• Korean Journal of Microbiology
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• v.22 no.3
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• pp.175-181
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• 1984

In order to develope a protoplast fusion system for industrial coryneform bacteria, the optimum conditions for the formation and regeneration of progoplast were examined for Brevibacterium flavum and Corynebacterium glutamicum and the protoplast fusion was performed. For the formation of the protoplast of B. flavum and C. glutamicum, the optimum time for penicillin G. treatment to obtain protoplast was mid-exponential growth phase ($O.D_{580}=0.6-0.8,\;8.0{\times}10^7-1.0{\times}10^8cell/ml$). At the optimum conditions (0.3units/ml penicillin G and $400{\mu}g/ml$ lysoyme for treatement), frequencies of protoplast formation and protoplast regeneration were 99% and 25%, respectively. Protoplast regeneration frequency was highest under the optimum conditions for the protoplast formation. Addition of 25mM $Mg^{2+}\;and\;50mM\;Ca^{2+}$ to the regeneration medium further increased the regeneration frequencies. The protoplast fusion frequencies of B. flavum and C. glutamicum in intraspecies fusion were $1.0{\times}10^{-8}\;and\;7.8{\times}10^{-4}$, of the regenerated protoplast respectively, when 33% of PEG (polythylene glycol) 6,000 was used as the fusing agent.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.359-364
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• 1995

Two lactic strains, Leuconostoc mesenteroides Lu5 and Pediococcus pentosaceus P1 isolated from Kimchi, were used to determine the optimum conditions for protoplast formation and regeneration. The maximum protoplast formation rate was obtained with both strains at early exponential growth phase and decreased rapidly during growth phase. For P. pentosaceus P1, 30 $\mu$g/ml of lysozyme treatment was sufficient to obtain over 90% of protoplast formation and 300 $\mu$g/ml for L. mesenteroides Lu5, showing great difference in sensitivity of these strains to lysozyme. For both strains, best results were obtained at pH 7, using 0.5 M sucrose as osmotic stabilizer. For regeneration of protoplast, the highest regeneration rate was obtained after 15 minutes of lysozyme treatment and declined drastically with prolonged digestion.

• Archives of Pharmacal Research
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• v.10 no.3
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• pp.158-164
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• 1987

To obtain a new strain of Ganoderma lucidum by protoplast fusion technique, its protoplast formation and regeneration were studied. Several factors affecting the protoplast formation and regeneration were investigated to find their optimum conditions. The mycelium was grown for four days on the cellophane membrane placed on G. Incidum complete medium (GCM). When various commercial lytic enzymes were examined for protoplast isolation, the combination of Novozym 234 and $\beta$glucuronidase was found to be effective. An osmotic stabilizer, 0.6 M sucrose in 20 mM phosphate buffer pH 5.8, gave the highest yield of protoplasts. Three-hour incubation in shaking incubator was most suitable for releasing protoplasts. To increase the protoplast yield, pretreatment with 2-mercaptoethanol was carried out. The regeneration frequency in GCM containing 0.6M MgSO$_4$ 7$H_2O$ was shown to be 0.66%.

• The Korean Journal of Mycology
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• v.21 no.1
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• pp.1-8
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• 1993

ABSTRACT: Factors responsible for protoplast formation and regeneration of Phytophthora capsici were examined. Protoplasts were successfully liberated from the mycelial culture by digestion for 6-9 hrs with Novozym 234 in 0.35 M $CaCl_2$, (pH 5.7) as osmotic stabilizer. Young rapidly-growing mycelium (24 hrs old) showed highest protoplast yields. High concentrations of Novozym 234 were effective in releasing protoplasts from the mycelium. The combination of 0.4 M mannitol and 0.1 M $CaCl_2$ was optimal osmotic stabilizers for protoplast regeneration. The synthetic Henninger media containing all nutritional elements gave the best regeneration rate. The protoplast regeneration was greatly inhibited in the media which were not supplement with amino acids or ${\beta}-sitosterol$. Certain amino acids such as L-aspartic acid and L-glutamic acid remarkably enhanced protoplast regeneration. However, the addition of microelements did not affect protoplast regeneration.

