원형질체 융합 방법을 이용한 전분 발효성 효모의 개발

전순배,배석,이기영,김수현,이진종,김상문,Chun, Soon-Bai,Bai, Suk,Lee, Kee-Young,Kim, Soo-Hyun,Lee, Jin-Jong,Kim, Sang-Moon 한국미생물학회 1990 미생물학회지 Vol.28 No.2

전분 분해능이 있는 담자균성 효모, Filohasidium capsuligenum의 종내 및 Saccharomyces cerevisiae와의 속간 원형질체융합을 시도하였다. F capsuligenum CBS 6122-2의 종내 융합율은 각각 $2.9\times10^{-3}$ (ade+mel trp), $8.5\times10^{-3}$(ade+met)이고, F. capsuligemum CBS 4381 (cys his)과 S.cerevisiae 262-12-1 (hom3 thr1), X2180-1A (ade thr)와의 속간 융합율은 각각 $8.8\times10^{-6}$, $9.5\times10^{-4}$이었다. DNA 정량, 핵 염색, 자외선 조사에 따른 생존력 비교 그리고 형질 분리 분석 결과 등을 통해 종내와 속간에서 핵 융합이 일어난 것을 알 수 있었다. 또한, 종내 융합체의 경우 세포당 $\alpha$-amylase와 glucoamylase 생성능이 양천형에 비해 1.6-2.1배의 증진 효과가 있었다. Intraspecific fusion frequency between Filobasidium capsuligenum CBS6122-2 ade and met trp or met strains was $2.9\times 10^{-3}$ and $8.5\times 10^{-3}$, respectively. And intergeneric fusion frequency between Folobadidium capsuligenum CBS 4318 (cys his) and Saccharomyces cerevisiae 262-12-1 (hom3 thr1) or X2180-1A (ade thr) was $8.8\times 10^{-6}$ and $9.5\times 10^{-4}$, respectively. Nuclear fusion appears to occru in fusants between intraspecies and intergenera as strongly suggested by DNA content, nuclear staining, comparison of survival rate to UV light and mitotic segregation analysis. It was also found that $\alpha$-amylase and glucoamylase activity from intraspecific hybrids was 1.6-2.1 fold increased when compared with that from thier parents.

Candida pseudotropicalis 융합세포의 유전적 분석

전순배,배석,Chun, Soon-Bai,Bai, Suk 한국미생물학회 1988 미생물학회지 Vol.26 No.2

The genetic analysis and characterization of protoplast fusion hybrids between complementary auxotrophic mutants of Candida pseudotropicalis were carried out. Nuclear fusion appeared to occur in fusion hybrids (e.g., F15 and F33), as strongly suggested by isolation of recombinants after mitotic segregation of parental genetic markers. This was confirmed by KNA content, nuclear staining and comparison of survival rate to UV light. After keeping fusion hybrids for approximately one year, the frequency of spontaneous mitotic segregation was $3.0\times 10^{-4}$ - $8.1\times 10^{-4}$ while that of induced mitotic segregation was $1.4\times 10^{-3}$- $1.7\times 10^{-3}$. These results suggested that they maintained stable karyogamy state. It was also found that the production of $\beta$-D-galactosidase from F15, F33 and F158 was somewhat increased when compared with that from either auxotrophic parents or wild type.

Candida pseudotropicalis의 원형질체 융합: 원형질체 형성 및 재생과 융합 조건

전순배,정기철,배석,Chun, Soon-Bai,Chung, Ki-Chul,Bai, Suk 한국미생물학회 1986 미생물학회지 Vol.24 No.3

Candida pseudotropicalis CBS 607에서의 야생형과 영양요구성 돌연변이 균주에 대한 원형질체 형성 및 재생과 상보적 영양요구성 균주간의 융합에 관해서 연구하였다. 야생형과 his tidine 혹은 adenine요구성 균주에서 원형질체 형성율은 96-100%이였으나 methionine과 tryptophan요구성 균주는 각각 52%와 72%였다. Myoinositol (0.5 mg/ml)을 첨가한 배지에서 전배양한 methionine과 tryptophan조 구성 균주는 원형질체 형성시 bovine serum albumin (BSA, 4 mg/ml)을 첨가하면 96-98%의 원형질체 형성율이 얻어졌다. 원형질체 재생 빈도는 18-20% 였으나. BSA와 myoinositol을 첨가하면 adenine요구성 균주(약 20%)을 제외한 나머지 영양요구성 균주에서는 재생율이 20% 이상 증가했다. 원형질체 융합에 대한 PEG와 $CaCl_2$,최적농도는 20%와 100mM이었고 최적 pH와 반 응시간은 각각 6.0과 30분이 있다. 상보적 영양요구성 균수의 융합빈도는 $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ 였으며 원행점체의 재생 증진으로 인해 융합율이 현저히 증가했다. Histidine요구성 균주와 tryptophan 요구성 균주을 융합시켰을 때 몇개의 융합세포를 얻었는데 측정된 DNA양과 액상의 차이로 보아 aneuploid나 diploid상태임을 알 수 있었다. Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

