CRP* 의존성 maltose 대사 촉진 유전자 sfs4의 클로닝 및 염기배열 결정

최용락,정희태,조무제,정수열 東亞大學校附設遺傳工學硏究所 1996 遺傳工學硏究 Vol.- No.3

CRP (cAMP receptor protein)은 cAMP와 결합하여 cAMP-CRP 복합체를 형성하여 전사조절의 조절인자로서 작용한다. crp 유전자에 변이를 도입하여 cAMP의 비존재 상태에서 cAMP-CRP와 비슷한 기능을 가진 crp* 유전자가 도입된 대장균 MK2001 (crp*¹, cya::km)을 숙주로 사용하여 cAMP 혹은 cGMP의 비존재하에서도 mal 유전자의 발현을 촉진시키는 유전자 sfs (sugar fermentation stimulation) 수 종을 클로닝하였다. 본 실험에서는 이미 밝혀진 nlp (Ner like protein) 유전자와 같이, sfs의 새로운 유전자를 탐색하여, 그 중 sfs4의 2126bp 전 염기배열을 결정하고, 잠정적인 sfs4의 promoter 영역에는 CRP 단백질과의 결합 DNA 공통 염기배열(5' AAT TGTGA ACACCA TCACC CGT 3')이 존재함을 확인했다. lacZ 융합 유전자를 작성하여 TP2010R1과 MK2001의 균주에서 cAMP를 첨가할 경우 각각 2.3배, 1.8배의 β-galactosidase 활성이 증가 하는 것으로 보아 sfs4는 cAMP-CRP에 의해 발현 조절을 받는 것으로 나타났다 In Escherichia coli, CRP forms a complex with cAMP and acts as a transcriptional regulator of many genes, including sugar metabolism operons. The E. coli MK2001, which is introduced the altered crp*¹, is functional in the expression of lac, ara and man, in the absence of cAMP. However, the expression of mal gene is fully activated by the addition of cAMP or cGMP. The object of the study is cloning of the sfs (sugar fermentation stimulation) genes, which was involved in regulation of mal gene expression with the altered crp*¹gene, and structural analysis and characterization of the genes at the molecular level. We have cloned 5 different E. coli genes which stimulate the maltose metabolism in a crp*¹, cya::km (MK2001) background. Newly identified genes were designated as sfs. One of the sfs genes (pPC1), located at the 53.2 min map position in the E. coli chromosome, was further analyzed. Expression of the genes, which is involved in maltose metabolism, malQ (amylomaltase), was increased to 5.8-fold in the presence of a plasmid, pAP5, containing the subcloned sfs4 gene. The nucleotide sequence of a common 2,126bp segment of the pPCM1 was determined and two open reading frames (ORF1 and ORF2) were detected. The ORF1 encodes the sfs4 gene and ORF2 encodes a truncated protein. Potential CRP binding site is located in the upstream of the putative promoter in the regulatory region. Expression of the cloned sfs4 gene was positively regulated by the cAMP-CRP complex.

대장균의 전사조절 유전자 nlp의 분자기구 해석

최용락,정수열,정영기,정정한 동의대학교 기초과학연구소 1994 基礎科學硏究論文集 Vol.4 No.1

An nlp (Ner like protein) gene from E. coli was previously cloned and sequenced. Here was show that expression of the sugar metabolism related genes, lacZ, malQ, and malP, increased 2.5- to 8.3-fold in the presence of a plasmid containing the nlp gene. This suggested that the nlp gene could induce maltose- and lactose-metabolism coordinately with crp*1 in the absence of cAMP. Using the nlp-lacZ fusion gene, it was possible to show the promoter of nlp was active in vivo. The overexpressed nlp gene product, a polypeptide of 10,000 daltons, was confirmed by SDS-polyacrylamide gel electrophoresis. The band shift assay revealed that the partially purified Nlp protein bound a specific DNA of the regulatory region of the nlp gene.

