• The Korean Journal of Mycology
• /
• v.20 no.1
• /
• pp.58-64
• /
• 1992

This research was focused on investigation of the general condition for protoplast formation of Trichoderma speues. for protoplast formation, the mycelia cultured in YM medium were collected from each growth phase and were treated with the Iytic enzymes. This procedure was carried out by all strains. The most optimal conditions of NOVOZYM 234 and DRISELASE were determined by T. saturnisporum IAM 12535 and T. longibruchiatum IBM 13107, respectively. The effect of osmotic stabilizers appeared ${KCI}>(NH_4)_2{SO_4}>NaCl>mannitol>{MgSO}_4$ and the optimal concentration of each osmotic stabilizer wns determined by 0.6-0.9 M. The optimal condition of DRISELASE for protoplast formation ; optimal pH 5.0, optimal concentration, 2%, optimal reaction time, 4 hours, and optimal temperature, $30^{\circ}C$. The optimal condition of NOVOZYM 234 for protoplast formation ; optimal pH 5.5, optimal concentration 1%, optimal reaction time 3 hours, and optimal temperature $30^{\circ}C$. The optimal culture period of mycelia for protoplast formation was between the initial and the middle exponential phase. Generally, DUSELASE was more effective than NOVOZYM 234 on protoplast formation except for T. longibruchiatum IAM 13107 and T. viride IAM 5141.

• The Journal of Natural Sciences
• /
• v.11 no.1
• /
• pp.55-63
• /
• 1999

In this study, the stabilizer, PH and lysis method for optimum condition of S. uvarum protoplast formation were investigated, and also enzyme activity and poly-P formation of intact cell and protoplast mere compared. Upon protoplast formation, incubation time of 5 hours in snail gut enzyme and 3 hours in drisielase were reignited. 0.8 Mole mannitol and 6 mole KCl were apt to protoplast formation. Protoplast was contained less 22-27 percentage in ALPase, 4-15 percentage in ACPase than intact cell. Accumulation of inorganic polyphosphate did not increase significently in protoplast compared with intact cell.

• Korean Journal of Microbiology
• /
• v.22 no.3
• /
• pp.175-181
• /
• 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
• /
• v.23 no.3
• /
• pp.359-364
• /
• 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.

• ALGAE
• /
• v.20 no.3
• /
• pp.239-249
• /
• 2005

Some marine coenocytic green algae could form protoplasts from the extruded protoplasm in seawater. The dissociated cell components of the coenocytic protoplasm could be reunited into live cells and, hence, the formation of new species by mixing protoplasms from different coenocytic cells has been predicted. Our results showed that an incompatibility barrier was present during protoplast formation in coenocytic algae to exclude foreign inorganic particles or alien cell components. No inorganic particles or alien cell components were incorporated into protoplast formed spontaneously in seawater. Even when the inorganic particles or alien cell and/or cell component were incorporated into protoplast in some experimental condition, they were expelled from the protoplast or degenerated within several days. A species-specific cytotoxicity was observed during protoplast hybridization between the protoplasms of Bryopsis spp. and Microdictyon umbilicatum. The cell sap of M. umbilicatum could destroy the cell components of Bryopsis spp., but had no effect on Chaetomorpha moniligera. Species C. moniligera and Bryopsis did not affect protoplast generation of either species. The wound-induced protoplast formation in vitro might have evolved in some coenocytic algae as a dispersal method, and the incompatibility barrier to alien particles or cell and/or cell component could serve as a protective mechanism for successful propagation.

• Microbiology and Biotechnology Letters
• /
• v.14 no.2
• /
• pp.175-179
• /
• 1986

In order to develope a protoplast fusion of the genus Cellulomonas having high assimilibility of cellulose, the optimum conditions for the protoplast formation of Cellulomonas flavigena NCIB 12901 was investigated and observed by means of Scanning Electron Microscope. The results suggested that the susceptibility of the cell wall by lysozyme treatment on protoplast formation was considerably depend on the cultural periods of the cells. Cells of C. flavigena at mid exponential phase could more efficiently convert to protoplast cells than those at late exponential phase did. The rate of the protoplast formation was 95%, even though the rate was over 99.9% on counting by indirect method after osmotic shock treatment, when cells of the organism at mid exponential phase were treated with lysozyme (400$\mu\textrm{g}$/$m{\ell}$) for 6 hours and observed by SEM. In the evaluation of protoplast formation of the genus Cellulomonas, direct method of the observation under Scanning Electron Microscope was much more reliable than the counting method of protplasts after osmotic shock treatment. Because defferences between the number of spheroplast and protoplast were not able to be figured out on counting the number of protoplast after osmotic shock treatment.

• KSBB Journal
• /
• v.9 no.2
• /
• pp.191-199
• /
• 1994

Protoplasts of both strains were produced by lysozyme digestion at $30^{\circ}C$ for 180min. Both strains were treated with $40{\mu}g$/ml of lysozyme in 30mM Tris-HCl buffer(pH 7.5) containing 10% sucrose at the late logarithmic growth phase. It was found that the efficiency of protoplast formation was high at $30^{\circ}C$ and pH 7.5 by measuring the decrease in absorbance. Optimum concentrations of sucrose $Ca^{2+}, \;Mg^{2+}$ for protoplast formation were determined to be 15%, 20mM and 6mM, respectively. Hydrolysis of cell wall and protoplast formation efficiency for L. plantarum showed better results than those for Leu. mesenteroides. The resistances to antibiotics erythromycin and chloramphenicols were chosen as the selection marker for the fusant between L. plantarum and Leu. mesenteroides. Production phase of protoplast in Leu. mesenteroides was also compared with L. plantarum in this paper.

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

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