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- 저자 : 임평옥(Pyung-Ok Lim) (검색결과 4 건)
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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.
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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.
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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.
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