설비의 개략적 투자비 추정기법

이영주(Young-Joo Lee),이용억(Yong-Eok Lee),장성용(Seong-Yong Jang) 한국철도학회 2011 한국철도학회논문집 Vol.14 No.2

다양한 스트레스에 유도되는 들잔디 ZjWRKY3, ZjWRKY7의 분석

김우남,송인자,강홍규,선현진,양대화,이용억,권용익,이효연,Kim, Woo-Nam,Song, In-Ja,Kang, Hong-Gyu,Sun, Hyeon-Jin,Yang, Dae-Hwa,Lee, Yong-Eok,Kwon, Yong-Ik,Lee, Hyo-Yeon 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.3

식용작물, 사료, 잔디를 포함하는 모든 작물은 건조, 염, 저온, 고온 등의 여러 가지 환경스트레스의 영향을 빈번히 받기 때문에 작물의 생산성이 떨어지게 된다. 식물은 환경스트레스 상황에서 스스로 벗어날 수 없다. 따라서 식물은 환경 스트레스를 극복하는 방향으로 진화하였다. ARF, ABI3, NAC, HSF, WRKY 같은 환경 스트레스에 반응하는 유전자들이 식물에서 보고되었다. 이 유전자들은 환경스트레스에 반응하는 전사인자로, 식물의 스트레스반응 경로에 연관되어 있다. OsWRKY76의 경우에는 저온 및 병원균에 대한 내성을 증가시켰고, AtWRKY28 의 경우 여러 가지 환경스트레스에 관련이 있는 것으로 보고되었다. 들잔디는 정원이나 골프코스에서 가장 흔하게 사용되는 잔디이다. 하지만 들잔디에서는 아직 WRKY 유전자가 알려지지 않았다. 본 연구에서는 들잔디로부터 1개의 WRKY domain을 포함하는 ZjWRKY3, ZjWRKY7 를 분리하였다. ZjWRKY3과 ZjWRKY7은 저온, 건조, 염 스트레스에 발현이 증가하였다. 들잔디의 갈색퍼짐병을 일으키는 R. solani의 감염이 ZjWRKY3과 ZjWRKY7의 발현을 증가시켰다. 또한 ZjWRKY3, ZjWRKY7이 Zjchi 유전자 promoter의 W-box에 결합하여 전사를 조절한다는 사실을 확인 하였다. 따라서 ZjWRKY3, ZjWRKY7 유전자는 전사인자로서 환경스트레스 및 병원균 관련 하위 유전자들을 조절할 것으로 예상된다. Many crops including cereals, tuber crops, feeds, and turf grasses are often damaged by various environmental stresses such as drought, salt, cold, and high temperature, causing the reduction of their productivity. Plants are sessile and cannot escape from environmental stresses. Thus, plants evolve in the direction of overcoming the environmental stresses. Some plant genes such as ARF, ABI3, NAC, HSF, and WRKY are known to respond to environmental stresses as they transcriptionally regulate the stress response pathways. For example, the OsWRKY76 gene contributes to the enhanced resistance to low temperatures and pathogenic infections. The AtWRKY28 also plays a role in environmental stresses. Zoysiagrass (Zoysia japonica Steud.) is popularly grown for gardens and golf courses. However, the function of the WRKY gene, another environmental stress-related gene, is not known in zoysiagrass. In this study, the ZjWRKY3 and ZjWRKY7 genes with one shared WRKY domain have been isolated in zoysiagrass. The expression of these genes increased in response to low temperature, drought, and salt stresses. Furthermore, the infection of the brown patch-causing Rhozoctonia solani induced the expression of ZjWRKY3 and ZjWRKY7. The corresponding proteins bind to the W-box of the Zjchi promoter, possibly regulating their transcriptions. The researchers suggest that the ZjWRKY3 and ZjWRKY7 genes transcriptionally regulate abiotic and biotic stress related downstream genes.

GMO 격리포장에서의 유전자변형 들잔디로부터 토착미생물로의 수평유전자전달 평가

배태웅,이효연,류기현,이태형,임평옥,윤필용,박신영,류기중,송필순,이용억,Bae, Tae-Wung,Lee, Hyo-Yeon,Ryu, Ki-Hyun,Lee, Tae-Hyeong,Lim, Pyung-Ok,Yoon, Pill-Yong,Park, Sin-Young,Riu, Key-Zung,Song, Pill-Soon,Lee, Yong-Eok 한국식물생명공학회 2007 식물생명공학회지 Vol.34 No.1

