• The Korean Journal of Mycology
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• v.16 no.2
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• pp.70-78
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• 1988

To obtain basic information for the genetic analysis and breeding of Flammulina velutipes, some factors affecting the release of protoplasts from the fungus were studied. Potato Dextrose peptone Agar medium was suitable for the growth of the mycelium and the protoplast formation of F. velutipes. The culture age for the high yields of protoplast was 5 days on PDPA. Few protoplasts were formed from the mycelium cultured on Mushroom minimum Media. The highest yield of protoplasts was obtained in enzyme solution containing Novozyme 234 plus cellulase CP at 10 mg $ml^{-1}$ concentration, while a half amount of protoplasts was obtained in enzyme solution containing Novozyme 234 only. The optimal reaction time of the mycelium in the Iytic enzyme mixtures was 3 hours. The best osmotic stabilizer for the protoplast formation of the mycelium was 0.6M sucrose without buffer at pH 6.2.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.467-471
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• 1986

Yeast cell wall lytic enzyme was purified from Fusarium moniliforme by ammonium sulfate fractionation and gel column chromatography. The lytic activity was found to consist of three enzyme activities which were resolved on Sephadex G-100. The first peak on chromatogram exhibited proteolytic, lytic and laminarinase activities, and the second had both lytic and laminarinase activities, whereas the third peak was shown to contain lytic activity only. Three enzyme activities showed the synergistic effect and reducing agents accelerated the yeast roil wall lysis. This indicates that lytic, proteolytic and laminarinase activity acted cooperatively in the lysis of intact cells. Tannic acid precipitate of crude enzyme constituted of three enzyme activities had a high lytic activity on viable yeast cell and has proved useful in yeast protoplast formation.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• Korean Journal of Microbiology
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• v.22 no.2
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• pp.103-110
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• 1984

The conditions for the protoplast fusion of auxotrophic mutants of Trichoderma koningii were determined. A preparation of commercial enzyme Driselase was used successfully to isolate protoplasts from the 18 hr old mycelium of T. koningii. The yields of protoplasts production were ranged from $0.3{\times}10^8$ to $2.5{\times}10^8$ protoplasts per mg of damp mycelium of various auxotrophic mutant strains. The regeneration frequencies from $9.3{\times}10^{-3}\;to\;2.0{\times}10^{-1}$ were obtained when the protoplasts from auxotrophic mutants were plated on the malt extract medium containing 0.6M $MgSO_4$, and 2% agar, and the optimal concentration of PEG for protoplst fusion was 30%. Exposure of protoplasts to PEG for 10 min was found to be sufficient to induce high frequency heterokaryon formation. Optimal pH of fusion mixture was determined as 5.5, and 1 mM of calcium chloride in fusion mixture was found to be sufficient to enhance protoplast fusion frequency. Under optimal condition, the fusion frequency of the cross between protoplasts from various auxotrophic mutants were $1.6{\times}10^{-2}\;and\;4.1{\times}10^{-2}$.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.139-139
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• 2005

• Archives of Pharmacal Research
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• v.12 no.4
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• pp.249-253
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• 1989

High producers or blocked mutants of aminoglycoside antibiotic-producing Streptomyces spp. were selected by application of an agar plug method and by culturing individual colonies in broth. The productivities of aminoglycoside antibiotic producing organisms were increased by selection of a high producer from colonies obtained by spreading spores of wild strain, or survived from treatment of a mutagen or from the colonies regenerated from protoplast-formation and cell-wall regenerations. Some mutagen treated colonies lost the ability to produce antibiotics (5-8%). Some A-factor negative and deostreptamine or streptidine negative mutants were obtained by N-methyl-N'-nitro-N-nitrosomethylguanidine (MNNG) treatment. Many of the survivors from the MNNG treatment lost the ability to produce antibiotics. Major colonies produced less amount of antibiotics ; only few survived colonies produced more antibiotics than the parent. Resistance of Streptomyces spp. against the antibiotics produced by itself was also markedly affected by mutagen treatment.

• Korean Journal of Microbiology
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• v.29 no.1
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• pp.8-15
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• 1991

Interspecific hybrids between the ASpergillus spp., A. awamori, A. usamii and A. oryzae, were obtained by nuclear transfer technique. Nuclei isolated from an auxotrophic mutant strain were transferred into the protoplasts of a recipient strain of different species. The frequency of interspecific hybrid formation by nuclear transfer was $2*10^{-5}$ $-7*10^{-4}$ In contrast, no interspecific hybrid was isolated by protoplast fusion. Among the hybrids tested, 10 strains showed increased activity of some or all components of cellulases, xylanases and amylase up to more than two times. Isozyme pattern of the hybrids were analyzed by polyacrylamide gel electrophoresis and isoelectric focusing followed by activity staining, which showed that some of the hybrids have isozyme patterns unidentical to either of the two parents. By measuring the DNA contents and the sizes ofthe conidia, the karyotypes of the hybrids were estimated to be aneuploid near to haploid, diploid or triploid. It was concluded that the unclear transfer technique is much more efficient in the formation of interspecific hybrids than protoplast fusion and is very useful for the improvement of Aspergillus strains.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.137-144
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• 1985

Studies were conducted on the conditions for preparation of yeast protoplasts utilizing Hansenula anomala var. anomala FRI YO-32 as well as Saccharomyces cerevisiae KFCC 32356 and a lytic enzyme from the snail Helix pomatia. The cell wails of the strain FRI YO-32 and S cerevisiae were found to be resistant to activity of the snail lytic enzyme if they were not treated with thiol compounds. Dithiothreitol was found to be more effective than 2-mercaptoethanol, but the latter was considered to be practical. As factors influencing the formation of yeast protoplast, it was considered to be concentration and incubation time of 2-mercaptoethanol or the lytic enzyme, growth stages in yeast cultivation, initial number of yeast cells, and concentration of osmotic stabilizer (KCI). Optimum conditions for the preparation of yeast protoplasts were determined.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.197-203
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• 1986

Morphology and ultrastructure of protoplast fusion mode in Streptomyces lavendulae were studied by scanning and transmission electron microscopy. The isolated protoplasts were stable in some degree in hypertonic solution except that several protoplasts showed irregular morphology. Fusion events were occurred as follows; contact zone, fusion zone and separation zone were appeared sequentially. After formation of the separation zone, cytoplasm and DNA from both parents were mixed eventually. In the contact zone, two menbranes were still separated by electron transparent space. The contact zone changed to fusion zone by formation of fusion membrane that phospholipid molecules of two membranes were rearranged. Thereafter, nonmembraneous separation zone was formed by disappearance of fusion membrane. These changes were characterized by successive changes in typical membrane structure in fusion areas and by a progressive loss of bispherical shape.

• 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.