대장균의 전사조절 유전자 nlp의 분자기구 해석

최용락,정수열,정영기,정정한 東亞大學校附設遺傳工學硏究所 1994 遺傳工學硏究 Vol.- No.1

대장균의 nlp(Ner like protein) 유전자를 클로닝하여 구조해석을 한 결과 전사조절 단백질인 Ner와는 61%의 높은 상동성을 가지고 있음을 이미 보고한 바 있다. 본 연구는 당 대사 관련 유전자의 발현 조절을 보고자 nlp 유전자를 도입시켜 tac promoter에 의해 대량 발현시킴으로서 lactose대사 관련 유전자 lacZ와 maltose 대사 관련 유전자인 malQ, malP의 유전자 발현이 2.5배에서 최고 8.3배 정도까지 증가됨을 확인하였다. 이는 nlp유전자가 cAMP비존재하에 crp*1와 상호작용하여 maltose및 lactose대사를 촉진시킴을 시사한다. nlp-lacZ 융합 유전자 산물을 Immunoblotting하여 분석한 결과 nlp의 promoter가 in vivo 상태에서 발현되어짐을 확인하였다. tac promoter와 IPTG에 의하여 nlp유전자 산물을 대량 발현시킨 결과 약 10,000Da의 산물을 SDS-poly-acrylamide gel 전기영동으로서 확인하였으며, 부분 정제된 Nlp단백질을 조절 영역의 DNA단편에 결합함으로서 전사조절에 관여하는 것으로 사료되어졌다. An nlp (Ner like protein) gene from E. coli was previously cloned and sequenced. Here we show that expression of the sugar metabolism related genes, lacZ, malQ and malP, increased 2.5- to 8.3-fold in the presence of a plasmid containing the nlp gene. This suggested that the nlp gene could induce maltose-and lactose-metabolism coordinately with crp*1 in the absence of CAMP. Using the nlp-lacZ fusion gene, it was possible to show the promoter of nlp was active in vivo. The overexpressed nlp gene product, a polypeptide of 10,000 daltons, was confirmed by SDS-polyacrylamide gel electrophoresis. The band shift assay revealed that the partially purified NIp protein bound a specific DNA of the regulatory region of the nlp gene.

Candida tropicalis로 부터 분리된 자율복제기점을 이용한 Candida maltosa와 Saccharomyces cervisiae에서의 shuttle-vector의 구축

최용락,조영수,차재영 東亞大學校附設遺傳工學硏究所 1994 遺傳工學硏究 Vol.- No.1

무포자 효모인 Candida속의 효모는 aikane계 화합물을 이용하며 cellubiose나 xylose를 동화하는 등 산업적으로 이용가능성이 높다. Candida tropicalis는 세포질 인자인 plasmid가 발견되지 않으므로 염색체 유래의 자율복제기점을 non-replicative plasmid인 YIp5에 삽입시켜 S. cevisiae YNN 27에서 분리하였다. 분리된 자율 복제 기점을 Hind III과 EcoR I제한 효소로 절단하고 각각 2.7kb와 2.3kb fragment를 회수한 후 동일 제한 효소로 절단한 YIp5와 YIp32에 ligation시켜 pIKS523, pIKS527및 pIKC27을 구축하였다. 특히, pIKC27은 2.7kb의 자율복제 기점을 가진 plasmid로서 S. cerevisiae DBY746과 C.maltosa J288에서 높은 형질 전환체를 나타내었다. 기존의 plasmid와 안정성을 비교하였으며, 여러 제한효소에 의해서 얻어진 결과로서 상세한 restrition map을 작성하였다. Candida species are special interest because of its ability to metabolize hydrocarbon, covet this material into single cell protein and assimilate cellobiose and xylose as substrate. Cadida tropicalis has no native plasmid similar to 2㎛ plasmid. Therefore, Candida tropicalis is regarded as a new yeast host. An 8-kb fragment was isolated from Sal I digest of Candida tropicalis IFO 0518 genomic DNA which conferred the property of autonomous replication in Saccharomyces cerevisiae YNN 27. The vectors for gene manipulation of Yeast, pIKS 523, pIKS 527 and pIKC 27, were constructed by combinding the ARS fragment and integration plasmid YIP 5 and YIP 32, respectively. One of the recombinant plasmids, pIKC 27 (9.4kb), was capable of autonomous replication in both S. cerevisiae DBY 746 to Leu+ at a frequency of 600 transformants per ㎍ DNA, and transformed Candida maltosa J288 to Leu+ at a frequency of 230 transformants per ㎍ DNA. These results indicate that the 2.7 kb ARS element was necessary for high frequency transformation and autonomous plasmid replication in both S. Cerevisiae and C. maltosa.