Bovine serum albumin, Myoinositol과 Ergosterol에 의한 Candida pseudotropicalis의 원형질체 재생 및 융합증진

전순배,배석,Chun, Soon-Bai,Bai, Suk 한국미생물학회 1987 미생물학회지 Vol.25 No.4

Candida pseudotropicalis의 영양요구성 돌연변이 균주들에 대한 원형질체 형성, 재생 그리고 융합에 있어서 bovine serum albumin (BSA), myoinositol 그리고 ergosterol의 증진효과를 연구하였다. 원형질체 형성율은 영양요구성 균주의 type에 따라 48%-98%였다. O. 5mg/ml의 myoinositol과 0.1mg/ml의 ergosterol을 배양액에 첨가하고 4mg/ ml의 BSA을 원형 질체 형성 완충용액에 첨가했을 때는 50~100%의 원형절체 형성윷을 얻었다. 영양요구성 균주의 생장배지에 myoinositol과 ergosterol을 동시에 첨가하고 원형질체 형성 완충용액에 BSA를 침가하면 2.2-3.0애의 원형질체 재생율 증진을 보여주었다. 원형질체 융합의 최적조건에서 상보적 영양요구성 균주간의 융합율은 $7.0\times 10^{-4}$ ~ $1.5\times 10^{-3}$ 이였으며 상기화합물을 처리하지 않고 얻은 융힐l율과 비교해서 1. 9-2. 3배의 증가를 보였다. 이러힌 결과로 본 연구에서 사용된 균주들에서는 상기화합물 처리 에 의해 원형질체 재생은 물론 세포융합율도 증진됨을 알 수 있었다. The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

榮山江 汽水域의 酵母에 關한 生態學的 硏究

전순배,Chun, Soon-Bai 한국미생물학회 1984 미생물학회지 Vol.22 No.1

1976年 3月부터 1977年 2月까지 滿 12個月동안 榮山江 汽水域 39個 場所로부터 採水한 156個 water sample로서 酵母의 季節的 分布와 度包配에 따른 이들 酵母의 分布型을 調査하였다. 酵母의 全體 平均値는 100ml당 52에서 487個의 生菌數를 보여주었고 가장 많은 個體數는 春季에 있었고 가장 적은 個體數는 夏季에 있었다. 14屬 83種에 해당하는 933個의 酵母와 한 種의 酵母樣 곰팡이가 分離되었는데 基中 Candida가 29%, Debaryomyces가 17.3%, Rhodotorula가 16%, Saccharomyces가 14%이었고 나머지 다른 屬들은 10%以內이었다. Debaryomyces hansenii와 Rhodotorula glutinis가 全水域에서는 물론 年中 優點種이었고 총 好氣性細菌集團의 크기, 陸水流入量, 그리고 몇가지 지형적 기후적 요소들이 汽水환경에서 酵母의 季節的 分布는 물론 種의 組成을 反映하는 같다. 酵母의 平均數, 種의 多樣性 特히 醱酵性 및 爲菌系形成 母酵들의 數는 감度가 감소함에 따라 增加한 反面 질산염 利用酵母들은 위와 相反된 경향을 보여주었는데 이는 汽水域에서 酵母의 分布型을 認識하는데 감度勾配가 바람직한 看破 요소로서 利用될 수 ㅣ있음을 시사하고 있다. With 156 water samples collected from 39 locations in the Yeong San River estuary during the 12month period from March 1976 to February 1977, the seasonal distribution of yeast and the distributional pattern of yeast on salinity gradient have been investigated. An overall average number of yeast ranged from 52 to 487 viable cells (c.f.u.) per 100ml water sample. The highest count of yeast was obtained in spring while the lowest value came in summer. 933 yeast and one yeast-like fungus pertaining to 14 genera and 83 species were recovered, of which Candida were 29%, Debaryomyces 17.3%, Rhodotorula Glutinis were dominant forms in all locations as well as throughout the year. The population size of total aerobic bacteria, the amount of terrestrial imputs, and some of geographical and/or climatic factor appear to reflect the seasonal distribution of yeast as well as the composition of yeast species in an estuarine environ. Average number of yeast, species diversity, and particularly the number of fermentative and pseudomycelium-producing yeasts increased with decreasing salinity whereas nitrate-utilizing yeasts showed opposite trend, suggesting that salinity gradient can be used as a feasible detector for the distributional pattern of yeast in estuarine habitat.

