Bacillus thuringiensis var. darmstadiensis 와 var. sotto의 내독소와 Bacillus thuringiensis var. kurstaki가 생산하는 아밀라제의 연구

이형환,김삼찬,황광현,임창로,조용칠,정인명 건국대학교 교육대학원 1991 敎育論叢 Vol.15 No.-

Endotoxin crystals produced by B.thuringiensis var. darmstadiensis cultured in modified GYS medium over 72 h were ovoidal in shape and separated using Renografin gradient centrifugation. The crystal protein bands were observed. When B.thuringiensis var sotto was grown in the modified GYS broth, it entered stationary phase at 8-9 h after inoculation. Proteinaceous crystals were banded at the position in 40% of Renografin gradient. The solubilized crystal proteins were formed by two bands, 130 Kd and 68 Kd in the SDS-PAGE. One plasmid was observed in B.thuringiensis var darmstadiensis and no plasmid in var. sotto in this study. Enzyme activity of amylase produced B.thuringiensis var kurstaki strain in the basal medium was 0.4 units per ml, and was enhanced to 0.6 units per ml in the basal medium contained soluble starch, Ca^++, Mg^++ and Mn^++ ions. Amylase production medium containing soluble starch was suitable for the amylase production and the highest activity appeared at 4-6 h after cultivation at 32℃. The amylase activity obtained by ethanol precipitation was 0.20 units per ml in 0.1 M phosphate buffer. The band of the molecular weight of 50 Kd was observed by SDS-PAGE analysis. Km value of the amylase for the soluble starch was 6.80 mg per ml.

Escherichia coli RY13의 Auxotrophic Mutant의 분리와 요구성 연구

李炯煥,姜判祚 건국대학교 교육대학원 1982 敎育論叢 Vol.2 No.-

Escherichia coli RY13 were cultivated in Nutrient broth at 37℃ and mutagenized with Nitrosoguanidine. From the mutagenized cells, 47 auxotrophic mutants were selected. Four mutants out of 47 auxotrophs did not require any amino acids at all, but 43 mutants out of them required all kinds of amino acids.

소(牛)성장호르몬 유전자 클로닝의 기초 연구

李炯煥 建國大學校基礎科學硏究所 1987 理學論集 Vol.12 No.-

소(牛)의 뇌하수체전엽에서 분비되는 소의 성장호르몬을 얻기 위하여 전엽 추출물을 PAGE 후에 젤의 각 밴드를 추출한 다음 RIA 방법을 이용하여 ??항체와 반응시켜 소의 성장호르몬양을 측정하였다. 측정결과 뇌하수체 gram당 6.69㎍의 성장호르몬이 있는 것으로 나타났다. 뇌하수체 분쇄물에서 RNA를 분리하기 위하여 agarose gel 전기영동을 한 결과 분자량이 0.08Kb에서 21.22Kbs의 크기를 갖는 8개의 밴드가 나타났고, 분쇄물을 다시 oligo dT 컬럼을 통과하여 총 mRNA를 얻었다. 이 컬럼을 0.1M NaCl과 NaCl이 없는 완충액으로 유출시킨 결과는 NaCl이 없는 유출액 중에 성장호르몬 mRNA의 양이 0.1M NaCl 유출액에서 보다 7배 정도가 많았다. mRNA 분획을 AMV reverse transcriptase을 이용하여 cDNA을 합성한 결과는 100㎕의 합성반응액에서 약 0.2㎍의 DNA을 얻었다. Bovine growth hormones were extracted from bovine pituitary glands and then measured by PAGE and RIA. Bovine growth hormone was reacted with ?? antibody. There were 6.69 ㎍ per gram of bovine pituitary gland. Growth hormone RNAs were extracted from the gland, which were electrophorized on 1.0 % agarose gel. There were formed about 8 different bands ranging from 0.08Kb to 21.22 Kbs. Total mRNAs were obtained by oligo dT chromatography, which were in vitro translated. In the eluent by elution buffer not contained NaCl, growth hormone mRNAs were presented 7 times more than 0.1 NaCl eluent. The mRNA eluent was used for template for cDNA synthesis by AMV reverse transcriptase. From 100㎕ of the reactant,about 0.2㎍ of cDNA were obtained.

