• Microbiology and Biotechnology Letters
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• v.21 no.2
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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.

• Korean Journal of Microbiology
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• v.22 no.3
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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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• v.29 no.1
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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.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.6-13
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• 1992

- Protoplast fusion between lincomycin resistant Lactobacillus casei KCTC 1121 and rifarnpicin resistant Lactobacillus delbrueckii JK-414 was attempted to obtain the improved strains. Protoplasts of L. casei and L. delbrueckii were produced by mutanolysin digestion at $42^{\circ}C$ for 15 min. L. casei cells were converted to protoplasts by treating with 5 $\mu g$ / m l of mutanolysin in 20 mM HEPES buffer (pH 7.0) containing 0.75 M sucrose at the middle logarithmic growth phase. In case of L. delbrueckii 1.0 M sucrose was used osmotic stabilizer. Regeneration of protoplast in both strains was efficiently accomplished on the regeneration medium containing 10% sucrose, 6 mM $MgC1_2, 6 mM CaC1_2$, and 2.5% gelatin. Protoplast fusion between L. casei and L. delbrueckii was carried out in the presence of 40% of PEG 4,000. The frequency of protoplast fusion was found to be about $3.2\times 10^4$. Acid production of L. casei was better than that of L. delbrueckii. Among fusants, F23 and F35 exhibited excellent lactic acid production. F23 and F24 exhibited the improved proteolysis compared to that of the parent strains and they had twice as much as DNA content of the parents.

• The Korean Journal of Mycology
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• v.21 no.4
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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.

• Korean Journal of Microbiology
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• v.29 no.2
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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.

• Korean Journal of Microbiology
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• v.24 no.2
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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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• v.3 no.1
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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.

• Korean Journal of Microbiology
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• v.22 no.1
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• pp.35-40
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• 1984

Attempts were made to optimise protoplast formation and regeneration methods to improve the protoplast fusion frequencies of Streptomyces coelicolor. The yields of protoplast formation and regeneration were varied with different growth phase of the culture. Maximum yields were obtained when cells were taken from the late logarithmic phase. Protoplast formation reached almost its maximum with lysozyme treatment at a concentration of 2mg/ml without any other lytic enzyme. A high frequency of protoplast regeneration was accomplished by overlay method: the method gave 14% recovery of regenerated protoplast versus 1.8% recovery for monolay method. A recombinant frequency of 1.8X10^-2 was obtained by protoplast fusion using PEG 1000(50% w/v).

• Korean Journal of Microbiology
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• v.26 no.1
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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.