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Journal of Biomolecular Structure &... 2023Diabetes mellitus (DM) is a global chronic disease characterized by hyperglycemia and insulin resistance. The unsavory severe gastrointestinal side-effects of synthetic...
Diabetes mellitus (DM) is a global chronic disease characterized by hyperglycemia and insulin resistance. The unsavory severe gastrointestinal side-effects of synthetic drugs to regulate hyperglycemia have warranted the search for alternative treatments to inhibit the carbohydrate digestive enzymes (e.g. α-amylase and α-glucosidase). Certain phytochemicals recently captured the scientific community's attention as carbohydrate digestive enzyme inhibitors due to their low toxicity and high efficacy, specifically the Withanolides-loaded extract of . That said, the present study evaluated the efficacy of Withanolide A in targeting both α-amylase and α-glucosidase in comparison to the synthetic drug Acarbose. Protein-ligand interactions, binding affinity, and stability were characterized using pharmacological profiling, high-end molecular docking, and molecular-dynamic simulation. Withanolide A inhibited the activity of α-glucosidase and α-amylase better, exhibiting good pharmacokinetic properties, absorption, and metabolism. Also, Withanolide A was minimally toxic, with higher bioavailability. Interestingly, Withanolide A bonded well to the active site of α-amylase and α-glucosidase, yielding the lowest binding free energy of -82.144 ± 10.671 kcal/mol and -102.1043 ± 11.231 kcal/mol compared to the Acarbose-enzyme complexes (-63.220 ± 13.283 kcal/mol and -82.148 ± 10.671 kcal/mol). Hence, the findings supported the therapeutic potential of Withanolide A as α-amylase and α-glucosidase inhibitor for DM treatment.Communicated by Ramaswamy H. Sarma.
Topics: Humans; Acarbose; alpha-Glucosidases; Molecular Docking Simulation; Molecular Dynamics Simulation; alpha-Amylases; Glycoside Hydrolase Inhibitors; Diabetes Mellitus; Hyperglycemia
PubMed: 35904027
DOI: 10.1080/07391102.2022.2104375 -
Journal of the Science of Food and... Aug 2023Black garlic (Allium sativum L.) melanoidins (MLDs) are produced by Maillard reaction under high temperature and high humidity, and has a variety of biological...
BACKGROUND
Black garlic (Allium sativum L.) melanoidins (MLDs) are produced by Maillard reaction under high temperature and high humidity, and has a variety of biological activities. The aim of this study was to analyze the structural characteristics and investigate α-amylase and α-glucosidase in vitro inhibitory activity of black garlic MLDs.
RESULTS
Spectroscopic and chemical analysis revealed that black garlic MLDs were heterogeneous macromolecular polymers with a skeletal structure similar to sugar chains. Molecular weight distribution and 3DEEM fluorescence showed that black garlic MLDs were composed of high-molecular-weight colorants with strong fluorescence properties. The polarity of black garlic MLDs was related to the fluorescence groups. The results of physicochemical properties proved that the polarity difference of black garlic MLDs was related to the elemental composition, resulting in differences in fluorescence, thermodynamic and apparent characteristics. MLDs with higher levels of fluorescent intensity (BG20 and BG40) had stronger inhibitory effects on α-amylase and α-glucosidase than BGW, and hydrolysis of fluorescent groups attenuated the inhibitory activity. The median inhibitory concentration (IC ) of black garlic MLDs against enzymes was positively correlated with the concentration, and the kinetic results detected non-competitive and mixed types of inhibition.
CONCLUSION
High-molecular-weight fluorescent components of black garlic MLDs played a crucial role in the inhibitory activities of α-amylase and α-glucosidase, and the inhibitory ability was positively correlated with concentration. Black garlic MLDs had the potential to block postprandial glucose rise. © 2023 Society of Chemical Industry.
Topics: Garlic; alpha-Amylases; alpha-Glucosidases; Glycoside Hydrolase Inhibitors; Chemical Phenomena
PubMed: 37060319
DOI: 10.1002/jsfa.12634 -
Preparative Biochemistry & Biotechnology Jul 2024sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified...
sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg 1.154 ± 0.1 mM, and 589 ± 3.5/(smM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(smM) of , / respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca Mg, Co, and Mn boosted α-amylase and cellulase activities. Cu and Ni both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.
Topics: alpha-Amylases; Cellulase; Bacillus; Hydrogen-Ion Concentration; Kinetics; Temperature; Enzyme Stability; Substrate Specificity; Molecular Weight; Bacterial Proteins; Starch
PubMed: 38141162
DOI: 10.1080/10826068.2023.2288574 -
Phytochemistry Dec 2023Ten previously undescribed iridoid constituents, viburnshosins A-E (1-5) and viburnshosides A-E (6-10), together with one known analogue (11), were isolated from the...
