Streptococcus thermophilus SKD 1006의 β-Galactosidase 유전자 구조

민해기,강국희,성문희 成均館大學校 科學技術硏究所 1992 論文集 Vol.43 No.2

The results can be concluded that the pSK 001 contains a gene for β-gal from Str. thermophilus SKD 1006, and expressed in E. coli JM 109. A 4.2 Kb of Hpa II-Hpa II fragment(lac Z of SKD 1006) of pSK 001 was subcloned into the corresponding site of pUC 19, and named pSK 002. A 1.2 Kb of Bgl II -Bgl II fragment of pSK 002 was ligated into the corresponding site of pUC 19, and named pSK 003. The nucleotide sequences of two portions(222bp close to 5' , 143 by close to 3' end) of Bgl II -Bgl II fragment of pSK 003 were determined by Sanger, Chen and Seeburg methods. The nucleotide sequences are identical to those of the gene for β-gal of Str. thermophilus A 054.

Bifidobacterium longum KCTC 3215의 β-Galactosidase 유전자의 Cloning 및 대장균에의 발현

민해기,강국희,성문희 成均館大學校 科學技術硏究所 1992 論文集 Vol.43 No.2

The β-D-galactosidase(β-gal) gene from Bifidobacterium longum KCTC 3215 was cloned to isolate and characterize it for potential use as a selection marker in food-grade cloning vector. Chromosomal DNA from Bif. longum KCTC 3215 was cleaved with the restriction enzyme Sau3AI and ligated to pBR 322 for transformation into E. coli JM109. A β-galactosidase positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactoside. This transformant possessed a single plasmid, designated pSK 100, which contained, in addition to the vector DNA, a 5.2 Kilobase(Kb) Sau3A1 insertion fragment coding for β-gal activity. An extract from JM 109(pSK100) contained a β-gal protein with the same electrophoretic mobility as the β-gal from Bif. longum KCTC 3215. A restriction enzyme map of pSK 100 was consisted of PvuII, HincII, PstI,AccI, EcoRI, etc.

α-Galactosidase 에 의한 Bifidobacteria 균수 측정에 관한 연구

강국희,이시경,백운화,민해기 成均館大學校 科學技術硏究所 1992 論文集 Vol.43 No.2

This method using the synthesis substrate of 5-bromo-4-chloro-3 indolyl-galactoside (X-a -Gal) was examined for the differential enumeration of Bifidobacteria and lactic acid producing bacteria. Bifidobacteria possess a high level of a-galactosidase activity. Bifidobacterium longum KCTC 3215 exhibited the highest a- galactosidase specific activity (8.57 units/mg protein). Determination of a -galactosidase activity using the PNPG procedure showed that Lactobacillus, Streptococcus, Pediococcus, and Leuconostoc strain had lower a-galactosidase activity as compared to Bifidobacteria. The X-a- Gal based medium is useful to identify Bifidobacteria among lactic acid producing bacteria since the enzyme action of a-galactosidase hydrolysis X-a-Gal substrate and releases indol which impacts a blue color to Bifidobacterial colonies on agar plates. All strains of Bifidobacteria appeared blue colonies on MRS agar medium supplemented with 100 uM X- a - Gal while colonies of other lactic acid producing bacteria appeared white or light blue.

김치로부터 분리한 Pediococcus halophilus에 의한 α-Glucosidase의 생산

강국희,민해기,홍승표 成均館大學校 科學技術硏究所 1992 論文集 Vol.42 No.2

Six strains able to efficiently produce α-glucosidase were isolated from kimchi by enrichment culture in APT medium. Among them, Strain No.2 was found to have levels of α-glucosidase activity. The morphological, physiological and biochemical characteristics of the strain No.2 were studied according to the methods of Bergey's manual. Based on the results obtained in these experiments, Stain No.2 was identified to be a similar species to Pediococcus halophilus. The optima conditions for α-glucosidase production were cultured 0.5% starch, 0.4% yeast extract, 0.4% trypticase peptone, initial pH 7.5 at 37℃ and 24 hours cultivation.

Bifidobacterium의 成長促進物質에 관한 硏究

姜國熙,張永斅,閔海基 成均館大學校 科學技術硏究所 1992 論文集 Vol.42 No.2

The objective of this study wsa to improve the usability of Bifidobacterium for the fermantation milk products. Thus, various biological materials were tested for the growth enhancing effects of Bifidobacterium in a synthetic medium. The organism used to conduct this study was Bifidobacterium bifidum SKD-2001, originationg from the feces of a breast-fed infant. Lactose, sucrose, peptone, L-cysteine HCI+vitamine K_1, tomato extract, and yeaset extract enhanced the growth of the test organism. The mixture consist of these materials as mentioned previously. This mixtute added in 12% reconstituted skim milk. The total viable counts of the test organism were 4.3 X 10^9/ml at 37℃, 48 hrs incubation anaerobically.

유럽공동체 국가들의 환경정책에 관한 연구 : 환경영향평가제도를 중심으로 비교분석 A Comparative Analysis of Environmental Impact Assessment

조해용,강순국 한국환경과학회 1997 한국환경과학회지 Vol.6 No.4

Environmental problems emerged as common global problems awaiting solution. Active collaborative efforts are required of nations and communities in order to solve environmental problems effectively. International collaboration occurs much more commonly these days than before, and European Community(EC) mumber countries are no exceptions. EC has established and executed the five-year programs on Environmental Policy Implementation, which explicated basic principles for environmental policy development, since 1973. EC programs tend to emphasize that the direction of the policies should reflect a change from a remedial approach to a preventive approach. Those programs have brought an awareness of the importance of Environmental Impact Assessment(EIA) to EC member countries. France installed an EIA system in 1976, which was the first among member countries. Several other member countries also established a system. EC decided that a common guideline was necessary, and therefore formulated the "European Community Guideline for Environmental Impact Assessment" in 1985. All member countries were required to legislate an EIA system within three years, according to the Guideline. This study will conduct a comparative analysis of the current EIA systems of different EC member countries. The findings of this study will provide helpful information on how to improve the efficacy of the Korean system.