The release of genetically modified organisms ($GMO_{s}$) into the environment has the potential risks regarding the possibility of gene transfer from $GMO_{s}$ to natural organisms and this needs to be evaluated. This study was conducted to monitor the possible horizontal gene transfer from herbicide-resistant zoysiagrass (Zoysia japonica Steud.) to indigenous microorganisms. We have first examined the effect of field-released GM zoysiagrass on the microbial flora in the gut of locust (Locusts mlgratoria). The microbial flora was analyzed through determining the 165 rDHA sequences of microorganisms. The comparison of the microbial flora in the gut of locusts that were captured at the field of GM zoysiagrass and of wild-type revealed that there is no noticeable difference between these two groups. This result indicates that the GM zoysiagrass does not have negative impact on microbial flora in the gut of locust. We then investigated whether the horizontal gene transfer occurred from GM zoysiagrass to microbes in soil, rhizosphere and faecal pellets from locusts by utilizing molecular tools such as Southern hybridization and polymerase chain reaction (PCR). When the total DNAs isolated from microbes in GM zoysiagrass and in wild-type zoysiagrass fields were hybridized with probes for bar or hpt gene, no hybridization signal was detected from both field isolates, while the probes were hybridized with DNA from the positive control. Absence of these genes in the FNAs of soil microorganisms as well as microbes in the gut of locust was further confirmed by PCR. Taken together, our data showed that horizontal gene transfer did not occur in this system. These results further indicate that frequencies of transfer of engineered plant DNA to bacteria are likely to be negligible.

Zea Mays L. 의 特殊成分과 澱粉의 X線 回析度

李容億,曺貞淳,申東旿 明知大學校 自然科學硏究所 1984 자연과학논문집 Vol.2 No.-

韓國産 메옥수수, 찰옥수수, 爆裂種에 대한 一般成分,無機質을 定量分析하고 또 胚芽油脂를 油出定量하고 그油脂의 物理,化學的 性質을 判斷하였다. 한편 各各의 녹말은 生 및 糊化시켜서 그의 X-線 회절도를 作成 比較 檢討하였다. 1.一般成分은 各各 Table 1에 表示한 바와 같다. 2.無機質成分은 各各 Table Ⅲ에 表示한 바와 같다. 3. 油脂의 物理化學的 性質 가.油脂의 色度差는 Table V에 表示한 바와 같다. 나.其他 粗脂肪, 比重, 굴절율, 요오드값, 산값, 검화값, 과산화물값, 불검화물값등은 Table Ⅳ에 表示한 바와 같다. 다.녹말에 대한 X-線 회절도의 試驗結果는 Table Ⅶ에 表示된 바와 같이 各 種間의 差異가 없었다. The general constituents and the mineral contents of White Dent Corn, Waxy Corn and Corn, all harvested in Korea, were first examined, and then the quality of each corn was examined through the test for the Physiochemical property of fat extracted from the germ of each corn. The result of qualitative comparison of starch, made by means of X-Ray Powder diffraction for the raw and the boiled starch of each corn, especially to find out any differences among the White Dent, the Waxy and the Pop Corn based on the point of starch, has reached the following conclusions; (1) The general consitutents and each content of the White Dent Corn are ; protein 10.3%, fiber 1.89%, Carbohydrate 69.93%, starch 64.5%, fat 5.36% and ash 1.65%. From it, it could be pointe out tthat the White Dent Corn contained more protein and ash than the others. Compared with the others, th Waxy Corn contained more Carbohydrate , showing its constitutents and contents as follows; protein 9.74f%, fiber 2.08%, Carbohydrate 71.04%, starch 65.5% fat 4.87% and ash 1.64%. As to the Pop Corn, however, it showed a relatively higher content of fat than the rest. Its constituents and each content were as follows ;protin 9.67%, fiber 2.19%, Carbohy-drate 69.39%, starch 64.0%, fat 6.62% and ash 1.64%. (2)Mineral Constituents; Regarding the mineral constiruents contained by the three kinds of corn which were sampled here for this study, the following results were noted; In case of the White Dent Corn, the content of P and Si were high, but the contents of Al and Zn were low. As for the Waxy Corn, the contents of Cu and Si were found less but the content of Al was found a little . Regarding the Pop Corn, however, the contents of its mineral constituents were higher in every respect than the other kinds of corn. Among them, especially , the contents of Si, Cu, Al, and Zn were a little higher. (3)Physiochemical Quality of Fat: The fat sampled here for the test was good enough with the one extracted through the method of pressure, oil pressing method, without any special refining process of high level.It was noted that the specific gravity of fat was 0.911 in the White Dent Corn, and 0.909 in the Pop Corn, which showed approximately 0.003 lower than the standard value specified b6 the Ministry of Health and Social Affairs. In the Waxy Corn, however, its value was almost as much as the specified level. As to the ditribution status of fat constituent, the Pop Corn contained the largest portion of all, showing that the Pop Corn 6.62%, the White Dent Corn 5.36% and the Waxy Corn 4.87% respectively. The iodine value of the sampled fat was 126 in the Waxy Corn, which belonged to the highest one of three kinds of Corn. And acid value of all the corn was generally a little higher than the standard required by the Ministry of Health and Social Affairs, since the fat was extracted from germ by means of ether and then was dried in the temperature of 90℃. In this case it was also noted that the Waxy Corn contained as much as the standard level. The White Dent Corn and the Pop Corn, however, wasd not up to the standard value. (4) Result of test for Starch X-Ray Power Diffraction: Starch, as its crystalline structure differs according to its kind, can be classified into three patterns such as A, B, and C. The raw starch (β-starch) sampled here belonged to A pattern and all the bolied starch (α-starch) to B pattern. When all the above were compared, all the sampled corn, White Dent, Waxy and Pop, did not show much difference in themselves.

