• 한국미생물·생명공학회지
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• 제21권2호
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• pp.107-112
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• 1993

The optimal conditions for protoplast fusion between the cells of kanamycin resistant Lactobacillus bulgaricus IFO 13593 and those of lincomycin resistant Lactobacillus helveticus IAM 12090 were investigated in this study. The highest fusion frequency of 9.1*10-4 was obtained when protoplast fusion was carried out for 0.5 min using 40% PEG 4000 solution as a cell fusion stimulant and subsequent post-PEG-incubation was undergone at 30 for 30 min in the PPI medium.

• 미생물학회지
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• 제22권3호
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• pp.175-181
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• 1984

In order to develope a protoplast fusion system for industrial coryneform bacteria, the optimum conditions for the formation and regeneration of progoplast were examined for Brevibacterium flavum and Corynebacterium glutamicum and the protoplast fusion was performed. For the formation of the protoplast of B. flavum and C. glutamicum, the optimum time for penicillin G. treatment to obtain protoplast was mid-exponential growth phase ($O.D_{580}=0.6-0.8,\;8.0{\times}10^7-1.0{\times}10^8cell/ml$). At the optimum conditions (0.3units/ml penicillin G and $400{\mu}g/ml$ lysoyme for treatement), frequencies of protoplast formation and protoplast regeneration were 99% and 25%, respectively. Protoplast regeneration frequency was highest under the optimum conditions for the protoplast formation. Addition of 25mM $Mg^{2+}\;and\;50mM\;Ca^{2+}$ to the regeneration medium further increased the regeneration frequencies. The protoplast fusion frequencies of B. flavum and C. glutamicum in intraspecies fusion were $1.0{\times}10^{-8}\;and\;7.8{\times}10^{-4}$, of the regenerated protoplast respectively, when 33% of PEG (polythylene glycol) 6,000 was used as the fusing agent.

• Mycobiology
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• 제29권1호
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• pp.15-18
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• 2001

Protoplast fusion is a useful technique for establishing fungal hybrids to overcome the natural barriers. The ultrastructure of protoplast and its fusion process were observed using a scanning electron microscopy(SEM) and a transmission electron microscopy(TEM). The protoplasts were variable in size from $0.5{\sim}15{\mu}m$ in diameter, and the mean diameter was about $3{\sim}5{\mu}m$. It was impossible to discriminate protoplasts of Lentinula edodes from protoplasts of Coriolus versicolor by size and surface structure. Big aggregates of the dehydrated protoplasts were observed, after polyethylene glycol 4000 treatment. Nucleus, mitochondria, lipid granules and various vesicles having granules were scattered in the cytoplasm. The vesicles were heterogeneous in size and vary from one protoplast to another. The fused membrane layer of the two protoplasts was observed. Time protoplast membrane contact and reorganization of membrane components were essential condition for protoplast fusion. Transmission electron micrograph showed fused protoplasts and flattening of the cells in the area of the membrane contact. We hope that our electron microscopic observations provide some insights into the understanding of the fusion process of protoplast in fungi.

• 한국미생물·생명공학회지
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• 제20권1호
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• pp.6-13
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• 1992

유산균주의 균주개량방법의 일환으로 protoplast fusion 방법을 사용하여 lincomycin에 내성을 나타내는 Lactobacillus casei KCTC 1121과 rifampicin에 내성을 나타내는 Lactobacillus delbruckii JK-414의 protoplast 형성과 재생, 융합에 대한 조건 및 융합주의 생리학적 성질 등을 검토하였다. Lactobacillus case와 Lactobacillus delbrueckii JK-414는 삼투압 안정제로 sucrose가 함유된 protoplast forming buffer에서 5$\mu g$/ml의 mutanolysin 으로 $42^{\circ}C$, 15분간 처리 했을 때 protoplast 형성율이 높게 나타났다.

• 한국균학회지
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• 제21권4호
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• 미생물학회지
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• 제29권2호
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• pp.123-129
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• 1991

In order to develop the protoplast fusion method of the strains of Fusarium, the interspecific protoplast fusion was attempted between Fusarium poae and F. sporotrichioides. Various auxotrophic mutants were isolated by the treatment of N-Methyl-N'-Nitro-N-Nitrosoguanidine. The optimal conditions for the formation and regeneration of protoplasts were examined and the characteristics of a fusant were studied. As a results, protoplasts were readily obtained from 18 hours cultured mycelia by the treatment of driselase for 3 hours and 0.6 M KCl as a best osmotic stabilizer at pH 6.0 for the formation of protoplast. Sucrose was the most suitable for the regeneration. Polyetylene glycol (M.W. 8,000) in $CaCl_{2}$-glycine solution was used to induce the protoplast fusion. The interspecific fusion frequency between protoplasts among the auxotrophic mutants of the two strains ranged from $2.7*10^{-2}$ to $5.7*10^{-3}$ . DNA content and cellulase activity were rather increased in the interspecific fusant. The lag phase of growth curve was slightly elongated in the fusant.

