• 약학회지
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• 제35권1호
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• pp.30-37
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• 1991

Streptomyces actuosus DMCJ-49 and Streptomyces minoensis DMCJ-144 produce the .alpha.-amylase inhibitor. Inerspecific protoplast fusion technique was used to increase the productivity of .alpha.-amylase inhibitor. Four auxotrophic mutants were obtained respectively from two strains by N-methyl-N'-nitor-N-nitrosoguanidine(3mg/ml) treatment. The optimum conditions for the protoplast formation of Streptomyces actuosus DMCJ-49 ade was as follows; 1.2% w/v of glycine, 3mg/ml of lysozyme, and 30 min of lysozyme treatment followed by 36 hr. incubation in the protop-last formation medium. In case of DMCJ-144-his those were 1.2%w/v, 3 mg/ml, 30 minutes and 60 hours, respectively. Regeneration was accomplished with hypertonic soft agar medium that contained 0.4M sucrose, 20mM CaCl$_2$, 50 mM MgCl$_2$ and low levels of phosphate. Fusion of protoplasts carrying different auxotrophic markers was achieved by treatment with polyethylene glycol. The optimum concentration of polyethylene glycol 1450 for the production of recombinants was 40%w/v. When the protoplasts was treated with 40% polyethylene glycol for 30 minutes, the frequency of recombinants was 6.5$\times$$10^{-3}$ and the $\alpha$-amylase inhibition activity of $ade^-his^-$ No. 4, which is the fusant with the most improved activity increased from 33 to 125 I.U./ml.

• Biomolecules & Therapeutics
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• 제1권1호
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• pp.26-30
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• 1993

Strain of treptomyces minoensis DMCJ-144 was tried to be improved so that it produces much more the $\alpha$-amylase inhibitor. Streptomyces minoensis DMCJ-144 was treated with 1 mg/mι (pH 9.0) of N-methyl-N'-nitro-N-nitrosoguanidine at $30^{\circ}C$ for 60 min and irradiated with UV light distanced 30 cm for 20 min. After mutagenesis, surviving colonies were cultured on the CM contaning acriflavine ($10{\mu}g/ml$) three times in order to enhance the mutability. And then through multi-level screening, colonies that ${\alpha}$-amylase inhibitor productibility. was Improved were selected by modified-blue value method. After third acriflavine treatment, $\alpha$-amylase inhibitory activities of selected colonies were found to be much better as compared with that of parent strain. One mutant strain showed 5.4 time inhibitory activity than the parent strain.

• 약학회지
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• 제36권4호
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• pp.390-396
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• 1992

Streptomyces minoensis DMCJ-144 isolated from soil produces the ${\alpha}-amylase$ inhibitor. Optimum culture conditions for ${\alpha}-amylase$ inhibitor production of the strain were determined in this experiment. The optimum composition of the culture medium was studied by supplementing various carbon sources, nitrogen sources, vitamins, and metal salts to the basal medium containing 1% glucose, 0.1% asparagine, 0.005% $MgSO_4{\cdot}7H_2O$, 0.005% $K_2HPO_4$, 0.005% NaCl. Other culture conditions such as the culture temperature, initial pH of the medium, aeration, and culture time were also investigated. When the strain was cultured in a 100 ml flask containing 20 ml of 2% glucose, 0.5% beef extract, 0.0002% riboflavin, 0.0002% thiamine HCI, 0.01% $ZnCl_2$, 0.005% $MgSO_4{\cdot}7H_2O$, 0.005% $CuSO_4{\cdot}5H_2O$, 0.005% NaCl, pH 7.2, 180 rpm at $30^{\circ}C$, the maximum production of the ${\alpha}-amylase$ inhibitor was observed after 5 days of the cultivation.