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Plasmid DNA pBR 322의 증폭과 Hydroxyapatite Chromatography에 의한 분리
오두환,유주현,신원철,정건섭,유승구 연세대학교 산업기술연구소 1984 논문집 Vol.16 No.1
The amplification and the isolation conditions of pBR322 plasmid DNA were investigated. For the amplification of pBR322 plasmid DNA, cells from logarithmic growth phase were most effective. The optimal conditions for the amplification of pBR322 plasmid DNA in Escherichia coli GM4 were obtained as follows; 50-150 ㎍/ml of chloramphenicol concentration, 6-8 hours of incubation time in the presence of chloramphenicol. Ampicillin and tetracycline had no effect on the amplification of pBR322 plasmid DNA. Approximately 1 mg of pBR322 plasmid DNA was purified from 3.5 ℓof Escherichia coli GM4 culture broth by hydroxyapatite chromatography. The purified pBR322 plasmid DNA was expressed in Escherichia coli C600.
윤기도,권동진,홍석산,김수일,정건섭 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4
To investigate the inhibitory effect of soybean and Korean traditional fermented soybean products on the chemically induced mutagenesis, we extracted soybean, Kanjang, Doenjang, Kochujang, and Chonkukjang with water, methanol and hexane. Inhibitory effect of extracts was assayed by the SOS chromotest using Escherichia coli PQ37 as a test strain. 4-nitroquinoline-1-oxide(4NQO), N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), and aflatoxin B_1(AFB_1) were used as mutagens. Methanol extracts showed relatively higher inhibitory effect than water and hexane extracts. Methanol extracts of soybea, Doenjang, Kochujang, and Chongkukjang showed inhibitory effect of 68.4, 96.3, 17.5, and 100.9%, against MNNG, and 28.6, 109.1, 41.3, and 101.8% against AFB_1., respectively. Doenjang methanol extract showed inhibitory effect of 51.0, 96.3, and 109.1% against 4NQO, MNNG, and AFB_1. Inhibitory effect of heat-treated Doenjang and Chongkukjang methanol extracts on the mutagenicity of MNNG and AFB_1 was remained over 95% of the inhibitory effect of heat-untreated extracts, demonstrating the heat stability of the potent antimutagenic activity.
Bacillus licheniformis GA9가 생산하는 키틴 분해효소의 정제 및 특성
황동호 ( Dong Ho Hwang ),홍성욱 ( Sung Wook Hong ),황형서 ( Hyung Seo Hwang ),정건섭 ( Kun Sub Chung ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.4
지렁이의 장내로부터 분리한 미생물 중에서 키틴 가수분해 활성이 우수한 미생물을 선발하였으며, 이를 동정하여 Bacillus licheniformis GA9으로 명명하였다. B. licheniformis GA9이 생산하는 키틴 분해효소의 정제는 배양상등액 40-60% 황산암모늄 침전, 음이온교환 크로마토그래피, 겔 크로 마토그래피를 사용하여 정제하였다. 최종적으로 정제한 키틴 분해효소는 45.2배로 정제되었고 효소단백질 회수율은 20.0%를 나타내었다. 정제한 키틴 분해효소의 분자량은 약 52.1 kDa으로 나타났으며, N-terminal amino acid sequencing 분석결과, 아미노산 서열은 D-S-G-K-N-G-K-I-I-R-Y-Y-P-IR로확인되었다. 키틴 분해효소의 최적반응 pH와 pH 안정성을 측정한 결과, pH 5.0에서 최대 활성을 나타내었으며 pH 5.0-6.0에서 안정성을 나타내었다. 키틴 분해효소의 최적반응 온도와 온도안정성의 경우, 40℃에서 최대 활성을 나타내었으며, 60℃까지 60%의 잔존 활성을 나타내었다. 정제한 키틴 분해효소는 10 mM Co<sup>2+ </sup>금속이온에 의해 효소활성이 증가하였으며, Fe<sup>2+</sup>과 Cu<sup>2+ </sup>금속이온에 의해 효소활성이 감소하였으나, EDTA 첨가시 감소한 효소활성이 일부 회복되었다. 정제한 효소의 K<sub>m</sub> 및 V<sub>max</sub>는 각각 4.02 mg/ml와 0.52 mg/min이었다. 