Candida kefyr의 ${\beta}$-D-galactosidase 合成誘導에 關한 硏究[I]

전순배,Chun, Soon-Bai 한국미생물학회 1984 미생물학회지 Vol.22 No.2

This examined some conditions for the induction of ${\beta}$-D-galactosidase synthesis in Candida kefyr CBS 834. The optimal pH, temperature, and inoculum size either for growth or${\beta}$-D-galactosidase synthesis were 5.5, $30^{\circ}C$ and above 0.2 at A610nm, respectively. Enzyme activity began to increase at 2h after the addition of inducer, and continued to increase linearly up to $2{\sim}3h$ before reaching stationary phase, and thereafter its activity was decreased. ${\beta}$-D-galactosidase was induced either by lactose or galactose but not either by glucose or ethanol. The greater activity of ${\beta}$-D-galactosidase on galactose than on lactose indicated that the former might be natural inducer for ${\beta}$-D-galactosidase synthesis. The rate of its induction as a function of lactose concentration showed that enzyme activity increased linearly above 4mM, while it was very low below that. Glucose represed the induction of ${\beta}$-D-galactosidase, and the period of adaptation to inducer from other carbon sources was relatively short.

Novozym 234에 의한 Kluyveromyces fragilis N100과 Candida pseudotropicalis CBS607의 원형질체 형성과 재생

배석,전순배,Bai, Suk,Chun, Soon-Bai 한국미생물학회 1984 미생물학회지 Vol.22 No.1

Novozym 234에 의한 K. fragilis와 C. pseudotropicalis의 원형질체 형성과 재생에 관한 연구를 하였다. intact cell을 원형질체로 전환시키는데 정지기의 세포보다 대수기에 있는세포가 효과적이었으며 osmotic stabilizer로는 $(NH_4)_{2}SO_4$가 두 종 똑같이 원형질체 형성이나 재생에 있어서 적합했고 최적 효소농도는 K. Fragilis에서는 3mg/ml, C. pseudotropicalis에서는 1~3mg/ml이었다. 반응 후 관찰된 세포 중의 원형질체 형성 수율은 K. fragilis에서 약 95%, C. pseudotropicalis에서 약 100%였으나 원형질체의 재생은 Glusulase와 비교해 볼 때 매우 낮았다. 그러나 세포 이물질이 없는 원형질체를 얻는데는 Novozym 234가 적합한 것 같다. Formation and regeneration of protoplasts by Novozym 234 from Kluyveromyces fragilis N100 and Candida pseudotropicalis CBS607 were studied. This enzyme was more effective on cells grown at exponential phase than those at stationary one to convert intact cells into protoplasts. As osmotic stabilizer, ammonium sulphate was suitable for not only protoplast formation but also regeneration in K. fragilis as well as in C. pseudotropicalis. Optimal enzyme concentration was 3mg per ml in K. fragilis and 1~3mg per ml in C. pseudotropicalis, respectively. After the exposure of K. fragilis cells to 3mg per ml of enzyme for 3hr at 30$^{\circ}C$ , approximately 95% of protoplast formation of all observed cells was obgained, while about 100% from C. pseudotropicalis under the same condition was produced. The regeneration frequency of protoplasts by this enzyme was much lower than that by snail enzyme(Glusulase) although Novozym 234 converted cells from above two species into protoplasts free of cell debris effectively, compared with Glusulase. Novozym 234 appears to be suitable for subcellular fractionation to obtain nuclei or other organelles rather than protoplast regeneration.

광양만 미생물 군집에 관한 환경요인분석

위인선,전순배,이종빈,나명석 ( In Sun Wui,Soon Bai Chun,Jong Bin Lee,Myung Suk Ra ) 한국환경생물학회 1993 환경생물 : 환경생물학회지 Vol.11 No.1

The factor analysis was applied to examine the effects of enviromental conditions on microbial populations in Kwangyang Bay from February to December, 1990. For this purpose, 17 abiotic and biotic parameters were determined for samples obtained from fifteen sites. Ecosystem in Kwangyang bay was regulated by 5 factors for four season. The microbial population was influenced mainly by the inflow fresh water, nitrogenous nutrients and water temperature. The overall size of microbial population was greater than that obtained in 1982.

