• Journal of Microbiology and Biotechnology
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• 제6권4호
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• pp.286-291
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• 1996

A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

• Biomolecules & Therapeutics
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• 제8권3호
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• pp.235-240
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• 2000

Antitumor effect of LC43, a protein-bound ploysaccharide (M.W. 43 kDa) that was purified from intergeneric protoplast fusant of Lentinus edodes and Coriolus versicolor, was elucidated against mouse sarcoma 180 cell in vitro and in vivo. By injecting LC43 into ICR mice bearing solid or ascitic sarcoma 180, tumor regression and survival rates were investigated. To examine the effects of LC43 on immunopotentiation activity. immunoorgan weight, B cell differentiation, T cell activity and macrophage activation were determined. LC43 showed antitumor effects against both solid tumor and ascitic tumor of sarcoma 180. It did not change significantly the immunoorgan weight but potentiated immune responses such as B cell differentiation and the release of superoxide anion from macrophages. These results suggest that the protein-bound polysaccharide of LC43 exhibited antitumor activities through the activation of immune-related cells and acted as an immunmodulator.

• 한국미생물·생명공학회지
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• 제17권1호
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• pp.1-7
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• 1989

Transformant TSD-14의 starch 발효능을 향상시키기 위해, 52$^{\circ}C$에서 60분간의 열처리로써 regeneration능을 제거한 TSD-14의 protoplast와 C, tropicalis RCT-40 (lys-)의 protoplast를 20 mM CaC1$_2$를 함유한 30% PEG를 fusogenlc agent로 하여 융합시키고 최소배지상에서 fusant를 선별한 결과 4.4$\times$$10^{-5}$ 빈도로 fusant를 얻었다. Fusant들의 탄소원 자화능을 조사한 결과 parental strains의 성질이 동시에 존재함을 알 수 있었으며 cell volume은 Parental Strains에 비해 약 1.5배정도 크게 나타났다. 또한 fusant는 유전적으로 매우 안정하였으며, parent인 TSD-14와는 달리 $\alpha$-1, 6-glucosidic linkage를 가수분해할 수 있었다. Fusant중 가장 우수 한 FSC-14-75 균주는 15%의 liquefied potato starch로부터 8.7%(v/v)의 ethanol을 생성하였고 또한 이때의 fermentation broth에 0.3%의 peptone을 첨가한 경우, ethanol 생성은 9.3%(v/v) 수준으로 증가하여 총당에 대해 86.0%의 발효율을 나타냈으며 산업적 이용가능성을 시사하였다.

• Applied Biological Chemistry
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• 제34권2호
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• pp.174-179
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• 1991

Brevibacterium flavum 과 Corynebacterium glutamicum 균주간의 원형질체 융합을 실시하였다. 원항질체 융합을 위하여, Brevibacterium flavum ATCC 21493과 Corynebacterium glutamicum ATCC 21831로부터 여러 변이주를 분리하고 융합의 최적조건을 검토하였다. 본 연구에서 저자 등은 Brevibacterium flavum 108-125와 Corynebacterium glutamicum 41-214A 균주 간의 이속간 원형질체 융합주 MWF 9031의 L-arginine발효능이 우수함을 확인하였다. 융합주 MWF 9031은 10% 포도당이 함유된 배지에서 32.5 mg/ml의 L-arginine을 생산하였다. 융합주의 생리적 성질은 두 모균주의 중간정도를 나타내고, 안정성이 60일 이상 유지되고 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.232-237
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• 1993

A strain of yeast which can convert starch directly to ethanol was developed by the intergeneric protoplast fusion between Schwanniomyces alluvius possessing $\alpha$ amylase as well as glucoamylase with debranching activity and FSC-14-75 which previously had been formed from a heterologous transformation and subsequent intergeneric protoplast fusion. Fusants were selected on minimal medium after protoplasts of auxotrophic mutant of S. alluvius fused with heat-treated protoplasts of FSC-14-75 in the presence of 30%(w/v) PEG and 20 mM $CaCl_2$. The fusion frequency was in the range of $10^{-6}$ order. All fusants tested were intermediate types of parental strains for carbon compound assimilation, and their cell volumes were approximately 1.1 times larger than FSC-14-75 and 1.8 times larger than S. alluvius. The fusants were unable to sporulate like FSC-14-75, while S. alluvius could sporulate. In flask scale the most promising fusant, FSCSa-R10-6, produced 7.83%(v/v) and 10.17%(v/v) ethanol from 15% and 20% of liquefied potato starch, respectively, indicating that the fermetation efficiency of each case increased 1.2 times and 1.6 times than that of FSC-14-75. The elution pattern on DEAE-cellulose chromatography showed that FSCSa-R10-6 has four distinct amylase peaks of which two peaks originated from S. alluvius and the other two from FSC-14-75. These results suggest that the enhanced fermentation efficiency of the fusant might be due to almost-complemented parental amylases.

