• Korean Journal of Microbiology
• /
• v.19 no.4
• /
• pp.192-198
• /
• 1981

Protoplasts from Trichoderma koningii were fractioned at varing digestion periods. There were distinct differences between the fractionated protoplasts in CMCase activity and protein content ; namely, protein contents and CMCase activities in the early-protoplasts were higher than the late-produced protoplasts. Reversion frequencies of the early-produced protoplasts. Reversion frequencies of the early-produced protoplasts (0-1hr.) and the late-produced protoplasts (1-2.5 hr.)were 1.25% and 0.56%, respectively. From these results it was assumed that the early-produced protoplasts were more active in physiological metabolism. Meanwhile, in osmotically stabilized liquid medium two distinct reversion patterns of these protoplasts were found. In the first, normal germ tube was developed from the opposite side of the protoplast after the production of an aberrent tube. In the second, reversion occurred through germination of protoplast, but this patterns was uncommon. It is suggested that the the physiological and morphological heterogeneities in protoplast reversion are related to the heterogeneous origins of protoplasts from highly differentiated mycelium.

• Korean Journal of Microbiology
• /
• v.24 no.2
• /
• pp.91-97
• /
• 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 Life Science
• /
• v.13 no.2
• /
• pp.143-149
• /
• 2003

This experiment was undertaken to investigate proper conditions for protoplast isolation and regeneration from the mycelia of Lyophyllum ulmnrium. Protoplast isolation and regeneration are influenced by a variety of factors such as enzyme, osmotic stabilizer, reaction time and age of mycelia. A combination of Novozyme 234 (10mg/ml) and cellulase Onozuka R-10 (10 mg/ml) with 0.6 M $MgSO_4$ was most effective for isolation of the protoplasts. The optimum reaction time of the mycelia with the lytic enzymes was 3.5~4 hours at $28^{\circ}C$ in shaking condition at 120 strokes per min. High yield of the protoplasts were obtained from its 4~5 days old mycelia on complete agar media. Its protoplasts were regenerated to normal hyphae. Regeneration media with 0.6 M sucrose were proper for regeneration of the protoplasts. Their regeneration frequency on complete agar media was 2.3~2.7%.

• Journal of Microbiology and Biotechnology
• /
• v.3 no.1
• /
• pp.24-30
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.29 no.1
• /
• pp.12-17
• /
• 2001

Aspergil-lus coreanus NR-15 and Aspergilus oryzae NR-2-5 from traditional Korean Nuruk were selected as parental strains producing starch hydrolysis enzyme. Xll(Arginine-) mutant from A. coreanus NR 15-1 showed high glu-doamylase activity and total acid productivity. Z6(Adenine-) mutant from A. oryzae NR2-5 showed the highest $\alpha$-amylase activity. Therefore, both XII and Z6 mutants were selected and investigated for the optimal conditions of protoplast formation for protoplast fusion. Mixture of equal amount of cellulase and driselase(10mg/ml each) was the most effective as lytic enzymes. The optimal pH and temperature for protoplast formation were 5.0 and $30^{\circ}C$, respectively. The most effective reaction for protoplast formation time was 4 hours. The maximum of protoplst for- mation of Xll mutant and Z6 mutant were $6.54$\times$10^{7}$ protoplasts/ ml and $3.04$\times$10^{ 7}$ protoplasts/ml, and the regen-eration frequencies of the protoplasts were 11.3% and 11.6%, respectively. The size of the protoplasts from X11 and Z6 mutants were 3~6 $\mu\textrm{m}$ and 4~9$\mu\textrm{m}$, respectively.