Str.thermophilus 510의 β-Galactosidase 유전자의 Cloning 및 대장균에의 발현

강국희,민해기,이호근,성문희 成均館大學校 科學技術硏究所 1992 論文集 Vol.42 No.2

Chromosomal DNA from Str. thermophilus 510 was purified and cleaved with the restriction enzyme PstI. Chromosomal DNA was ligated to pBR322 for transformation into E. coli JM109. A β-galactosidase positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactoside. This transformat possessed a single plasmid, designated pSK 001, which contained, in addition of the vector DNA, A 7.0Kb Pst I insertion fragment coding for β-gal activity. A restriction enzyme map of pSK 001 was consist of Hind Ⅲ, EcoR I, Bgl Ⅱ, Sal I, Pvu Ⅱ, etc. An extract from E. coli JM109 (pSK001) contained a β-gal protein with the same electrophoretic mobility as the β-gal from Str. thermophilus 510.

비만세포주에서 Histamine 유리에 관한 연구

김형룡,국윤아,신민,이홍수,원진숙,김형민,이영미,정헌택,이혜숙,안녕형 원광대학교 생명공학연구소 1995 생명공학연구소보 Vol.3 No.1

Bifidobacterium angulatum으로부터 α-galactosidase의 정제 및 특성

강대중,민해기,강국희 成均館大學校 科學技術硏究所 1993 論文集 Vol.44 No.1

α-galactosidase는 (α-D-galactoside galactohydrolase: EC 3.2.1.22)는 다당류나 올리고당인 melibiose, raffinose, stachyose와 guar gum 등의 α-galactosidic 결합을 하고 있는 당을 분해하는 효소이다. 장내세균중 우익균이며 우세균주인 Bifidobacteria로부터 α-galactosidase의 효소학적 특성을 알아보기 위하여 배양여액으로부터 ammonium sulfate분획, 핵산의 제거, Sephadex G-200 gel filtration 및 DEAE-Sephadex A-50 ion exchange chromatography 등의 4단계 정제과정을 거쳐 정제한 결과 약 8배로 정제되어 단일 단백질로 분리하였다. 또한, 효소분해산물을 알아보기 위하여 TLC에 의하여 당분해 및 올리고당의 생성을 알아보았다. 정제효소의 활성 최적온도는 37℃, 최적 pH는 7.0이었다. 효소활성이 K^+, Zn^2+과 같은 금속이온과 8-Hydroxyquinoline, Sodium Bisulfite 등에 의해선 그다지 저해되지 않았으나, Hg^+, Cu^2+과 p-Chloromercuribenzoic acid에 의해선 저해되었다. 효소의 분자량이 301, 995, 합성기질인 PNPG에 대한 K_m은 0.069mM, V_max는 347.22μmol/min.mg protein이었다. To elucidate enzymatic properties of α-galactosidase (EC 3.2.1.22) from Bif.angulatum. α-galactosidase was purified to homogeneity by ammonium sulfate fractionation, protamine sulfate precipitation, sephadex G-200 gel filtration, and DEAE-sephadex A-50 ion exchange chromatography. The purified α-galactosidase was found tobbe homogeneous by SDSpolyacrylamide gel electrophoresis. The molecular weight of this enzyme, estimated by sepadex G-200 gel filtration, was about 301,995. The optimum conditions for the enzyme reaction was pH 6.5 to 7.0 at 37℃. The purified enzyme was stable at 45℃ or below and in buffer at pH 6 to 7.0. The activity was inhibited by mercury, copper ion, and p-chloromercuribenzoic acid. The kinetics of this enzyme, with p-nitrophenyl-α-D-galactoside as substrate, was determined : K_m was about 0.069mM and V_mas was 347.22 μmoles/min mg protein.

Bandom Amplified Polymorphic DNAs 기법을 이용한 Bifidobacteria의 신속한 동정

왕지원,민해기,장영효,강국희 성균관대학교 생명과학자원연구소 1995 生命資源科學硏究 Vol.2 No.1

Random Amplified Polymorphic DNAs (RAPD) has been applied to the identification of human intestinal anaerobic Bifidobacteria. Recently, The RAPD technique has been used to analyse DNA polymorphism of genomic DNA from eukaryotes such as plants and animals(1, 3, 4, 10, 11). But there were only several reports on the polymorphism of prokaryotic genomic DNA by using this method(2,7). Total 100 random sequence primers were used in screening of species-specific primer. The oligonucleotide primers were ten bases long and designed to have average G+C contents of 60 mol %. Eight species of Bifidobacteria from human feces were selected. According to conventional method genornic DNA were extracted from each bifidobacterium and then RAPD-PCR was performed. Four primers have produced distinct electrophoretic band patterns, RAPD markers. Especially arbitrary primer # 101 showed species-specific DNA polymorphism for each of Bifidobacterial genomic DNA tested. The arbitrary primer # 101 showed good reproducibility when the PCR conditions were optimized. And also primer # 156, # 174, # 183, have produced amplified PCR products but not for all of the strains tested. Better DNA polymorphism was observed when two arbitrary primers were used in combination.