•Important factors affecting cooked starch digestion determined by Plackett–Burman experiment.•The optimal proportion of HPA and ctMGAM in a starch digestion process determined by response surface methodology.•The inhibitory effects of different inhibitors evaluated by in vitro enzymal starch digestive system.•The inhibitory effects of inhibitors confirmed by in vivo assay.In human, digestion of cooked starch mainly involves breaking down of α-amylase to α-limit dextrins and small linear malto-oligosaccharides, which are in turn hydrolyzed to glucose by the gut mucosal maltase-glucoamylase (MGAM). Human pancreatic α-amylase (HPA), amino- and carboxyl-terminal portions of MGAM (ntMGAM and ctMGAM) catalyze the hydrolysis of α-d-(1,4) glycosidic linkages in starch, playing a crucial role in the production of glucose in the human lumen. Accordingly, these enzymes are effective drug targets for the treatments of type 2 diabetes and obesity. In this study, a Plackett–Burman based statistical screening procedure was adopted to determine the most critical factors affecting cooked starch digestion by the combination of HPA, ctMGAM and ntMGAM. Six factors were tested and experimental results showed that pH and temperature were the major influencing factors, with optimal pH and temperature at 6.0 and 50 °C, respectively. Surprisingly, ntMGAM had no significant contribution to the glucose production from starch digestion compared to the HPA and ctMGAM. The optimal proportion of HPA and ctMGAM in a starch digestion system was further determined by response surface methodology. Results showed a maximum starch digestion (88.05%) within 0.5 h when used HPA:ctMGAM=1:9 (U). The inhibitory effects of various inhibitors on the cooked starch digestion by HPA1/ctMGAM9 were evaluated by determining their half maximal inhibitory concentration (IC50) values. Acarviostatin II03 showed the highest inhibitory activity, with 67 times higher potency than acarbose. Moreover, acarviostatin II03 could significantly depress postprandial blood glucose levels in mice, better than that by acarbose. These findings suggest that our in vitro enzymatic system can simulate in vivo starch digestion process, and thus can be used to screen and evaluate α-glucosidase inhibitors.

KGLP-1, a 31-amino acid glucagon-like peptide-1 (GLP-1) analogue, has a great therapeutic potential for anti-diabetes. In this work, a strategy for expression and purification of functional KGLP-1 peptide has been established. KGLP-1 cDNA was fused with glutathione S-transferase (GST), with an enterokinase cleavage site in the fusion junction. The recombinant fusion protein GST–KGLP-1 was affinity purified via the GST-tag, and then digested with enterokinase. The resulting GST part as well as the enzymes were eliminated by ultra-filtration followed by size exclusion chromatograph. The yield of purified KGLP-1 was approximately 12.1 mg/L, with purity of 96.18 %. The recombinant KGLP-1 was shown to have similar bioactivity as native GLP-1 when evaluated in a Chinese hamster ovary cell line expressing a GLP-1 receptor-egfp reporter gene.

The search for the effective and safe α-glucosidase and α-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350–190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit α-glucosidase or/and α-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human α-glucosidase and α-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active α-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin I03 by MS and NMR spectrometry. Its IC50 value was 1.25 and 12.23 μg/ml against human intestinal N-terminal maltase-glucoamylase and human pancreatic α-amylase, respectively.

A novel amino-oligosaccharide, named SF638-1, was isolated from the culture filtrate of the Streptomyces strain PW638. Its chemical structure was determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance spectroscopy. The novel compound was a mixed inhibitor of human pancreatic α-amylase, with a Ki value in the same order of magnitude as that of the α-amylase inhibitor, acarbose. SF638-1 inhibited starch hydrolysis and glucose transfer in vitro, and suppressed postprandial blood glucose elevation in vivo. These results suggest that SF638-1 may be a potent antidiabetic agent.

In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtMGAM) of small intestinal maltase glucoamylase (MGAM) are α-glycosidases that catalyze the hydrolysis of α-(1→4) glycosidic linkages in the process of starch digestion, and are considered to be the main therapeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccharides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, voglibose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic α-amylase (HPA) provide us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper, DNJ was the most effective inhibitor against MGAM; the Ki values for the two catalytic domains were 1.41 and 2.04 μM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-03 and 3-03 inhibition constants, Ki, for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02 and 6.08 μM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate specificities and inhibitor tolerance despite their structural relationship.