Bacillus thuringiensis가 생산하는 Exotoxin이 흰쥐에 미치는 영향

李炯煥 건국대학교 1982 學術誌 Vol.26 No.2

This experiment was carried out to investigate the production of exotoxin by Bacillus thuringiensis serotype 1 and 3ab, and its effects on mice and black goat kidney cells. The difference of exotoxin productivity between Bacillus thuringiensis serotype 1 and 3ab was hard to recongnize. Micrococcus flavus was ascertained as a suitable test organism for determining the productivity of exotoxin by plate agar assay method. However Excherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were not susceptible to the exotoxin. The non-concentrated supernatant of the serotype 1 and 3ab from 48 hour-culture, administered orally, intraperitoneally, subcutaneously, nasally and intracerebrally, did not have any toxic effects on mice and this was the same case with the 5x concentrated supernatant. Black goat cells treated with 10% concentration of nonconcentrated supernatant of the serotype 1 and 3ab from 48 hour-culture failed to demonstrate any cytotoxic effects.

Bacillus sphaericus ts-D1290 단백질의 전기영동적 분석

서정희,이형환,김영희 한국산업미생물학회 1990 한국미생물·생명공학회지 Vol.18 No.5

Bacillus sphaericus ts-D1290 치사돌연변이주의 특성을 연구하기 위하여 제한온도와 허용온도에서 생산하는 단백질의 차이점을 연구하였고, 모기유충에 치사력을 가진 단백질을 분리하여 모기유충에 치사되는지를 조사하였다. ts-D1290 균주를 42℃에서 배양했을 때는 30℃배양보다 균체의 단백질 함량이 약 1/5로 감소하였고, 42℃에서 4시간 배양 후 30℃로 배양하였을 때는 약 1/3로 감소하였다. 그리고 단백질의 패턴은 ts-D1290 균주를 42℃에서 전배양 후 30℃로 하강 배양하였을 때는 30℃에서 전배양하여 42℃로 상승배양하였을 때와는 달리 분자량 221kd 단백질이 나타나지 않았지만 분자량 150kd 단백질은 30℃ 전 배양하였을 때와 같이 현저하게 나타났다. 두 균주에 80㎍/㎖ 농도의 chloramphenlcol을 첨가하여 42℃에서 배양하였을 때 정상균주는 30℃에서 배양한 대조군보다 단백질 함량이 1/2로 감소하였고, ts-D1290 균주는 30℃에서 배양하였을 때는 첨가하지 않았을 때보다 분자량 150kd 단백질이 약하게 나타났다. ts-D1290과 정상균주가 생산한 150kd단백질은 모기유충에 치사성을 나타냈다. Bacillus sphaericus ts-D1290 was characterized by SDS-PAGE produced by the mutant at 30℃ and 42℃. The total amount of proteins produced by the mutant at 42℃ decreased to one-fifth of those at 30℃; however, when the culture was shifted down from 42℃ after 4 to 30℃, the total amount of protein decreased to one-third and the 221 kd protein did not appear, but the 155 kd appeared remarkably. When the mutant and the wild type strain were cultured in the media containing 80㎍ per ㎖ of chloramphenicol at 42℃, the wild type strain synthesized half amounts of the total proteins than those at 30℃, and the mutant produced one-tenth of the total protein amounts. When the both strains were cultured in the media containing chloramphenicol, the 155 kd protein was produced was produced in lesser amounts than those without chloramphenicol. The 150 kd protein showed lethal activity to Culex pipiens 3rd instar larvae.

