[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 K(i) 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, K(i), 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.
No preview · Article · Dec 2011 · Carbohydrate research
[Show abstract][Hide abstract] ABSTRACT: Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
[Show abstract][Hide abstract] ABSTRACT: Targeting the important enzyme in human glucose metabolic pathway, we established a high throughput screening model for human pancreatic alpha-amylase inhibitors.
Pichia pastoris expression system was used to clone and express the human pancreatic alpha-amylase; we established the alpha-amylase inhibitor screening model using the catalytic properties of enzyme; this model was applied in screening of actinomycete' metabolites; the taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree.
We cloned and expressed the intact gene of human pancreatic alpha-amylase successfully; the high-throughput screening model of alpha-amylase inhibitors was established; nearly 2000 actinomycete' metabolites were screened, 14 alpha-amylase inhibitor producing strains were obtained finally, and showed taxonomically rich diversity.
The alpha-amylase inhibitor high-throughput screening model had high practical value for developing new hypoglycemic drugs.
No preview · Article · Aug 2011 · ACTA MICROBIOLOGICA SINICA
[Show abstract][Hide abstract] ABSTRACT: Co-composting of chicken manure, straw and dry grasses was investigated in a forced aeration system to estimate the effect of aeration rates on NH3, CH4 and N2O emissions and compost quality. Continuous measurements of gas emissions were carried out and detailed gas emission patterns were obtained using an intermittent-aeration of 30min on/30min off at rates of 0.01 (A1), 0.1 (A2) and 0.2 (A3) m3min−1m−3. Concentrations of CH4 and N2O at the low aeration rate (A1) were significantly greater than those at the other two rates, but there was no significant difference between the A2 and A3 treatments. CH4 and N2O emissions for this mixture could be controlled when the composting process was aerobic and ammonia emissions were reduced at a lower aeration rate. Comparison of CH4, N2O, NH3 emissions and compost quality showed that the aeration rate of the A2 treatment was superior to the other two aeration rates.
[Show abstract][Hide abstract] ABSTRACT: Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.
Full-text · Article · Nov 2010 · Journal of Structural Biology
[Show abstract][Hide abstract] ABSTRACT: Targeted at the important enzyme in human glucose metabolic pathway, the purpose of this paper is to establish alpha-glucosidase inhibitors high throughput screening model.
Pichia pastoris expression system was used to clone and express the human alpha-maltase glucosidase. Using the catalytic properties of enzyme to establish alpha-glucosidase inhibitor screening model. This model was applied in screening of actinomycete metabolites library. The taxonomic status of positive strains were analyzed by constructing 16S rRNA phylogenetic tree.
The N-terminal catalytic domain of human alpha-maltase glucosidase was successfully cloned and expressed for the first time. The high-throughput screening model of alpha-glucosidase inhibitors was established. A natural product library containing metabolites from nearly 2000 actinomycetes was screened, 20 alpha-maltase glucosidase inhibitor producing strains were obtained finally, of which, 19 strains initially identified as Streptomyces, and showed taxonomically rich diversity.
The alpha-glucosidase inhibitor high-throughput screening model has high practical value, this work laid the foundation for developing new hypoglycemic drugs.
No preview · Article · Aug 2010 · ACTA MICROBIOLOGICA SINICA
[Show abstract][Hide abstract] ABSTRACT: An absorbent mixture of magnesium hydroxide (Mg(OH)(2)) and phosphoric acid (H(3)PO(4)) was added to compost mixtures of pig manure with cornstalk in different molar ratios (T1, 1:1; T2, 1:2; T3, 1:3) in order to examine its effect on controlling ammonia losses during composting. Based on the principle of struvite precipitation, and with an unamended trial as control (CK), an in-vessel composting experiment was conducted in fermenters (60L with forced aeration) in which the absorbent mixture was added with proportions of 3.8%, 7.3% and 8.9% of dry weight for T1, T2 and T3, respectively. The results showed that the total nitrogen loss was reduced from 35% to 12%, 5% and 1% of initial N mass, respectively. In the final compost, the total nitrogen content in T1, T2 and T3 was improved by 10, 14, 12gkg(-1), and NH(4)(+)-N in T1, T2 and T3 was improved by 8, 9, and 10gkg(-1), respectively, compared with the unamended trial. The results of the germination index test showed that the maturity of treatment T2 was best among the four treatments in the final compost, followed by T1, CK and T3. The results of X-ray diffraction (XRD) confirmed the formation of magnesium ammonium phosphate hexahydrate (MgNH(4)PO(4).6H(2)O:MAP) in the T1, T2 and T3 compost. Based on these results, the adsorbent mixture of Mg(OH)(2)+H(3)PO(4) could control nitrogen loss effectively during composting via struvite crystallization. However, an excess of phosphoric acid (1:3) had a negative influence on composting properties. The pH value decreased which led to reduced microorganism activity, and which finally resulted in reduced biodegradation of the organic matter.
No preview · Article · May 2010 · Waste Management