Ten previously undescribed iridoid constituents, viburnshosins A-E (1-5) and viburnshosides A-E (6-10), together with one known analogue (11), were isolated from the branches of Viburnum chinshanense. Their structures were unambiguously elucidated by a comprehensive analysis of 1D and 2D NMR data, together with HRESIMS spectroscopic data. The absolute configurations of compounds 1-10 were assigned by means of the calculated ECD spectra. Interestingly, compounds 2 and 3 are the first iridoids with an unusual C-3-C-7 oxo bridge. Compounds 4, 5, and 10 displayed remarkable inhibitory effects against α-amylase (IC: 38.42, 37.65, and 21.64 μM, respectively) and α-glucosidase (IC: 12.97, 19.34, and 25.71 μM, respectively), comparable to those of the positive control acarbose (IC: 39.75 and 23.66 μM, respectively). The interaction modes of compounds 4 and 10 with two enzymes were analyzed by molecular modeling.
Topics: alpha-Glucosidases; Iridoids; Glycoside Hydrolase Inhibitors; Viburnum; Molecular Structure; alpha-Amylases
PubMed: 37820889
DOI: 10.1016/j.phytochem.2023.113893 -
Biochimie Jun 2024Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This...
Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical "gene fishing" and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified "Enchytraeus-Eisenia type" α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.
Topics: alpha-Amylases; Animals; Evolution, Molecular; Phylogeny; DNA, Complementary; Annelida; Oligochaeta; Amino Acid Sequence
PubMed: 38242278
DOI: 10.1016/j.biochi.2024.01.008 -
Plants (Basel, Switzerland) Aug 2023The rising predominance of type 2 diabetes, combined with the poor medical effects seen with commercially available anti-diabetic medications, has motivated the... (Review)
Review
The rising predominance of type 2 diabetes, combined with the poor medical effects seen with commercially available anti-diabetic medications, has motivated the development of innovative treatment approaches for regulating postprandial glucose levels. Natural carbohydrate digestion enzyme inhibitors might be a viable option for blocking dietary carbohydrate absorption with fewer side effects than manufactured medicines. Alpha-amylase is a metalloenzyme that facilitates digestion by breaking down polysaccharides into smaller molecules such as maltose and maltotriose. It also contributes to elevated blood glucose levels and postprandial hyperglycemia. As a result, scientists are being urged to target α-amylase and create inhibitors that can slow down the release of glucose from carbohydrate chains and prolong its absorption, thereby resulting in lower postprandial plasma glucose levels. Natural α-amylase inhibitors derived from plants have gained popularity as safe and cost-effective alternatives. The bioactive components responsible for the inhibitory actions of various plant extracts have been identified through phytochemical research, paving the way for further development and application. The majority of the findings, however, are based on in vitro investigations. Only a few animal experiments and very few human investigations have confirmed these findings. Despite some promising results, additional investigation is needed to develop feasible anti-diabetic drugs based on plant-derived pancreatic α-amylase inhibitors. This review summarizes the most recent findings from research on plant-derived pancreatic α-amylase inhibitors, including plant extracts and plant-derived bioactive compounds. Furthermore, it offers insights into the structural aspects of the crucial therapeutic target, α-amylases, in addition to their interactions with inhibitors.
PubMed: 37631156
DOI: 10.3390/plants12162944 -
Journal of Biomolecular Structure &... Nov 2023α-Amylase catalyses the hydrolysis of glucosidic bonds in polysaccharides such as starch, glycogen and their degradation products. In the present study, the...
α-Amylase catalyses the hydrolysis of glucosidic bonds in polysaccharides such as starch, glycogen and their degradation products. In the present study, the three-dimensional structure of fenugreek () α-amylase was determined using a homology modeling-based technique. The best predicted model was deposited in PMDB server with PMDB ID PM0084364. The phylogenetic tree was created using the UPGMA method with 8 homologous protein sequences, was utilized as the target protein. Alignment of the phylogenetic tree identified two primary functional groupings (A and B). α-Amylase from the target genome (Acc. No: GHNA01022531.1) was clustered with (Acc. No: XP003589186.1), (Acc. No: XP004499059.1), (Acc. No: XP020231823.1), (Acc. No: NP001316768.1) and (Acc. No: P17859.1), in group A cluster, while (Acc. No: Q40015) and (PDB ID: 3WN6) were in cluster B. The molecular dynamics simulations were performed to understand the molecular basis and mode of action of α-amylase. Additionally, a geometry-based molecular docking technique was used to evaluate potential binding interactions between the modeled structure of α-amylase and maltose. The results show that Trp, Glu, Arg, His, Tyr, Asp, Phe and Asp from α-amylase enzyme is involved in the binding to the substrate maltose. Our study provides a 3D model of α-amylase and aids in understanding the atomic level molecular underpinnings of the mechanism of α-amylase interaction with substrate maltose. Ca are essential for the stability of domain B since they are connected to it. Ca site ligands are Asp, Glu, Thr, Asp and Gly residues. HIGHLIGHTSIn analysis, gene prediction of α-amylase was carried from .Analysis of the structure of α-amylase was carried out using homology modelling.Calcium binding sites and their interactions with α-amylase were visualised using BIOVIA DISCOVERY STUDIO 2019.The molecular interaction between α-amylase and maltose was studied in using a molecular docking-based method.To give the required simulation parameters, RMSD, RMSF, and Total Energy were calculated using BIOVIA DISCOVERY STUDIO 2019.[Figure: see text]Communicated by Ramaswamy H. Sarma.
