• Korean Journal of Food Science and Technology
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• v.25 no.4
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• pp.366-372
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• 1993

This study mainly designed to high quality of mirin production by using protopast fusion. In order to enhance the acid carboxypeptidase (ACPase) activity by the method of protoplast fusion. In order to enhance the acid carboxypeptidase (ACPase) activity by the method of protopalst fusion, the mutants, Aspergillus oryzae 9-12 and Aspergillus shirosamii IFO 6082-60 were selected by mutation among various mutants. Protoplast of Aspergillus oryzae 9-12 and Aspergillus shirousamii IFO 6082-60 were formed effectively by incubation of the mixtures of chitinase (10mg/ml), cellulase (10mg/ml) and zymolase 20T (5mg/ml). For protopalst fusion, the mixture of two mutant were fused to effective under the optimum conditions by solutions containing 30% PEG 6,000, 0.01M $CaCl_2\;2H_2O$, 0.6M KCl and 0.05M glycine. Fusion frequency was 0.71% and fusant, F-50 appeared ACPase activity of 20,800 unit/g which has 1.5 times higher than that of each mutants.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.6
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• pp.964-969
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• 1995

For the effective utilization of cellulosic biomass, conidial protoplast fusion between Aspergillus niger MAN-831(${\beta}-glucosidase$) and A. wentii MAW-538(CMCase and avicelase), which produced potently cellulolytic enzymes was carried out. Optimal conditions for formation and regeneration of protoplast were conidiospore age-5 dyuas. $2-DG-30\mu\textrm{g}/ml$, preincubation time-4 hours, osmotic stabilizer-0.7M KCl, novozyme(7mg/ml)＋driselase(2.5mg/ml) and reaction time of enzyme-5 hours. Optimal conditions for protoplast fusion were obtained by treatment of protoplasts with 15mM CaCl2 and 25% polyethylene glycol 4000(pH 6~7) as fusogenic agent at $36^{\circ}C$ for 25~30 minutes. The frequency was then $7.94{\times}10^{-4}$. CMCase, avicelase and ${\beta}-glucosidase$ activity of fusant F-208 strain was 1.5, 1.3, 1.2 times higher than those of parental strains, respectively.

• Korean Journal of Pharmacognosy
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• v.27 no.3
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• pp.231-237
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• 1996

The antitumor components of the protoplast fusants of Lentinula edodes and Ganoderma lucidum were examined for immunological activity to elucidate the mechanism of their antitumor activity. They did not show any direct cytotoxicity against tumor cells. But being examined for immunopotentiation activity, they increased the number of colonies in the bone marrow stem cells to 3.0 times. They also increased the activities of the acid phosphatase in activated macrophages to 2.1 times and the secretion of nitric oxide in RAW 264.7 to 2.2 times, respectively. They activated the components of the alternative complement pathway. In humoral immunity. they increased the activities of the alkaline phosphatase in differentiated B cells to 1.6 times and the number of plaque forming cells to 1.8 times, respectively. In cellular immunity, they restored the depressed response of delayed type hypersensitivity in tumor bearing mice to normal level.

• Korean Journal of Food Science and Technology
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• v.22 no.6
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• pp.611-617
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• 1990

This study was conducted to breed a high vitamin $B_{12}$ producer by the fusion of protoplasts between Bacillus natto and Bacillus megaterium. Auxotrophic mutants of Bacillus natto SH-34 ($thr^-try^-rif^r$) and Bacillus megaterium BK-13 ($arg^-ade^-lys^-str^r$) which showed high protease activity and production of vitamin $B_{12}$, respectively, were isolated for the fusion experiment. Protoplasts were induced by incubating the cells with lysis solution containing $500{\mu}/ml$ lysozyme, and the ratio of protoplast and regeneration formation were ranged from 99% and 67%, respectively. Fusion frequencies of fusants between Bacillus natto SH-34 and Bacillus megaterium BK-13 were appeared in the ranges of $1.0{\times}10^{-5}$ under the treatment of 30% PEG 6000 containing 3% PVP. The fusant, MNF-72 showed the highest product yield of $7.85{\mu}g/g-cell\;vitamin\;B_{12}$ in production medium. For the improvement of productivity, the immobilization of fusants with sodium alginate was carried out. In batch and continuous fermentation systems, the productivity were determined to be $0.58{\mu}g/ml.hr\;and\;0.80{\mu}g/ml.hr\;vitamin\;B_{12}$ under optimum condition, respectivity.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.88-93
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• 1989