암모니아 및 아질산성 질소 산화세균의 분리 및 특성

이용석,유주순,정수열,박춘수,최용락 한국농화학회 2003 Applied Biological Chemistry (Appl Biol Chem) Vol.46 No.1

본 연구는 폐수 중의 질소 제거를 위한 생물학적 처리용 미생물 개발을 위한 목적으로 질소의 산화 능력이 뛰어난 균주를 분리하였다. 분리된 세균 중에서 질소 산화능과 생육 속도가 뛰어난 CH-N 균주를 선별하였으며, 생리, 생화학적인 특성 조사에 의해 Bacillus sp로 추정되어 Bacillus sp. CH-N이라 명명하였다. 분리 균주는 0.5% glucose가 포함된 초기 pH가 7.0인 암모니아 및 아질산성 질소 함유 배지에서 30시간 배양 후 가가 85%와 90%의 암모니아성과 아질산성 질소가 단시간에 질소의 감소율을 나타내었다. 폐수 및 생활하수에 분리 균주를 이용한 결과, 수질 속의 암모니아성 질소가 단시간에 크게 감소시키는 효과를 확인하였다. 균주를 고정시킨 담체의 질소산화 효과를 시험하고자 Bacillus sp. CH-N을 고정시킨 세라믹 담체를 이용한 결과, 배양 2일 후에는 암모니아성 질소가 전부 제거되었다. In order to improve the system for biological nitrogen oxidizing process in sewage and wastewater, a bacterium having high abilities to oxidize of nitrogen was isolated from wastewater and polluted soils. The strain was identified to Bacillus sp. CH-N, based on the physiological and biochemical properties. Characteristics and oxidizing ability of both ammonia and nitrite were examined for the strain, Bacillus sp. CH-N. The strain showed the oxidizing rate about 80% to 90% on the sewage and wastewater after 48 h culture. The nitrogen oxidizing rate was increased in proportion to the initial concentration of glucose. The microorganism, Bacillus sp. CH-N cell immobilized on ceramic carrier were evaluated for the oxidation of ammonia in culture media.

중금속 제거 및 pH 상승을 위한 폐달걀껍질의 재활용

조영수,최영락,손희정,김은호 東亞大學校生命資源科學大學附設 農業資源硏究所 1997 農業生命資援硏究 Vol.6 No.1

In the present, batch test was conducted to evaluate the neutralization and adsorption of heavy metals from wastewater using waste egg shells. Neutralization and removal rate of heavy metals were excellent in the increase of waste concrete amounts and a small size. It seemed that adsorption efficiencies of heavy metals were influenced by solubility. As a result on the experiments of Freundlich isotherm, the adsorption capacities(k) were Cr 3.11, Cu 2.61, Mn 3.02 and Pb 0.95, respectively and the adsorption intensities(1/n) were Cr 0.35, Cu 0.44, Mn 0.4 and Pb 0.41, respectively. In view of these results, it showed that wastes containing the similar compositions as waste egg shells could utilize the neutralization and adsorption of heavy metals in wastewater.

정수처리공정의 침전지에서 부착조류의 특성과 이취발생에 관한 기초연구

성낙창,최용락,김은호 東亞大學校附設 農業生命科學硏究所 1999 農業生命資援硏究 Vol.8 No.1

The purposes of this study were to investigate species of attached algae, and determine these characteristics and relations of odor origin for estimating its source in tap water. Oscillatoria sp. and Mougeotia sp. were colonized at the upper and lower of sedimentation basin in water treatment plant, respectively. In the results that they were analysed to collect colonies of Oscillatoria sp. and Mougeotia sp, they did not detected inodorous Mougeotia sp. but Oscillatoria sp. of some decayed odor did geosmin 3.7ppt and Dimethyl-disulfide with trace. 20species compounds were found in each of sample that were incubated in room temperature for 5days, 4species of these were THMs originated tap water and concentration was 35㎍/ℓ, and 16species compounds were by algac. Especially, sulfur compounds of 10species such as Dimethyl-sulfide were initially not detected but did very much. Also, Indole and Naphthalene were small detected. Considering characteristics of decayed decomposed sampling, generated byproduct for decayed algae of 2species turned out to be nearly identical. If estimating course of odor generation in tap water, chlorine tolerative algae formed big colony to be attached in sedimentation basin and decay was advanced with varying anaerobic condition inside colony's algae. Therfore, a lump of algae detached in suspended state flowed in filter bed, and owing to decayed colony's algae, generated inodororus material could inflowed tap water.