• 미생물학회지
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• 제24권2호
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• pp.91-97
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• 1986

Intra and interspecfic fusants were produced by the protoplast fusion of auxotrophic mutants from Trichoderma koningii ATCC 26113 and Trichoderma reesei QM 9414. It was found that 0.6M $MgSO_4\;and\;0.6M\;NH_4Cl$ was the best osmotic stabilizer for the preparation of protoplasts from the mycelium of T. koningii and T. reesei respectively. However, $MgSO_4$ was the most suitable one for the regeneration of the protoplasts from both species. The intraspecific protoplast fusion frequencies between the auxotrophic mutants from T. reesei were $1.8{\times}10^{-2}\;to\;5.1{\times}10^{-1}$. Interspecific protoplast fusion frequencies between the auxotrophic mutants from T. koningii and T. reesei were $3.6{\times}10^{-3}$\;to\;8.4{\times}10^{-2}. Interspecific complementing fusants, however, were not alwats produced. Fusants obtained from interspecific potoplast fusion were spontaneously segregated into various strains including parental types, non-parental auxotrophic hybrids, and prototrophic hybrids on complete plate. Interspecific hybrids revealed to have partially enhanced celluloytic activities.

• Journal of Microbiology and Biotechnology
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• 제3권1호
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• pp.24-30
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• 1993

In order to develop a novel yeast which produces the charateristic aroma of soy sauce, a protoplast fusion between Zygosaccharomyces rouxii WFS4 and Torulopsis versatilis IAM 4993 was carried out. Auxotrophic mutants as selective markers were obtained from Zygosaccharomyces rouxii and Torulopsis versatilis by treatment of N-methyl-N -nitro-N-nitrosoguanidine. The conditions of the protoplast formation and the regeneration for fusion were examined. The protoplast fusion using polyethylene glycol 4000 led to the fusion frequency of $4~5{\times}10^{-7}\;cells/ml$. Among fusants, a fusant ST723-F31 presented the best results in terms of the aromaticity of fragrance, the growth pattern, the resistance against salt and the degree of growth according to pH. It makes easy to control the production and the balance of aroma components so that it gives a good flavor, shortens the fermentation period and, simplifies the preparation process when using a bioreactor into which fusant is immobilized.

• 미생물학회지
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• 제22권1호
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• pp.35-40
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• 1984

Streptomyces속 균주개발의 수단으로서 이용할 목적으로 원형질체 융합방법의 확립을 시도하였다. 특히 융합빈도를 높이고 실험을 간편화하는데 역점을 두었다. 원형질체의 형성 및 재생빈도는 균의 배양시간에 따라 변하였는데 대수기에서 수확한 균체로부터 가장 높은 빈도의 수율을 얻었다. 원형질체의 형성은 다른 용균효소를 사용하지 않고 Lysozyme 단독처리 만으로도 충분히 가능하였고 원형질체의 세포막 재생은 Monolay법 보다는 Overlay법이 훨씬 좋은 결과를 주었다. Monolay법은 1.8%, Overly법은 14%의 재생빈도를 나타냈다. 본 실험에서 PEG1000 (50% W/V)를 사용한 원형질체 융합방법으로 얻은 S. coelicolor의 재조합체의 빈도는 $1.8 {\times} 10^{-2}$이었다.

• 미생물학회지
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• 제26권1호
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• pp.37-43
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• 1988

This research was focused on investigation of the condition for protoplast formation and regeneration of protoplast fusion between Saccharomyces cerevisiae which has fermentation ability and Candida cariosilignicola which can grow at high temperature and utilize methanol. The results obtained were as follows; The highest production was collected in exponential growth phase. Ninety-nine% protoplast formation of C. cariosilignicola was obtained in glycin-NaOH buffer (pH10.0) containing Zymolyase 0.5mg/ml at $35^{\circ}C$ for 1hr incubation. The highest regeneration was produced when protoplast wuwpension containing 0.5% soft agar in buffered 50mM $CaCl_{2}$ was poured as a soft overlay onto 2% agar plates. Equal amuont of protoplast suspension of two strains was mixed and centrifuged. The subsequent pellet was added to 2ml of 35% polyethylene glycol (MW 4,000) containing 50mM $CaCl_{2}$, and incubated at $30^{\circ}C$ for 10min. Then 0.1ml of the suspension of aggregated protoplast was immediately covered with minimal medium and incubated at $40^{\circ}C$ for 5-7 days. As results, $SC_{1}$, $SC_{2}$, and $SC_{3}$ fusants were obtained. The physiological characteristics of fusants produced by protoplast fusion were; $SC_{1}$, and $SC_{2}$ utilized maltose, galactose, methanol, potassium nitrate. $SC_{3}$ utilized all the above materials except galactose.