Alpha-Amylase 저해제 생성균 Streptomyces actuosus DMCJ-49와 Streptomyces minoensis DMCJ-144의 종간 융합에 의한 균주 개량

Strain Improvement by Interspecific Fusion of Streptomyces actuosus DMCJ-49 and Streptomyces minoensis DMCJ-144 producing alpha-Amylase Inhibitor

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''-nitro-N-nitrosoguanidine(3 mg/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 protoplast formation medium. In case of DMCJ-144-his- those were 1.2%w/v, 3mg/ml, 30 minutes and 60 hours, respectively. Regeneration was accomplished with hypertonic soft agar medium that contained 0.4 M sucrose, 20 mM CaCl2, 50 mM MgCl2, 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 6.5X10-3 and the alpha-amylase inhibition activity of ade-Xhis- No. 4, which is the fusant with the most improved activity increased from 33 to 125 I.U./ml.