• Journal of Microbiology and Biotechnology
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• v.6 no.4
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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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• v.8 no.3
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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.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.1-7
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• 1989

To enhance the capability of starch fermentation of the transformant TSD-14, the heat treated protoplasts of TSD-14 were fused with the protoplasts of C. tropicalis (lys$^-$) in the presence of 30% (w/ v) PEG and 20 mM CaC1$_2$. Fusants were selected by nutritional complementation on minium medium and the fusion frequency was 4.4$\times$10$^{-5}$. All fusants tested were possessed of complemented traits concerning carbon compound assimilation, and the cell volumes of the fusants were approximately 1.5 times larger than the parental strains. The fusants were genetically very stable, and were able to hydrolyze alpha 1,4-glucosidic linkage as well as alpha 1,6-linkage of starch contrary to one of parents TSD-14, The most promising fusant FSC-14-75 produced 8.7% (v/v) of ethanol from 15% liquefied potato starch medium, but the result was enhanced to 9.3% (v/v) by addition of 0.3% peptone. The corresponding fermentation efficiency was 86.0%.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.174-179
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• 1991

Protoplast fusion was carried out between Brevibacterium flavum and Corynebacterium glutamicum. For the protoplast fusion, various mutants were isolated from Brevibacterium flavum ATCC 21493 and Corynebacteriurn glutamicum ATCC 21831. The optimum conditions for protoplast fusion of these mutants were examined. In the present work, the authors obtained a fusant, MWF 9031, by the intergeneric protoplast fusion between Brevibacterium flavum 108-125 and Corynebacterium glutamicum 41-214A, which was excellent in L-arginine fermentation. Fusant MWF 9031 was found to accumulate a large amount of L-arginine reached 32.5 mg/ml with a medium containing 10% glucose. The fusant possessed intermediate characteristics between the parental strains and the stability was found to retain for 60 days.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.5
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• pp.779-784
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• 1995

The effective production of 5'-GMP(5'-Guanylic acid) by immobilized 5'-GMP producing fusant RC102(intergeneric protoplast fusion between Brevibacterium ammoniagenes ATCC21263 and Corynebacterium glutamicum ATCC21171) was investigated. The Fusant RC102 was immobilized by entrapping in -carrageenan, agar, polyacrylamide or Ca-alginate. 3% k-carrageenan was selected as the most suitable matrix. In the production of 5'-GMP using the immobilized whole cells of fusant RC102, the optimum conditions were $32^{\circ}C$, pH 8.0, $30\mu\textrm{g}/L\;of\;Mn^{2+},\;1{\times}10^{-6}%\;of\;Zn^{2+}$. In order to use fermentation medium containing CSL(Corn Steep Liquor) plentiful in $Mn^{2+}$, the optimum conditions of penicillin G, D-cycloserine and POESA(polyoxyethylene stearylamine) for production of 5'-GMP were 0.8unit/ml, 0.8unit/ml, 0.8unit/ml and 5mg/ml, respectively. Cationic surfactant, POESA was effective and superior to the antibiotics, penicillin G or D-cyloserine in 5'-GMP productivity. The condinuous fermentation using immobilized fusant RC102 showed that 5'-GMP productivity was stable for more than 15 days.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.325-330
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• 1991

In order to develop yeast strains which can effectively produce ethanol from cellulosic hydrolyzates, protoplast fusion between Candida sp. KM-09-135 and Saccharomyces cweoisiue SM-07 was carried out and obtained the excellent fusant KMS-23. Fusant KMS-23 showed the optimal growth temperature and ethanol productivity at $37^{\circ}C$, and assimilated xylose, cellobiose, maltose and raffinose as fermentative sugars. Cell size of the fusant was about 1.2 times greater than that of KM-09-135 and 1.5 times SM-07. DNA content of fusant was 1.3 times higher than that of SM-07 and similar with KM-09-135. Fusant KMS-23 produced 2.57% (v/v) ethanol from saccharified wheat bran containing 6.44% (w/v) of reducing sugar, which was 1.3 times higher than parent strains under the same conditions.

• Korean Journal of Microbiology
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• v.31 no.3
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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.

• 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.