Topics: Trigonella; Molecular Docking Simulation; alpha-Amylases; Phylogeny; Maltose; Plant Extracts
PubMed: 36369783
DOI: 10.1080/07391102.2022.2144458 -
Plants (Basel, Switzerland) Jun 2024Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of...
Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from . The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via -mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.
PubMed: 38891347
DOI: 10.3390/plants13111539 -
Food Research International (Ottawa,... Nov 2023The objective was to assess aspartame excretion in saliva and the salivary insulin, total protein (TP), and alpha-amylase (AMI) levels in response to the ingestion of... (Randomized Controlled Trial)
Randomized Controlled Trial
The objective was to assess aspartame excretion in saliva and the salivary insulin, total protein (TP), and alpha-amylase (AMI) levels in response to the ingestion of sweetened beverages (sodium cyclamate, aspartame, acesulfame, and sucrose). Fifteen healthy participants were included in a single-blinded trial with the intake of Diet soft drink, Regular soft drink, Water + sweeteners, Low sucrose content (3.5 g), and Water (blank) in 5 different days. In each day, saliva was collected at T0 (fasting), T1 (15 min after test-drink intake), T2 (30 min), T3 (60 min), and T4 (120 min) for the measurement of salivary aspartame (HPLC), TP, AMI (ELISA assays) and insulin levels (chemiluminescence). Chi-square, Friedman, ANOVA and Spearman correlation tests were applied. The late-perceived sweet/sour residual flavor was reported at a frequency of 80%, 60% and 20% after ingestion of artificially sweetened drinks, beverages with sucrose, and plain water, respectively (p < 0.05). Aspartame was detected in saliva after artificially sweetened drinks intake, with highest area under the peak for the Diet soft drink (p = 0.014). No change was observed for TP and AMI levels during the 120 min. Insulin levels increased 1 h after soft-drinks ingestion (regular and diet), while the levels did not change for Low sucrose content and Water + sweeteners test-drinks. Salivary aspartame correlated with insulin levels only after Diet soft drink intake (rho ≥ 0.7; p < 0.05). As aspartame can be detected in saliva and swallowed again until completely excreted, these results contribute to the knowledge of the biological fate of artificial sweeteners and the study of health outcomes.
Topics: Humans; Aspartame; Sweetening Agents; Sugar-Sweetened Beverages; Insulin; Single-Blind Method; alpha-Amylases; Sucrose; Water
PubMed: 37803739
DOI: 10.1016/j.foodres.2023.113406 -
Carbohydrate Polymers Nov 2023Hydrolysis of highly concentrated soluble starch (60%, w/w) was performed using sequential α-amylases from Bacillus stearothermophilus (T, 0.2%, w/w) and Bacillus...
Hydrolysis of highly concentrated soluble starch (60%, w/w) was performed using sequential α-amylases from Bacillus stearothermophilus (T, 0.2%, w/w) and Bacillus amyloliquefaciens (B, 0.1%, w/w) to identify their possible action patterns. We found that T reduced the average molecular weight (Mw) of soluble starch from 52,827 Da to 31,914 Da and significantly affected its branched chain length. Compared with soluble starch, the chains with DP 6-12 and DP ≥ 13 in the T samples were diminished by 46% and 96%, respectively. This resulted in an attenuation in the proportions of exterior and inner chains, as well as low iodine binding capacity of the hydrolysates. In contrast, a slower decrease in the average Mw of soluble starch occurred after TB incubation, and the level of DP 6-12 further lowered, causing a gradual decline in the iodine binding capacity of the hydrolysates. Gathered data revealed an unusual action pattern of sequential α-amylase treatment at high substrate concentrations. Bacillus stearothermophilus α-amylase exhibited more pronounced endo-hydrolysis of amylopectin, whereas the attack of Bacillus amyloliquefaciens α-amylase on the exterior chains was enhanced in amylopectin residues. These findings suggest that the synergy of various α-amylases is an effective strategy to promote the dextrinization of highly concentrated starch and finely modify the molecular structure of starch.
Topics: Starch; alpha-Amylases; Amylopectin; Hydrolysis; Bacillus amyloliquefaciens; Iodine
PubMed: 37659787
DOI: 10.1016/j.carbpol.2023.121190