Detection and Cloning of α-Glucosidase Inhibitor Gene of Streptomyces sp. BWA 65 and Its Potential as an Anti Hyperglycemic in Mice (Mus musculus)

Abstract

Diabetes mellitus is a metabolic disorder characterized by the presence of hyperglycemia due to defective insulin secretion, defective insulin action or both. One therapeutic approach for treating diabetes is to decrease the post-prandial hyperglycemia. This is done by preventing the absorption of glucose through the inhibition of the carbohydrate-hydrolysing enzymes α-glucosidase and α-amylase in the digestive tract. Actinomycetes have been known as source of commercialized acarbose, an α-glucosidase inhibitor. Acarbose is keto analog moieties of the C7N aminocyclitols. Sedoheptulose 7-phosphate is converted into 2-epi-5-epi-valiolone via the activity of sedoheptulose 7-phosphate cyclase, at the first step of the biosynthesis of C7N aminocyclitol. This research aimed to detect and clone sedoheptulose 7-phosphate cyclase gene and to investigate the capability of crude α-glucosidase extract from Streptomyces sp. BWA 65 in lowering blood glucose levels in mice. Detection of sedoheptulose 7-phosphate cyclase gene was done by using Polymerase Chain Reaction (PCR) with designed primers C7N aminocyclitol. The primer used was designed on the basis of the known sequence of sedoheptulose 7-phosphate cyclase (acbC) from that of Actinoplanes sp. SE50/100 which was then cloned by T-Vector pMD20. The in vivo experiment was conducted by using thirty mice, by the oral glucose tolerance test (OGTT) and induction of streptozotocin diabetes methods. The result showed that there was similarity of nucleotide series sedoheptulose 7-phosphate cyclase of Streptomyces sp. BWA 65 wich have 100 % sequence similiarity with DNA fragment of sedoheptulose 7-phosphate cyclase Actinoplanes sp. complete acarbose (acb) gene cluster, strain SE50/110, accses number Y18523.4 reported in the GenBank analysis through Blast Program. The result indicated that the designed primer was able to amplify the sedoheptulose 7-phosphate cyclase acarbose although the accomplishment in amplifying the gene was still up to 300 bp. The result for in vivo experiment by oral glucose tolerance test (OGTT) showed that α-glucosidase extract from Streptomyces sp. BWA 65 had potency about 75 percent in decreasing blood glucose levels postprandial compare to acarbose and for induction of streptozotocin diabetes showed that α-glucosidase extract from Streptomyces sp. BWA 65 had an effect in decreasing blood glucose levels diabetic mice hyperglycemia better than the acarbose concentration examined. The in vivo experiment indicated that α-glucosidase inhibitor from Streptomyces sp. BWA 65 had potential as antidiabetic in mice.