Volvariella volvacea의 營養菌絲 및 擔子器 細胞의 核分裂에 關한 硏究

張鶴吉,李炯煥 건국대학교 1981 論文集 Vol.13 No.1

The studies were carried out to examine the nuclear behaviour and morphological variation in the vegetative mycelia and basidia at various stages on the life cycle of Volvariella volvacea(Bull ex Fr.) Sing. Nuclear division of vegetative mycelia: 1.Somatic mycelia of vegetative cells were multinucleate. Sizes and shapes of the nuclei diff tired from those of various stages and nuclear division. 2.The number of nuclei per cell ranged from one to more than thirty. 3.In vegetative mycelia, nuclear behaviour clearly demonstrated that somatic nuclei usually divide mitotically and asynchronously. Interphase, prophase, metaphase, anaphase and telophase nuclei are seen. 4.No spindle has been resolved, and the mechanism of anaphase separation is still unknown. Clamp connections are definitely absent. Nuclear division of basidia: 1.Hyphae and cells Occurring in the hymenium can be classified morphologecally basidia, bas-idioles(paraphysis), and cystidia. 2.All nuclei present in the fruiting body tissues are of uniform shape and size. The haploid chromosome number is shown to be nine. 3.Meiosis occurs regularly after nuclear fusion and various stases of meiosis can be clearly distinguished. From uninucleate basidiospores are formed.

Bacillus sphaericus ts-U311의 단백질 합성변화의 전기영동적 분석

임헌길,황성희,이형환 建國大學校基礎科學硏究所 1992 理學論集 Vol.17 No.-

Bacillus sphaercus 1593 K-5와 ts-U311의 총 세포 단백질을 전기영동적인 방법으로 비교했다. 1. B. sphaercus 1593 K-5와 ts-U311의 총 단백질간에는 분자량과 등전점에서 많은 차이가 있었으나,동일 균주에서는 성장온도 변화에 따른 단백질 양상에서 거의 차이가 없었다. 2.B.sphaericus 1593 K-5는 분자량이 최고 225Kd에서 최저 13Kd에 걸쳐 총 26개 밴드가 분리되었고,ts-U311 은 최고 220K 달톤에서 최저 13.5K달톤까지 24개 밴드가 분리되었드며, 두 균주의 단백질은 분자량이 150,94,77,57,40,32,19Kd인 곳에서 존재유무와 양에 차이가 있었다. 3. B.sphaericus 1593 K-5 ts-U311은 pH 4.2에서 pH 4.8 사이에서 등전점을 가지는 단백질이 이차원젤상에 나타나지 않았으나, B.sphaericus 1593 K-5는 진하게 염색된 4번과 5번 단백질 점이 나타났다. 4. 이차원젤 상에서 두 균주의 단백질 종류의 차이점은 다음과 같다. 4번,5번,12번,19번,20번,21번은 B.sphaericus 1593 K-5에 존재하나 ts-U311에는 없었고, 36번,37번,38번,39번은 ts-U311에는 존재하지만 B.sphaericus 1593 K-5에는 없었다. 또한,3번,6번,27번,28번,29번,35번은 단백질의 양에서 차이가 있었고,여러 곳에서 전개위치가 약간씩 다르게 나타났다. 5.B.sphaericus 1593 K-5의 단백질 중에서 분자량이 150Kd인 것이 독소인 것으로 확인되었고 ts-U311에서는 이 단백질이 없었다. 생체검정시험에서도 B.sphaericus 1593 K-5는 강력한 독성을 보였지만 ts-U311은 전혀 독성이 없었다. Protein syntheses by B. sphaericus 1593 wild type strain and ts-U311 mutant were studied by gel electrophoretic analysis. The protein patterns of the two strains were different in the molecular weight bands of 150,94,77,57,40,32 and 19 kilodaltons by SDS-polyacryl-amide gel electrophoresis analysis. In the isoelectric-focusing the proteins from B. sphaericus ts-U311 were not banded in the pH ranges 4.2 to 4.8 In the analysis of two-dimentional polyacrylamide gel electrophoresis the wild type strain had number 4,5,12,19,20 and 21 sptted proteins, but the ts-U311 did not. Number 36,37,38 and 39 spotted proteins were produced from the proteins of ts-U311,but the wild type strain did not. The 150 Kd protein from B.sphaericus wild type strain was toxic to the larvae of Culex pipiens, but it was not produced from the B.sphaericus ts-U311.

