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.

Limulus Amoebocyte Lysate법에 의한 원유와 살균유의 세균수 측정

姜國熙,王智怨,朴興洙,李圭鍾,金敬民 成均館大學校 科學技術硏究所 1993 論文集 Vol.44 No.2

LAL method for the determination of Gram-negative bacterial Lipopolysaccharides(LPS) in milk is proposed and its value for bacteriological quality control is investigated. The relationship between the LPS contents and total colony count(cfu/ml), which covers the Gram-negative bacteria, was determined in raw milk samples. The coefficient of correlation between Gram-negative bacterial counts(cfu/ml) and LPS contents was r^2 =0.969. And, the correlation coefficient between total bacterial counts(cfu/ml) and LPS contents was r^2 =0. 914. It was also investigated how much the LPS contents were in UHT milks from l0 different dairy industrial companies in Korea. It varied from 92 ng LPS/ml to 938 ng LPS /ml.

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.

김밥과 그 재료의 세균수 측정

강국희,김혜란,고애경,김경민,최선규 성균관대학교 생명과학자원연구소 1994 生命資源科學硏究 Vol.1 No.2

We have investigated the distribution of bacteria in kimbab and its materials. The total bactria counts were over 3×10 exp(6)/g(n=30) when the kimbabs were delivered to restaurant and it exceeded the prieliminary legal level 1 × 10 exp(6)/g even though it should be negative for coliforms. In order to look into the cause of bacterial contamination in kimbabs, we examined the content materials of kimbabs. The bacterial counts were founded 10^4-10^8/g for Kim, 10^4-10^8/g for sausage, 10^4-10^6/g for spinach, 10^3-10^7/g for carrot, and 10^3-10^6/g for danmugi, respectively. From these results it could be concluded that the bacterial contamination in Kimbabs are due to kim, spinach, carrot, and sausage. Therefore, so as to reduce the bacterial contamination for kimbab, the sanitary manufacture and storage of kim and the refrigeration of content materials should be went abreast.

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.

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.

당귀(Angelica acutiloba)와 감초(Glycyrrhizauralensis)의 열 추출액이 장내 세균의 성장에 미치는 효과

강국희,김경민,정재록 성균관대학교 생명과학자원연구소 1997 生命資源科學硏究 Vol.4 No.2

Bifidobacteriurn longum, Bifidobacterium adolescentis, Clostridium butyricum, Escherichia coli and Eubacterium limosum were cultured on the modified PYF medium containing 0.5% of glucose, heating extract(w/w) of Angelica acutiloba or Glycyrrhiza uralensis, and anaerobically incubated for 48hrs at 37℃. As a control, the same intestinal microorganisms cultured on the PYF medium with no additives and they were compared with other treatments. The addition of the heating extract of Angelica acutiloba increase the growth but optical density was lower than addition of glucose for the growth of intestinal microorgamisms which were investigated in this study. However, the heating extract of Glycyrrhiza uralensis slightly inhibited the growth for most intestinal microorganisms(Bif. longum, Bif. adolescentis, Cl. butyricum, and Eu. limosum) and it had a significant inhibition effect on the growth of Eu limosum when they were compared with control.

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.

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.