또한 키틴 분해효소는 생명공학, 생물의약, 농업, 식품영양 등 다양한 산업분야에서 응용이 가능하다. A bacterium producing a large amount of chitinolytic enzyme was isolated from the intestinal tract of earthworm. The isolate was identified as Bacillus licheniformis by 16S ribosomal RNA analysis and designated as B. licheniformis GA9. The enzyme was purified by 40-60% ammonium sulfate precipitation, diethyl-aminoethyl groups exchange chromatography, and gel filtration chromatography. The molecular weight was estimated to be 52.1 kDa and the N-terminal amino acid sequence was D-S-G-K-N-G-K-I-I-R-Y-YP- I-R. The optimum activity of the purified chitinolytic enzyme was shown at pH 5.0 and 40℃, and the enzyme was stable in the ranges of 20-50℃ and pH 5.0-6.0. Enzyme activity was increased by Co<sup>2+, </sup>while it was inhibited by Cu<sup>2+ </sup>and Fe<sup>2+</sup>. But it was recovered by chelating metals with ethylenediaminetetraacetic acid. The K<sub>m</sub> and V<sub>max</sub> values of the purified enzyme were 4.02 mg/ml and 0.52 mg/min, respectively. The chitinolytic enzyme characterized in this study has potential applications in areas such as biotechnology, biomedicine, agriculture, and nutrition.
Secretion of Bovine $\beta$-Casein by Saccharomyces cerevisiae
Chung, Kun-Sub,Rafael, F.R.,Oh, Sang-Suk,Richardson, T. The Korean Society for Microbiology and Biotechnol 1991 Journal of microbiology and biotechnology Vol.1 No.1
Yeast expression plasmids containing an appropriate leader sequence and bovine $\beta$-casein cDNA were constructed to produce $\beta$-casein for the study of its functional characteristics. Two kinds of expression systems for $\beta$-casein were constructed using pCGYl444 as a precursor plasmid. This plasmid is a yeast-E. coli shuttle vector which contains the chelatin promoter. The plasmid pISB202 contains the invertase leader sequence and $\beta$-casein gene. The plasmid pDEB303 contains the original bovine $\beta$-casein leader sequence gene. These two plasmids were introduced into S. cerevisiae AB116 which is a strain deficient in the major yeast proteinases. Each clone was grown in minimal media for 24 h before induction by $CuSO_4$. The cells were thus grown under expression conditions. Both strains harbouring pISB202 and pDEB303 expressed bovine $\beta$-casein. The $\beta$-casein was detected using immunochemical staining after western blot. Secretion of $\beta$-casein was detected in the culture broth. The estimated amount of secreted $\beta$-casein was approximately 50 ${\MU}g$/l.
Secretion of Bovine β-Casein by Saccharomyces cerevisiae
Chung, Kun Sub,Rafael, F. R.,Oh, Sang Suk,Richardson, T. 한국미생물 · 생명공학회 1991 Journal of microbiology and biotechnology Vol.1 No.1
Yeast expression plasmids containing an appropriate leader sequence and bovine β-casein cDNA were constructed to produce β-casein for the study of its functional characteristics. Two kinds of expression systems for β-casein were constructed using pCGY1444 as a precursor plasmid. This plasmid is a yeast-E. coli shuttle vector which contains the chelatin promoter. The plasmid pISB202 contains the invertase leader sequence and β-casein gene. The plasmid pDEB303 contains the original bovine β-casein leader sequence gene. These two plasmids were introduced into S. cerevisiae AB116 which is a strain deficient in the major yeast proteinases. Each clone was grown in minimal media for 24h before induction by CuSO_4. The cells were thus grown under expression conditions. Both strains harbouring pISB202 and pDEB303 expressed bovine β-casein. The β-casein was detected using immunochemical staining after western blot. Secretion of β-casein was detected in the culture broth. The estimated amount of secreted β-casein was approximately 50㎍/ℓ.