• Applied Biological Chemistry
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• v.28 no.1
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• pp.13-18
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• 1985

To investigate the fusion of bacterial protoplasts, two auxotrophic mutants of Bacillus subtilis were isolated after treatment with nitrosoguanidine. Two auxotrophs were uracil-auxotroph and tryptophan-auxotroph. Back mutation frequencies of $ura^-$ and $trp^-$ -auxotrophs were $2.4{\times}10^{-8}$ and less than $1.0{\times}10^{-8}$, respectively. The optimal pH and temperature of protoplast formation with lysozyme were 6.5 and $30^{\circ}C$. The optimal lysozyme concentration was $200{\mu}g/ml$. The regeneration frequency of protoplast was 3.3%.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• KSBB Journal
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• v.6 no.1
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• pp.63-68
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• 1991

An auxotrophic regulatory mutant of Corynebacterium glutamicum ATCC 21674 produced 2.1-3.4g/1 of phenylalanine with 2.9-4.4g/l of tyrosine in the batch shake flask fermentations. At higher sugar concentration, the production of both amino acids was lower than that at low sugar concentration. There was a pronounced effect of temperature on the amino acid production. At $30^{\circ}C$, much higher levels of phenylalanine and tyrosine were produced than those at $37^{\circ}C$. The pH decrease in the shake flask fermentation was so fast that it was impossible to maintain a constant pH with calcium carbonate as a buffering agent. Even though the strains we have used are reported as tyrosine auxotrophs, they produced tyrosine and were able to grow on the minimal medium where no tyrosine was present.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.1
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• pp.11-18
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• 1997

This study was to provide the basic data in improving protoplast formation and regeneration of antagonistic bacteria against phytopathogenic fungi and pest. The antagonistic rhizobacterium, BS 101, against Rhizoctonia solrmi and Fusurium oxyspomm was isolated and identified as Bacillus subtilis. Another bacterium for protoplast formation and regeneration was B. thuringiensis subsp. kurstcJtiHD-l (BT 37669) which have insectcidal toxin in the orders Coleopteria, Dipteria etc.. Auxotrophic mutants, BS 1013 and BT 69, were isolated by treating with NTG 300 ug/ml for 40 min. at $37^{\circ}C$, and with NTG 300 ug/ml for 30 min. at $37^{\circ}C$, respectively. The BS 1013 and BT 69 were converted to protoplas by treating with lysozyme 300 ugh1 for 30 min. at 37C, and lysozyme 9 mglml for 60 min. at $37^{\circ}C$, respectively. The fequencies of the protoplast formation of BS 1013 and BT 69 were 90.00 and 92.83% respectively, after 1~2 day at $37^{\circ}C$. The regeneration kequencies of the protoplasts BS 1013 and B T 69 were 0.52 and 0.10%, respectively, after 4~6 days at $37^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.23-30
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• 1994

Intergeneric hybrids formed between Aspergillus oryzae var oryzae and Penicillium verruculosum F-3 were obtained by nuclear transfer technique. Several auxotrophic mutants isolated fromconidio-spores of the two strains mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were 3$\times $10$^{-5}$ $~$1$\times $10$^{-5}$. From observation of genetic stability, conidial size, DNA content, nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrid posses the 1.3$~$2.2 fold higher amylase activities than those of parental strains. It was also revealed that some hybids had different isozyme patterns compared to those of parental strains by amylase activity assays.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.45-49
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• 1998

Two kinds of Mutants of Corynebacterium glutamicum, which were resistant to branched chain amino acid analogues, were obtained for L-leucine production; C. glutamicum LT26 resistant to 4-azaleucine and $\alpha$-amino-$eta$-hydroxyvaleric acid, and from which C. glutamicum LT3811-70 resistant to DL-4-thiaisoleucine were derived. Accumulation of L-leucine in the culture broths of these mutant strains, C. glutamicum LT26 and LT3811-70, were much higher than those of their parent strains even though they were non-auxotrophic mutants. Enzymatic analyses were performed to measure the activities of $\alpha$-acetohydroxy acid synthase (AHAS) and $\alpha$-isopropylmalate synthase (IPMS), which were the key enzymes for the L-isoleucine, L-valine and L-leucine biosynthetic pathways branching from a common precursor. In C. glutamicum LT26 and LT3811-70, AHAS and IPMS were found to be derepressed and desensitized to L-leucine. In addition, in C. glutamicum LT3811-70, IPMS was further more derepressed by L-leucine and AHAS was more desensitized by L-isoleucine and L-valine compared to its parent strain, C. gIEitamicum LT26.

• 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.26 no.4
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• pp.290-296
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• 1998

Interspecific hybrids between Aspergillus oryzae var. oryzae and Penicillium chrysogenum (Tyr$\^$-/), high alkaline protease producing fungi, were obtained by nuclear transfer technique. Nuclei isolated from the wild type Aspergillus oryzae var. oryzae strain were transferred into auxotrophic Penicillium chrysogenum mutants and selected the new strains showing an increased protein degrading capability. Maximum production of protoplasts were obtained by 1% Novozym 234 at $30^{\circ}C$ for 3 hours and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6M KCl. Frequencies of hybrid formation by nuclear transfer were 1.3${\times}$10$\^$-3/∼2.8${\times}$10$\^$-3/. They could be suggested as an aneuploid by the observation of genetic stability, conidial size, DNA content, and nuclear strain. The hybrids showed 1.1～2.2 fold higher alkaline pretense activities than parental strains.

• The Korean Journal of Mycology
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• v.18 no.2
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• pp.77-83
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• 1990

Factors influencing the fusion frequency of protoplasts were investigated with auxotrophic mutants of Pleurotus florida and Pleurotus ostreatus. Immediately after the polyethylene glycol (PEG) solution was added, the protoplasts adhered firmly and shrank. During the subsequent dilution with 0.6 M sucrose, the protoplasts regained their normal size and larger bodies were observed. Interspecific heterokaryons were obtained by fusion of the nutritionally complementing protoplasts. Hyphae of the heterokaryotic fusants formed true clamp connections. The optimum conditions were a total of 1 to 15 million protoplasts per ml, 30% polyethylene glycol 8000 solution with adjustment to pH 8.0 and 0.6 M sucrose stabilized regeneration medium. Other parameters such as $CA^{++}$, glycine, exposure time and temperature influenced mainly the viability of the protoplasts.

• The Korean Journal of Mycology
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• v.27 no.6 s.93
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• pp.399-405
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• 1999

Intergeneric hybrids between Saccharomyccopsis fiburigera KCTC 7393 and Saccharomyces cerevisiae KCTC 7049 (tyr-, ura-) were obtained by nuclear transfer technique. Nuclei isolated from the wild type S. fiburigera strain were transfered into auxotrophic S. cerevisiae mutants and new strains showing an increased starch degrading capability were selected. Maximum production of protoplasts was obtained from the treatment with 0.1 % Novozym 234 at $30^{\circ}C$ for 90 min, and most effective osmotic stabilizer for the isolation of protoplasts was 0.6 M KCl at pH 5.8. The frequency of protoplast regeneration was 14.64% under the conditions. Genectic stability, conidial size, DNA content, and nuclear stain suggested that the fusants were aneuploidy. The specific activity of ${\alpha}-amylase$ was observed to increase about $1.2{\sim}1.9$ folds.