To improve a new yeast strain capable of converting xylan to ethanol directly, we tried protoplast fusion between xylose fermenting yeast (Candida sp. X-6-41) and xylan assimilating yeast (Crypto-coccus sp. XB-33), finally selected the most promising two fusants (XFU-1 and XFU-2). As the optimum conditions for protoplast formation, the yeast cells were cultured to exponential phase in YPD and YPX containing 0.6M KCI, respectively, and then treated with zymolyase (0.25mg/$m\ell$), cellulase(4mg/$m\ell$) and 100mM 2-mercaptoethanol at pH 8 and 3$0^{\circ}C$. The protoplasts of parental auxotrophs were fused in the presence of 20mM CaCl$_2$and 40% polyethylene glycol(M.W.4000). The physiological and morphological characteristics of the fusants, such as assimilation of carbon sources, cell size, growth rate, xylanase activity and xylan fermentation ability were investigated. Xylanase activity of fusants that cultured in chemically minimal medium was higher than that of fusants that cultured in completed medium, because xylanase producing activity of xylose fermenting yeast(X-6-41) was inhibited by isoleucine.

• 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 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.3
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• pp.160-163
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• 1992

Killer toxin from killer yeast fusant FWKS 260 developed by protoplast fusion between the wild killer yeast and alcohol-fermenting yeast was purified by ammonium sulfate fractionation. Amicon PM I0 concentration. Sephadex G-200 and Scphadcx G-75 column chromatography. The purified killer toxin showed a single band by SIX-polyacvlamide gel electrophoresis. The protein part of killer toxin was active site. which was found by treating the proteolytic enzyme such as pronase E and pepsin to killer toxin. The killer toxin was stable at pH 2.0-5.0 and 20$^{\circ}$C. but inactivated with increasing temperature. The molecular weight was determined to be approximately 13.000 according to the results obtained from the SDS-polyacrylamide gel electrophoresis. It was confirmed that the purified killer toxin is glycoprotein by showing a red single band after st'tining with Schiffs reagent.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.142-149
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• 1988

This experiment was carried out to obtain a hybrid with potent ethanol fermenting ability, by means of protoplast fusion between a thermophilic strain (D-71) of Saccharomyces cerevisiae and an osmotolerant strain (SR-S) of Zygosaccharomyces rouxii. The conditions for formation of protoplasts from both strains and for their fusion and regeneration were studied. Favorable conditions for formation of protoplasts from Saccharomyces cerevisiae D-71 were : treatment of the cells at late-exponential phase with 2-mercaptoethanol (l% v/v) for 10 minutes in the presence of 0.5M sorbitol, then incubation for 60 minutes in the set medium containing Zymolyase-20T (4mg/$m\ell$) ; and from Zygosaccharomyces rouxii SR-S were : treatment of the cells at mid-exponential phase with 2-mercaptoethanol (1% v/v) for 10 minutes in the presence of 0.5M or 1M mannitol, then incubation for 120 minutes in the set medium containing Zymolyase-20T(4mg/$m\ell$). The protoplasts of parental cells were fused in the presence of 20mM CaCl$_2$, 0.5M sorbitol and 40% of polyethyleneglycol (M.W 4000), then fusants obtained were selected as regenerated colonies which embedded and grown in the minimal medium containing 3% of agar. The frequencies of fusant formation were 1.2$\times$10$^{-6}$ to 9.1$\times$10$^{-6}$ for the regenerated protoplast.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.275-281
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• 1988

To develop cellulase and xylanase-producing strain by protoplast fusion, alkalophilic Bacillus sp. F204 and K17 were treated with NTG(N-methyl-N'-nitro-N-nitrosoguanidine) and isolated anti-biotics resistant strains of S20 (Km$^r$ , Cm$^r$) and G70 (Str$^r$). The frequency of protoplast formation was about 95% when cells of mid-log phase were treated with 200$\mu\textrm{g}$/ml Iysozyme at 37$^{\circ}C$ for 30-45 minutes. Under addition of 0.4-0.5M sodium succinate, 0.5% casamino acid, 1.5% polyvinylpyrrolidone, 25mM MgC1$_2$ and 50mM CaC1$_2$ to the regeneration medium, the regeneration frequency of Bacillus sp. F204 and K17 was 24.9% and 26.2%, respectively. The fusion frequency was 6.6$\times$10$^{-6}$ in the presence of 30% polyethylene glycol 6000 containing 50mM $Ca^{++}$ at 45$^{\circ}C$ for 5 minutes. Cellulase complex and xylanase activities of fusant were compared with parental strains.