• Applied Biological Chemistry
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• v.42 no.1
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• pp.20-28
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• 1999

To improve Bacillus strains producing biopolymer, conditions for protoplast formation and regeneration were investigated in biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. Bacillus subtilis K-1 mutant (SM-2) and Bacillus coagulans mutants (CM-12) were marked auxotrophic and antibiotics-resistant (SM-2) and an antibiotics-resistant mutants, respectively. To formate protoplasts derived from the mutants, conditions were established as follows. For B. subtilis mutant SM-2, its culture in mid-logarithmic phase was added with penicillin G (1.0 unit/ml) and further reacted for 1.5 hr. Cells were collected and then treated in lysis fluid (pH 7.0) containing 0.4 M sucrose and lysozyme $25\;{\mu}g/ml$ for 40 min at $37^{\circ}$. Protoplast formation was very successful (99.6%) and the ratio of cell wall regeneration was 2.4%. For Bacillus coagulans mutant CM-12, its mid-logarithmic phase culture was treated with penicillin G (0.3 unit/ml) and glycine (0.5%) for 1hr. Cells were collected and then resuspended in lysis buffer (pH 7.0) containing 0.6 M lactose and lysozyme $(300\;{\mu}g/ml)$ for 30 min at $37^{\circ}$. Protoplast formation was also successful (90.8%) and cell wall regeneration ratio was similar to SM-2 (2.2%). To improve regeneration frequency, regeneration medium was obtained as followed condition,. Cell wall regeneration was improved 2-4 folds with 5.1% for B. subtilis SM-2 and 10.3% for B. coagulans CM-12 when protoplasts mixed with soft top agar(0.4%) was overlaid onto trypticase soy broth medium containing 0.4 M sucrose, 0.7% casamino acid, 1% PVP, 25 mM $MgCl_2,\;25\;mM\;CaCl_₂$ and 1.5% agar.

• Korean Journal of Soil Science and Fertilizer
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• v.3 no.1
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• pp.55-59
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• 1970

In an effort to determine the bio-synthesis in the soybean as investigate to the variance of each substance: Glutamic acid, Aspartic acid and its amides during the growth of younger soybean plants. 1. The variance-curve of Gultamic acid and Aspartic acid as the acidic amino acids in the cotyledons was appeared the peak the first half period at Glutamic acid and the latter half at Aspartic acid in the growth of soybeans, and was received the symmetrical impression centering around the stage of adult leaf-development. But, in the embryonic organ, it appears the peak at both part, in the developmental stage of adult leaf and also appears near phenomena of increase and decrease in the variation-curve of metabolites. 2. It's amides-Gultamine and Asparagine-appears the peak at the developmental stage of adult leaf in the both cotyledons and embryonic organ, and rapid increase in the cotyledons were very impressed compare with the decrease at fallen stage of cotyledons in the embryonic organs. 3. In the relation of variance at Glutamic acid and Aspartic acid, both substance were discovered the fact of translocation from cotyledon to embryonic organ, and Glutamic acid could supposed that bear the charges of outrider substance in other amino acid as the Glutamic acid-self and major basic function for receiving the ammonia as the nitrogen contain constituent of plant. In the case of Glutamine, formation-mechanism of ammonia which develops due to its hydrolysis in the latter period of soybean growth, suggested that was forfeit its function till instance of fallen cotyledons. 4. In the relation the Aspartie acid and Asparagine, Aspartic acid which begins to decrease from seed-state was supposed that bear sufficiently the charge of outrider substance in the formation of Asparagine other than translocated to embryonic organ from cotyledon. And, formation-theory of Aspartic acid which suppose as formational substance from Kreb's cycle were recognized from latter period of soybean growth, and then, rapid accumulation of Asparagine's amounts were supposed that adapt to two theory: Theory which consider to transformation as Asparagine state for pressing to less than noxious weight the concentration of ammonia developing from the cells, and was formate and accumulate as ammonia or carbohydrates containing excess in the cotyledons.