Bacillus subtilis β-glucanase유전자의 효모세포에서의 발현

이영호,이희무,이형환 건국대학교 생명과학연구원 1995 생명과학지 Vol.2 No.-

The clone pABC1 inserted the β-glucanase gene of Bacillus subtilis was transformed and expressed in a polyploid yeast cell, S. uvarum OE2. The yeast/pABC1 produced the enzyme intracellularly, however not extracellularly. When the S. uvarum OE2 was disrupted, the cell extract showed β-glucanase activity which meant the expression of β-glucanase gene in S. uvarum OE2. To make a β-glucanase extracellular secretion in S. uvarum OE2, the promoter of β-glucanase region derived from B. subtilis in the pABC1 was substituted with the promoter of the alcohol dehydrogenase 1 gene in the pAMC1 vector originated from S. cerevisiae and named pABC1. The S. uvarum OE2 transformed with pABC1 extracellularly secreted the β-glucanase.

大腸菌의 炭水化物 利用에 관한 硏究

徐貞姬,申一燮,李炯煥 건국대학교 1982 論文集 Vol.15 No.1

Growth of Escherichia coli K12 has been measured in the minimal stilts media contained monosaccharides (glucose, galactose and fructose, separately), discacharides (saccharose, lactose and maltose, separately) and combined with the both compounds. E. coli K12 has utilized glucose very fast than other saccharides. Saccharose could not be utilized by E. coli. Growth of E. coli was synergistically increased in the minimal media contained galactose and lactose. Except saccharose, the two disaccharides, maltose and lactose combined with the three monosaccharides increased the growth rate and prolonged growth of E. coli.

Bacillus sphaericus D1216-Spo- 균주의 증식과 핵산합성의 특성

김수영,김재수,이형환 建國大學校基礎科學硏究所 1993 理學論集 Vol.18 No.-

Bacillus sphaericus 1593 K-5와 이의 무아포 돌연변이주인 D1216-Spo-에 항생제 chloramphenicol 또는 nalidixic acid를 첨가한 배지와 첨가하지 않은 배지에서 30℃, 42℃, 30→42℃, 42→30℃로 배양하고, 이들의 성장과 DNA 생합성의 속도를 측정하고 비교했다. 30℃에서 두 균주는 유사한 성장속도를 보였으나 42℃에서는 1593 K-5가 약간 빠른 성장을 보였다. 온도상승실험에서의 성장속도는 D1216-Spo-이 약간 빨랐으나, DNA와 RNA의 생합성은 1593 K-5가 1.5배 이상 생성했다. 온도하강실험에서, 42℃에서의 4시간의 성장속도는 온도상승실험의 30℃에서보다 약 절반 정도였고, 30℃로 온도하강후에 두 균주는 빠르고 긴 대수성장기를 보였다.Chloramphenicol을 첨가하면 성장과 DNA생합성의 속도가 온도에 상관없이 chloramphenicol을 첨가하지 않은 것의 절반 정도였다. Nalidixicacid를 첨가하면 DNA 생합성의 속도는 2~3배 정도 감소하지만, 성장속도는 약간만 감소하였다. Bacillus sphaericus 1953 K-5and its D1216-Spo- mutant were cultured at 30℃, 42℃, 30→42℃,42→30℃ with or without antibiotics(chloramphenicol, nalidixic acid)and then their growth curves and nucleic acid(DNA, RNA) biosynthesis rates were measured. At 30℃both strains showed similar growth rate, and at 42℃ the 1593 K-5 grew slightly faster, and at temperature shift-up(30→42℃)the D1216-Spo- grew slightly faster, but the 1593 K-5 synthesized about 1.5times more DNA and RNA after 6 hours. At temperature shift-down(42→30℃) the growth rates of two strains during 4 hours at 42℃were about a half than that of 30℃ at temperature shift-up, and after temperature shift to 30℃ two strains showed fast and long logarithmic growth phase. When chloramphenicol was added, growth rate and DNA biosynthesis rate of two strains were a-half than culture without chloramphenicol regardless of the growth temperature. When nalidixic acid was added, DNA biosynthesis rate of two strains was reduced 2-3 times,but the reduction of growth rate was slight