• Korean Journal of Microbiology
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• v.36 no.4
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• pp.259-266
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• 2000

Spheroplast cell fusions were performed with Flavimonas oryzihabitans degrading aniline and Pseudomonas sp. degrading 4-chlorobiphenyl to develope the new fusant degrading aniline and 4-CBP and its characters were investigated. F. oryzihabitans was induced to antibiotic marker ($Cm^r$ by NTG treatment for the fusants selection. The results of spheroplast formation and regeneration frequencies of the strains treated with lysozyme-EDTA were 99% and 5.0~6.6%, respectively. Fusion products were treated with 40% (v/v) PEG 6000 and fusion frequency was $3.16{\times}10^{-4} $. The DNA content of fusant, F22 was approximately 2-fold compared with parents. The fusant was stable, and showed the mixed biochemical characteristics of the parent strains. F22 was similar to parent for cell growth pattern and degrading capacity on 5 mM aniline but cell growth rate of F22 was 1.5-fold higher than that of the parent on 10mM aniline. However 4-CBP degrading ability of F22 was slightly lower than that of parental strain.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.105-110
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• 1988

The production of single cell protein using the amylolytic fusant obtained from cell fusion between Hansenula anomala and Saccharomyces cerevisiae was studied. The fusant12 strain was selected for single cell protein production from starchy materials among five fusants. Optimum nitrogen source and its concentration for the growth of fusant12 were ammonium sulfate and 0.1%, respectively. Optimum concentration of soluble starch and optimum pH of the basal medium were lord and pH 5.6, respectively. Autolysis of fusant12 was effectively carried out by addition of 5% (v/v) ethyl acetate to the cell suspension and liquidization for 30 min before incubation for 24 hr at 3$0^{\circ}C$. Coculture of fusant12 and non-amylolytic yeast, Torulopsis candida YA-l5, resulted in the increase of the mass as compared to the monoculture of fusant12. The cell mass on tapioca medium was increased about 2.5 times as on soluble starch medium. The content of crude protein and nucleic acid of the dry cell were 39% and 5.8%, respectively.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.311-318
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• 1986

Glucoamylase and ethanol productivities of HSDD-170 and HSDM-119 formed by S. cerevisiae and S. diastaticus protoplast fusion were investigated. For the production of the glucoamylase, soluble starch as carbon source, yeast extract and C. S. L as nitrogen source added into the basal medium were favorable. The production of the enzyme reached at maximum after cultivation of the fusant for 4 days at 3$0^{\circ}C$, aerobically. The properties of glucoamylase produced by fusants were very similar to those produced by S. diastaticus as based on optimum temperature, pH stability. In alcohol fermentation from starch, strain HSDD-170 fermented starch faster than either of its parental strains. The maximum of alcohol yield in 15% of liquefied potato starch was 7.5% (v/v).

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.104-111
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• 1987

As a trial method of breeding L-lysine producing strains, the intraspecific protoplast fusion bet-ween Brevibacterium flavum ATCC 21528R and Brevibacterium flavum ATCC 21529S and the intergeneric protoplast fusion between Brevibacterium flavum ATCC 21528R and Corynebacterium glutamicum ATCC 13058S were performed. The optimum conditions for protoplast formation of these strains were examined and the effect of plasma expander on regeneration and/or fusion was also observed. Both fusants No. CH23 and No. CH4l showed higher productivity of L-lysine than those of parental cells under the optimum cultural conditions at a rate of 21％ and 8.9％, respectively. And, activity of several enzymes in L-lysine biosynthetic pathway including aspartokinase, a rate-limiting enzyme, was determined. Besides, metabolic control mechanism of L-lysine biosynthesis in fusant No. CH23 and in No. CH41 was investigated to compare with that of parental strains.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.164-169
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• 1988

Protoplast susion between Zygosaccharmoyces rouxii M-12 and Saccharimyces cerevusuare M-43 were investigated for breeding of a new brewing yeat strain for soy sauce. Auxotrophic mutants of Zygosaccharomyces rouxii ZRM-83 ($Met^-,\;Thr^-$) and Saccharomyces cerevisiae SCM-46 ($Lys^-,\;Arg^-$) were selected by treatment of 3.0% ethylmethane sulfonate and nutritional complementary method. Protoplast of both strains were more effective by treatment of 0.05mg/ml zymolase 20T for 60min. Fusion effeciency was much higher by treatment of 30% PEG 6,000 for 30min and fusion frequencies were $10^{-4}{\sim}10^{-5}$. These fusants originated from two protoplasts had properties of big cell size and much DNA content.