• 한국식품영양과학회지
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• 제24권5호
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• pp.779-784
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• 1995

5'-GMP(5'-Guanylic acid) 생산 융합균조 fusant RC102를 고정화하여 5'-GMP을 연속적으로 생산하고자 하였다. 균체 고정화용 담체로서 k-carrageenan, polyacrylamide, Ca-alginate 및 agar 등 4가지 담체에 고정화한 결과 최적 담체로서 3% k-carrageenan을 선정하였으며, 이담체에 고정화한 균체를 이용하여 5'-GMP 연속적으로 생산하였다. 고정화 균체에 의한 5'-GMP 생산의 최적 온도 및 pH은 $32^{\circ}C$, pH 8.0, 미량온소로서 $Mn^{2+}과\;Zn^{2+}$의 농도는 각각 $30\mu\textrm{g}/L,\;1{\times}10^{-6}%$이었다. $Mn^{2+}$이 풍부하고 값싼 CSL(Corn Steep Liquor)을 배지로서 사용하기 위해서 penicillin G와 D-cycloserine 및 POSEA(polyoxyethylene stearylamine) 농도 최적 농도는 각각 0.8unit/ml, 0.8unit/ml, 그리고 5mg/ml으로 나타났으며, 항생물질 보다 계면활성제가 효과적이었다. 이상의 최적 조건에서 고정화 fusant RC102는 15일간 안정되게 5'-GMP를 생산하였다.

• 한국미생물·생명공학회지
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• 제19권4호
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• pp.325-330
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• 1991

섬유성 당화액에서의 ethanol 생산에 적합한 균류를 개발할 목적으로 Candida sp. KM-09-135와 Saccharomyces cerevisiae SM-07의 이속간의 원형질체 융합을 행하여 우수한 융합체로 인정된 KMS-23 균주를 얻었다. 융합주 KMS-23은 37'C에서 생육 및 ethanol 생성능이 가장 좋았고, 발효당으로 xylose, cellobiose, maltose 및 raffinose도 이용하였다. 세포의 크기는 KM-09-135 및 SM-07에 비해 각각 1.2배, 1.5배 증가하였으나 DNA 함량은 약간의 증가만 보여 완전한 2배체의 융합주가 아닌 것으로 나타났다. 환원당으로서 6.44(w/v)의 당을 함유한 밀기울 당화액에서 융합주 KMS-23은 동일조건하에서 KM-09-135보다 약 1.3배 높은 2.57(v/v)의 ethanol을 생산하였다.

• 미생물학회지
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• 제31권3호
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• pp.218-223
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• 1993

Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

• 한국미생물·생명공학회지
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• 제15권2호
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• pp.104-111
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• 1987

Lysine 생산균주를 개량하기 위한 시도로서 Brevibacterium flavum ATCC 21528R 과 Brevibacterium flvum ATCC 21529S의 동종간 및 Brevibacterium flavum ATCC 21528R 과 Corynebacterium glutamicum ATCC 13058S 와 의 이속간 원형질체 융합을 실시하였다. 이들 균주들에 대한 원형질체 형성의 최적조건을 조사하고 재생과 융합에서의 plasma expander의 효과를 검토하였다. 융합주 No. CH23과 No. CH41은 최적 배양조건 하에서 L-lysine 생산성이 모균에서보다 각각 21％와 8.9％ 향상된 것이었다. L-Lysine 생합성 회로상의 중심효소인 asparto-kinase를 포함한 주요효소의 활성을 측정하였고, 융합주 No. CH23과 No. CH41에서의 L-lysine 생합성 대사제어를 친주와 비교하였다.