• Microbiology and Biotechnology Letters
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• v.21 no.2
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• pp.101-106
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• 1993

The optimal conditions for the production and regeneration of L. helveticus protoplasts were examined. The protoplast formation of L. helveticus was most efficient obtained when the cells grown to mid and late logarithmic phase in MRS medium were used. The maximum number of protoplasts was obtained when lysozyme and mutanolysin were used to lysis the cell wall in 20mM HEPES buffer (pH 7.0) containing 1M sucrose. Regeneration was accomplished with a complex medium containing 10% sucrose, 10mM MaCl2, 20mM CaCl2, 5% gelatin and 0.5% bovine serum albumin. The regeneration frequency of the protoplasts was 10-20% after 5 days of incubation at 30C.

• Korean Journal of Food Science and Technology
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• v.16 no.3
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• pp.363-367
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• 1984

Conditions for efficient formation and regeneration of protoplasts of Streptococcus lactis ATCC 11454 were investigated. Addition of 20mM DL-threonine into growth medium, growth phase and lysozyme concentration had significant effects on protoplast formation. Approximately, 20% regeneration efficiency was obtained by optimizing the medium composition and modifying the plating procedure.

• Microbiology and Biotechnology Letters
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• v.15 no.2
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• pp.89-94
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• 1987

The optimal conditions for the protoplast formation and regeneration of Streptomyces mitakaensis have been investigated. S. mitakaensis cells were converted to protoplast by treating with 0.1 mg/$m\ell$ of lysozyme in phosphate-tris buffer (pH 7.2) to the cells grown at the late logarithmic growth phase in the GBYN medium (gycerol 20g, beef extract 5g, yeast extract 5g, NaCl 5g in 1 liter of distilled water) contained 0.5% glycine. Cell regeneration from protoplast was accomplished in 10 days post inoculation on the R2 regeneration agar medium and at 3 days post inoculation on the H2 regeneration liquid medium. The efficiency of the regeneration was 0.l% in 3 days at 35$^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.145-149
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• 1985

Studies were conducted on the conditions for yeast protoplast regeneration in Hansenula anomala var.anomala FRI YO-32 and Saccharomyces cerevisiae. Protoplasts lysed when suspended in hypotonic solutions of KCI, and the least degree of osmolysis was shown in the hypertonic solution containing 1.4M KCI for the strain FRI YO-32 or 0.8M KCI for S. cerevisiae. It was considered that the concentration of agrar and KCI, and protoplast plating method were the main factors influencing regeneration of yeast protoplasts. Yeast protoplasts were regenerated very favorably when embedded in the complete protoplast regeneration media containing 3％ agar as well as 0.4M KCI for the strain FRI YO-32 or 1.0M KCI for S. cerevisiae. It was shown from the relationship between protoplast formation and regeneration that the higher extent of protoplast formation, the lower extent of protoplast regeneration.

• The Korean Journal of Mycology
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• v.23 no.4 s.75
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• pp.305-309
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• 1995

To investigate optimal conditions for the protoplast formation and regeneration of Phanerochaete chrysosporium, preparations of three enzymes were used to liberate protoplasts from its 20 hrs-old mycelium on cellophan membrane covered agar media. Novozym 234 alone with 0.6M sucrose was the most effective for isolation of protoplasts from the mycelium with 3hrs incubation time at $39^{\circ}C$ in shaking condition of 120 rpm. The poly-R medium stabilized with 0.6M mannitol was the best for regeneration of the protoplasts.

• Korean Journal of Microbiology
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• v.22 no.1
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• pp.35-40
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• 1984

Attempts were made to optimise protoplast formation and regeneration methods to improve the protoplast fusion frequencies of Streptomyces coelicolor. The yields of protoplast formation and regeneration were varied with different growth phase of the culture. Maximum yields were obtained when cells were taken from the late logarithmic phase. Protoplast formation reached almost its maximum with lysozyme treatment at a concentration of 2mg/ml without any other lytic enzyme. A high frequency of protoplast regeneration was accomplished by overlay method: the method gave 14% recovery of regenerated protoplast versus 1.8% recovery for monolay method. A recombinant frequency of 1.8X10^-2 was obtained by protoplast fusion using PEG 1000(50% w/v).