韓國産 高酸價 米糠油의 脫色에 關한 硏究

李容億,金恩卿,盧長淑 명지대학교 1983 明大論文集 Vol.14 No.-

The rice bran produced in the process of rice milling is only ingredient of edible oil and fat in Korea. If and when the rice bran is left negligently for a long time, a good quality of edible oil cannot be obtained. It is not only because the loss of the rice bran is resulted in the process of refining from starch degeneration, discoloration, and generation of free fatty acid or unsaponifiable matter but because the decolorization processing becomes much more difficult. In this connection, the experiment on the decolorization of the high acid value rice bran oil was made with clay. Although remarkable results were not yielded, continuous research is needed. If successful, it will contribute to the development of domestic medical ingredients with consequent extraction and refining of orizanol or inositol etc.

고사리의 Shikimic acid 分析과 動物實驗(2) : Analysis of shikimic acid from bracken fern and the effect of bracken fern to mice

李容億,曺貞淳 명지대학교 1983 明大論文集 Vol.14 No.-

Study on the poisonous component of bracken fern. The investigator carried out the experiments for the purpose of finding out the effect of the bracken fern(BF) to mice. In this paper results about histological, nutritional and microbial researches of the bracken fern are reported. All experiments were carried out with bracken fern from Jae judo in 1979. First, those substances which were dried after boiling for 20 min dipping in water for 21hr. and extracted them from the water. After that, they were analyzed by AOAC method and spectrography method. The results showed that the backen fern of Jaejudo contains more fat and less protein, compared with that of Caernarvonshire and Sooweon. In respect to the minerals, K and Na are contained at a large amount and Si, Ca, Al, Fe, Ti, As and Mg at a relatively small amount. Evans, I.A. and Osman, M.A. in 1974 reported that the substance which caused carcinogenic effect was organic substance. The above team especially carried out the experiments to find out shikimic acid(3α, 4α, 5β-trihydroxy-1-cyclohexene-1-carboxylic acid). The edible portion of bracken fern was analyzed to find out shikimic acid by following methods. Sample A is drying fresh bracken fern, sample B is drying after boiling for 20 min and dipping in water for 21 hrs and sample C is drying after boiling for 10 min. Sample A showed 0.18 wt% of shikimic acid and sample of B,C showed value lessthan 0.001 wt% by the metohd of gas-liquid chromatography. To know the effect of shikimic acid to enteric bacteria, feces from each mouse weretested. So their enumerated bacteria were identified by API 20E kit and compared with the control mouse feces. All these identifications were carried out by means of selective media and API 20E kit. The identified bacteria were Escherichia coli, Enterobacter agglomerance, Enterobacter cloacoe, Citrobacter diverus-Levinea and Serratia rubidaea. Escherichia coli was dominant in control mice but it was reduces greatly in the treated mice. And in comparision with extracted components, pure shikimic acid showed the inhibitory effect on Escherichia coli. To find the effect when bracken fern were fed to mice, total 40 mice were divided into eight groups and feeding methods were pouring of BF powder and standard diet. In the former method, there was an increase in body weight of mice, but in the later method, not as former do. As a result of taking much fern by infusion method caused the result of not only decrease of weight but also much change around their months. That is, there was a phenomena like loss of bristle and the reduction of skin fur. In spite of the reduction of appetite, there was no change in the properties like degree of hardness, colour, smallness of feces in comparision with those of control. After above experiment, the histological changes were searched after six months feeding, the paraffin section of the tissues around around mouth and intestine were mainly stained by hematoxylen-eosin. The change of the skin tissues were the infiltration and the increase of the squamous-cells. The disapperance of the hair sheath was caused by the necrosis of the hair root, the infiltration of its cell, expention and hyperaemia of the blood vessels. And the symptoms like the edema and the tissue infiltration were developed in the hair root around the mouth. The increase of the giant cells in the spleen was rapid in contrast to controls and the differention of the nucleus was high. In the tissue of lungs, there was increase of lymphoid cells around the blood vessel and the small bronchus. Cell infiltrations were observed in blood vessel and glomerulus area in kidney. Noticeable facts were the enlargement of blood capilary and the hyperaemia of the tissues in lung, kidney, liver and stomach. It was generally accepted that the development of cancer like edema and carcinoma appeared in long term experiment i.e. at least six months, so the results of this experiment could be understood as an early symptom of above developments. And it might be thought as an important result that in skin tissue there were infiltration of squamous-cell and regressive changes around their mouths Consequently the appearance of phenomena like alopecia of elastic hair, skin and development of alopecia spots assumed to be the important results.