GENETIC IMPROVEMENT OF ANTIDIABETIC ALPHA-GLUCOSIDASE INHIBITOR PRODUCING STREPTOMYCES SP

摘要

Objective : This study aims to control type 2 of diabetes mellitus by a hypoglycemic substance that extensively produced by Streptomyces bacteria. The antidiabetic action of this substance depends on prevention of starch hydrolysis and then the liberation of glucose monomers via an inhibition of α-glucosidase as one of starch hydrolyzing enzymes. Methods : The strains of marine actinomycetes were isolated on starch nitrate agar, and then qualitatively and quantitatively screened to prevent starch hydrolysis. The most potent strain was identified by classical and genetical methods. The genetic improvement of the most potent strain was carried out by using UV radiations at different exposure periods per second. The optimization of environmental conditions was studied to obtain the maximum activity of the α-glucosidase inhibitory protein, which purified and electrically separated to determine its molecular weight. Results : Among 55 marine actinomycetes, only 7 strains were found have antidiabetic activity. This activity was assayed spectrophotometrically at 400 nm, where p -nitrophenyl-α-d-glucopyranoside and acarbose were used as a substrate and a positive control respectively. The most potent strain which marked as AD-7 was identified as Streptomyces coelicolor , which exposed to the genetic improvement using UV radiations to obtain a highly activity of an inhibitory protein at 10 s of the exposure period. The activity and stability continued for 5 d at 37 °C. The maximum activity and stability of an improved inhibitory protein were obtained with optimization of environmental conditions included inoculum size (10 6 cfu/ml/300 μl), incubation period (14 d), agitation speed (160 rpm), incubation temperature (30 °C), and pH (8.5). An inhibitor was purified and separated at 34 KDa. Conclusion : Alpha-glucosidase inhibitory protein as a powerful hypoglycemic agent was extracted from the filtrate of S. coelicolor . The mutant strain of the latter had been produced most active and stable inhibitory protein, which prevents the starch hydrolysis via an inhibition of α-glucosidase enzyme for 5 d at 37 °C.