유당 자화 효모간의 원형질체 융합

최원기,전순배,이용규,배석,이진종,이향,Choi, Won-Ki,Chun, Soon-Bai,Lee, Yong-Kyu,Bai, Suk,Lee, Jin-Jong,Lee, Hyang 한국미생물학회 1988 미생물학회지 Vol.26 No.3

유당함유 울질로부터 알코올을 생산하기 위해 Saccharomyces cerevisiae X 2180-1 A, Candida pseudotropicalis ATCC 8619와 CBS 607 사이에 속간 및 종내 원형질체 융합을 시도하였다. S. cerevisiae X2180-1A(ade rho)와 C. pseudotropicalis CBS 607 (his met)간의 속간 융합율은 $1.0\times 10^{-5}$ C. pseudotropicalis ATCC 8619와 CBS 607 사이의 종내 융합율은 $3.9\times 10^{-6}$ - $1.0\times 10^{-4}$ 이었다. 이는 bovme serum albumin, myoinositol 그리고 ergosterol을 처리하지 않고 얻어진 원형질체 융합율과 비교해서 약 2-3.5배의 증가를 보여 이들 화합물이 속간 및 종내 융합을 증진시킨 것으로 사료된다. DNA 함량, 핵염색, UV 조사에 따른 생존력 비교 그리고 형질분리 후 교잡형 출현 등을 통해서 속간 (S. cerevisiae와 C. pseudotropicalis)과 종내 ( C psemdotropica/is strains) 융합체에서 핵융합이 일어난 것을 알 수 있었다. 또한 양친형과 비교해서 알코올 생성능이 증가된 종내 융합체를 얻었다. Intergeneric or intraspecific protoplast fusion between Saccharomyces cerevisiae X-2180-1A, Candida pseudotropicalis ATCC 8619 and CBS 607 was attempted to produce ethanol from lactose containing materials. Teh intergeneric fusion frequency between Saccharomyces cerevisiae X-2180-1A (ade rho) and Candida pseudotropicalis CBS 607 (his met) was $1.0\times 10^{-5}$. These values exhibited approximately 2-3.5 fold increase when compared with fusion frequency obtained without the treatment of bovine serum albumin, myoinositol and ergosterol, suggesting that these compounds may improve intergeneric of intraspecific protoplast fusion. Nuclear fusion appears to occur in fusants between intergenera(S. cerevisiae+C. pseudotropicalis) and intraspecies (C. pseudotropicalis strains) as strongly suggested by DNA content, nuclear staining, comparison of survival rate to UV light and isolation of recombinants after mitotic segragation. It was also found that alcohol production from intraspecific hybrids was somewhat increased when compared with that from their parents.

Penicillium verruculosum으로부터 Cellobiohydrolase의 정제 및 특성에 관한 연구

조남철,김강화,전순배,정기철,Cho, Nam-Chul,Kim, Kang-Hwa,Chun, Soon-Bai,Chung, Ki-Chul 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.5

효소생성유도물질로서 cellobiose octaacetate를 포함하는 P. verruculosum 배양상등액으로부터 소단위체 분자량이 60,000(I) 과 66,000(II) 인 두 종류의 cellobiohydrolase와 KG-flock을 함유하는 배지로부터 소단위체 분자량이 76,000(III)인 또 하나의 cellobiohydrolase를 분리정제하여 그들의 일반적 특성을 검토하였다. 이들의 자연상태의 분자량은 소단위체 분자량의 2~3배 였으며 모두 당단백질로서 당함량은 cellobiohydrolase I, II 및 III가 각각 8.6%, 4.2% 그리고 8.5%였다. 세 효소 모두 pH 4.5~5.0과 온도 $50{\sim}60^{\circ}C$에서 높은 활성도를 나타냈으며 Avicel, cotton, 여지 등의 결정성 섬유소 뿐만 아니라 CMC, PNPC 등에도 분해활성도를 나타내었다. Cellobiohydrolase I, II 및 III의 Avicel 분해 비활성도는 각각 0.07, 0.10, 그리고 0.07 unit per mg. of protein이었으며 그 분해생성물은 거의 cellobiose였다. PNPC 기질에 대한 효소반응에 있어서 포도당에 의해서는 활성도가 억제되지 않은 반면 10~20mM의 cellobiose에 의해 50% 이상의 억제효과를 보였다. Three cellobiohydrolase I, II and III, from culture solution of Penicillum verruculosum grown in liquid media containing cellobiose octaacetate or KC-flock, as the sole carbon source has been extensively purified and characterized. Molecular weights of the purified enzymes, I, II and III, were estimated as 60,000, 66,000 and 76,000 on 10% polyacrylamide gel containing sodium dodecyl sulfate and have 8.6, 4.2 and 8.5% of carbohydrate contents, respectively. The three enzymes showed hydrolytic activities towards crystalline celluloses such as Avicel, cotton and filter paper and soluble cellulose such as carboxymethylcellulose. Specific activities of cellobiohydrolase, I, II and III against Avicel as a substrate were 0.07, 0.10 and 0.07 unit per mg. of protein, respectively. The optimum pHs and temperatures of three enzymes were 5.0 and $50^{\circ}C$. All enzyme produced extensively cellobiose from Avicel, and the enzymatic hydrolysises of ${\rho}$-Nitrophenyl-${\beta}$-D-cellobioside were inhibited by cellobiose, but not by D-glucose.