• Applied Biological Chemistry
• /
• v.28 no.1
• /
• pp.13-18
• /
• 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 the East Asian Society of Dietary Life
• /
• v.5 no.1
• /
• pp.93-99
• /
• 1995

For the improvement of the L-lysine productivity of Brevibacterium flavum and Corynebacterium glutamicum, fusants were induced by interspecific protoplast fusion of Bacillus subtilis with C. glutamicum and B. flavum. The following results were obtained through protoplast formation of strains condition of protoplast fusion, characteristics of the fusants, and the productivity of lysine form starch. B. flavum BF-5 and C. glutamicum protoplasts were made by the treatment of 0.3unit/$m\ell$ of penicillin G at the early stationary growth phase for 2 hours followed by incubation with 10mg/$m\ell$ of lysozyme at 37$^{\circ}C$ for 120 min. When a mixture of the protoplast was treated with 30% PEG(M.W.6,000) solution containing 50mM CaCl2 at optimal conditions, the intergeneric fusion frequency between protoplasts of C. glutamicum CG-2 and B. subtilis BD 224 was 7.1${\times}$105. The genetic properties on the L-lysine producing fusants were compared with those of parental strains. As a results, the intergeneric fusants were completed in each auxotrophic requirement, resistances for S-(2-amino-ethyl)-L-cysteine and kanamycine were confirmed. And one of fusants selected, FBB-41 were found to be genetically stable fusants. The aspartokinase activity of FBB-41 strain increased than that of the parent strain.

• Microbiology and Biotechnology Letters
• /
• v.22 no.2
• /
• pp.143-151
• /
• 1994

In the course of the study on strain inprovement by protoplast fusion, Lactobacillus acidophilus 88 protoplasts production and regeneration conditions were investigated. This strain produced a bacteriocin that revealed strong inhibitory activity against various indicator strains, especially L. helveticus CNRZ 1096. Protoplasts of L. acidophilus 88 strains were very efficiently obtained by treatment with 125 $\mu $g/ml lysozyme in a protoplast forming buffer containing 20 mM N-2 hydroxy-ethtl-piperazine-N'-2-ethane-sulfonic acid(HEPES, pH 7.0) and 1M sucrose at 37$\circ $C for 30 min. Hovever, treatment with mutanolysin was not effective for the production of L. acidophilus 88 protoplasts under the same conditions. High protoplast yield was obtained form the cells at the middle to late logarithmic growth phase in the de Man, Rogosa and Sharpe(MRS) medium. Regeneration was efficiently accomplished with the MRS medium containing 10% sucrose.

• Archives of Pharmacal Research
• /
• v.20 no.3
• /
• pp.206-211
• /
• 1997

The optimal conditions for the production and regeneration of the protoplasts from Lentinula edodes were studied. Protoplast formation from the mycelia of L. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at $30^{\circ}C$ and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or $MgSO_{4}$ More than 90% of the protoplasts contained nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was $3-5{\mu}m$ and it had a well defined cell structure.

• Korean Journal of Microbiology
• /
• v.27 no.4
• /
• pp.422-429
• /
• 1989

Auxotrophic mutant were isolated from wild types by the treatment with NTG as a mutagen, and the conditions of protoplast formation for them were established. The protoplasts of killer yeast Saccharomyces cerevisiae K52 were formed to the level of above 70% when cells grown for 20 hr in PM medium were treated with 200 unit/ml Lyticase 50,000 at $30^{\circ}C$ for 60 min after pretreatment of 50 mM 2-mercaptoethanol in 10mM potassium phosphate buffer (pH 7.5) containing EDTA and 0.6 M sorbitol for 15 min. Also, the protoplast of the recipient S. cerevisiae S 29 were formed to the level of above 85% as it was cultured to the log phase of 24 hr in PM medium under the same conditions. The fusion frequency between the protoplast of killer yeast S. cerevisiae K 52 and the protoplast of recipient S. cerevisiae S 29 was reached to $8.2\times 10^{-6}$ when the hypertonic regeneration medium embeded with the fused protoplasts after mixing the parental protoplasts to 10$^{8}$ cells/ml in SP buffer containing 20 mM $CaCl_{2}$ and 30% PEG 6,000 for 15 min at $30^{\circ}C$ were incubated.