조류 부산물중 2-MIB의 제거에 관한 기초연구

성낙창,최용락,김은호 東亞大學校附設 農業生命科學硏究所 1999 農業生命資援硏究 Vol.8 No.1

This experiments were performed using O₃, ClO₂and Granular activated carbon in order to remove 2-MIB(2-Methylisobornelo) causig odor in water. In Freundlich equilibrium experiments, 2-MIB was well adsorbed in PAC comparatively and raw water containing TOC was less adsorbed than distilled water by effect of TOC. Jar tests with coconut-based PAC were showed high removal efficiency of 2-MIB in proportion to contact time and concentration of PAC. Oxidation capacity of O₃was demonstrated to be superior to that of ClO₂. Prepared comparatively highly initial concentration as 1991ppt of raw water was treated with post-O₃in condition of 1ppm and 12min. of contact time and then its residual concentration was approximately 569ppt(70% removal efficiency), and the 2-MIB removal efficiencies of the Post-O₃water through coal, coconut shell, wood and zeocarbon based GAC(granular activated carbon) column were 95.8%, 89.5%, 88.4% and 93.7%, respectively. Coal based carbon have most effective adsorption capacity but wood based carbon have a little lower adsorption capacity than others. In GAC column experiment, TOC, UV-254, 2-MIB and so on ewere removed most effectively in case of A(coal) and for this experiment intervals, the change of water quality(UV-254, TOC) was not happened by input of odor substance(2-MIB).

sfs유전자의 malE유전자 발현 촉진

정수열,이희영,최용락 東亞大學校附設遺傳工學硏究所 1995 遺傳工學硏究 Vol.- No.2

CRP에 변이가 도입된 crp*¹ 유전자를 통하여 cloning된 sfs(sugar fermentation stimulation)유전자 중 nlp와 sfs4 유전자가 maltose 및 maltodextrin의 운반에 관여하는 유전자들 중에서 MalE 단백질을 증가시킴을 immunoblotling을 통하여 확인했다. 즉 MalE 단백질은 periplasmic에 존재하면서 cytoplasmic membrane에 존재하는 MalFGK 단백질과 반응하여 cytoplasm내로 maltose, maltodextrin을 운반하는 MBP(maltose binding protein)로 밝혀져 있다. Most crp* mutants isolated so far were capable of fermenting lactose despite the absence of cAMP, however, they could ferment other sugar such as maltose in the presence of cAMP. nlp and sfs4 genes, which stimulated the maltose metabolism in a crp*¹, cya::km(MK2001) host system. Especially, MalE protein(malE gene: MBP: maltose binding protein), that is transport protein with maltose and maltodextrin in periplasmic, were increased in the cloned with nlp and sfs4 genes.

Cellulose 생합성균주 Acetobacter sp. CBⅡ의 분리 및 특성

이승진,유주순,최용락 동아대학교 환경문제연구소 2000 硏究報告 Vol.23 No.1

The strain of cellulose-producing CBⅡ was isolated from the traditionally fermented persimmon vinegar, and identified as Acetobacter sp. CBⅡ with respect to physiological and biochemical characteristics. The isolated strain was cultivated statically in broth culture, a thick cellulose pellicle was formed. It was found that the strain was fluorecenced with UV light when the strain was cultivated agar medium contained Tinopal. The strain produced acetic acid from ethanol, and then decomposed acetate to CO₂ and H₂O. Capacity of cellulose biosynthesis from isolated strain was compared with Acetobacter xylinum ATCC 53582. The cellulose produced by the isolated strain was purified and cegraded by HCl. The released sugar was separated on thin layer chromatograpy(TLC) consistent with the degradation product of native cellulose.