• Journal of The Korean Society of Grassland and Forage Science
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• v.18 no.2
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• pp.133-142
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• 1998

This study was conducted to investigate the effects of antagonistic bacteria and pathogenic fungi on the growth of tall fescue(Festuca arundinacea Schreb.) in continuous cropping soil(CCS) and non-continuous cropping soil(NCCS). Tall fescue was established by seeding into pots of 11 cm in diameter and 9 cm in depth containing 1 : 1 mixture of soil and vermiculite, and cultivated at pots with antagonistic bacteria and pathogenic fungi in a vinyl house. The bacteria used in this study were Bacillus subtilis and hsants. B. subtilis was isolated and identified kern forage rhizosphere soil and fusants were isolated through cell hsion from B. subtilis and B. thwingiensis. B. subtilis was named as B. subtilis 101 and hsants were named as F-3, F-7 and F-8. In dark culture experiment, tall fescue inoculated with the antagonistic bacteria lived longer than that of control in both CCS and NCCS. However, tall fescue of CCS lived shorter than that of NCCS. Dry weight of tall fescue inoculated with the antagonistic bacteria was higher than that of tall fescue inoculated with pathogenic hngi in both CCS and NCCS(P< 0.05), and the antagonistic bacteria showed positive effects on the growth of tall fescue. However, Dry weight of tall fescue was decreased by the inoculation of the pathogenic b g i in both CCS and NCCS(P< 0.05).

• Korean Journal of Food Science and Technology
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• v.21 no.1
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• pp.154-163
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• 1989

This studies were designed to improve the productivity of L-lysine by protoplast fusion and immobilized system of fusants using strains of Brevibacterium flavum ATCC 21528, Brevibacterium lactofermentum ATCC 21086 and Corynebacterium glutamicum 820. Mutants were isolated with concentration method of $300{\mu}g/ml$ penicillin-G after treatment of $250{\mu}g/ml$ N-methyl-N-nitro-N-nitrosoguanidine. B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;AEC^r)$ and C. glutamicum 57-5$(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ were isolated from mutants. Protoplasts were induced by being incubated with $500{\mu}g/ml$ lysozyme of lysis solution for 6 hr and the ratio of protoplast formation and regeneration were ranging from 97-99% and 33-37%, respectively. Fusion frequencies of fusants of BBFL 21, BCFG 37 and BCLG 59 were shown in the range from $1.25{\times}10^{-6}\;to\;5.83{\times}10^{-7}$ under the optimum conditions. The fusant BBFL 21 showed the highest productivity of $411.1\;ng/ml{\cdot}hr$ L-lysine in the lysine productivity broth at $30^{\circ}C$ for 72hr. In the immobilization systems, fusant BBFL 21 was employed in various polymer matrices such as sodium alginate, polyacrylamide, agar and ${\alpha}-carrageena$. The immobilization of sodium alginate showed the highest productivity of $413\;ng/ml{\cdot}hr$ L-lysine in the batch system. Continuous fermentation of immobilization system by using tube fermentor was produced the highest productivity $416.7\;ng/ml{\cdot}hr $ L-lysine under optimum condition.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.546-552
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• 1992

Ethanol-tolerant strain, S. eerevisiae BUI a26 ($alc^r thr^-$) and gJucoamylase-producing strain, S diastatieus AI5a6 (STA+ hom-) were prepared by means of genetic manipulation, Protoplast fusion was carried out to introduce STA gene from AI5a6 strain to BUla26 strain, Protoplast formation was shown at 0,8 M sorbitol and 200 Jig/ml to 400 Jig/ml zymolyase treatment for 2 hours incubation, Fusion frequency was $ 3.25 {\times} 10^{-3}$ to the regenerated protoplast number using PEG 6000 for 90 min incubation. The excellent fusants with genotype of STA- $alc^r thr^-$ hom+/STA+ ($alc^s thr^+$ hom- (2n), F7 and FIO, were selected by ethanol-tolerant, ethanol fermentation, and glucoamylase production tests, Glucoamylase production of AI5a6 showed 2,7 units, but 4.2 or 8.4 units for F7 or FIO fusant at $30^{\circ}C$, Ethanol fermentation from 32% glucose by BUla26 was 14,0%(v/v) in fermentaion medium for 5 days incubation, but 14.5% or 15,0% for F7 or FIO strain, respectively. Ethanol fermentation from 5% starch was 2,0% by F7, or 1.8% by FIO strain in fermentation medium for 5 days fermentation.

• Korean Journal of Microbiology
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• v.31 no.4
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• pp.318-325
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• 1993

The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at $37^{\circ}C$ for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were $2.0{\times}10^{-5}~3.6{\times}10^{-5}$. All tested recombinant clones were very stable on further propagation.

• The Korean Journal of Mycology
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• v.20 no.4
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• pp.324-336
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• 1992

On the five interspecific protoplast fusants of Ganoderma lucidum and G. applanatum was the antitumor test performed. The fusant F-2 was selected, to examine the cultured mycelia (protein bound polysaccharide) as antitumor components. When a dose of 20 mg/kg/day of each components purifed from F-2 fusant was, i.p., injected into ICR mice, the inhibition ratio of Fr. II against the solid form of sarcoma 180 increased to 1.5 times as compared with that of their parents. When Fr. II was examined for immunopotentiation activity, it increased the amount of the superoxide anion in activated macrophages to 1.2 times and the count of hemolytic plaque forming cells in the spleen to 4.3 times as compared with that of each control group. Its chemical analysis showed 85.2% polysaccharide which consisted of glucose, galactose, mannose, fucose and xylose, and 0.39% protein of 15 amino acids. The content of hexosamine was 0.39% and the molecular weight of Fr. V was $5.6{\times}10^4$ dalton.