Α-amylase Molecule Research Articles

Statistical Characterization of Food-Derived α-Amylase Inhibitory Peptides: Computer Simulation and Partial Least Squares Regression Analysis.

α-Amylase inhibitory peptides are used to treat diabetes, but few studies have statistically characterized their interaction with α-amylase. This study performed the molecular docking of α-amylase with inhibitory peptides from published papers. The key sites, side chain chargeability, and hydrogen bond distribution characteristics were analyzed. Molecular dynamics simulated the role of key sites in complex stability. Moreover, partial least squares regression (PLSR) was used to analyze the contribution of different amino acids in the peptides to inhibition. The results showed that, for the α-amylase molecule, His201 and Gln63, with the highest interaction numbers (INs, 15, 15) and hydrogen bond values (HBVs, 11.50, 10.33), are the key sites on α-amylase, and amino acids with positively charged side chains were important for inhibitory activity. For the inhibitory peptides, Asp and Arg had the highest HBVs, and amino acids with charged side chains were more likely to form hydrogen bonds and exert inhibitory activity. In molecular dynamics simulations, peptides involving key binding sites formed more stable complexes with α-amylase than α-amylase alone, suggesting enhanced inhibitory effects. Further, PLSR results showed that amino acids close to the N-terminus of the inhibitory peptide, located in the third and fifth positions, were significantly correlated with its inhibitory activity. In conclusion, this study provides a new approach to developing and screening α-amylase inhibitors.

Antidiabetic and antioxidant potentials of Abelmoschus esculentus: In vitro combined with molecular docking approach

This study was designed to determine the in vitro antidiabetics and antioxidant ability of Abelmoschus esculentus methanolic extract (MEAE) using α-amylase and α-glucosidase inhibition model. α-amylase and α-glucosidase binding interaction with MEAE was also determined by molecular docking. Methanolic extract of A. esculentus was prepared and then total phenolic content (TPC) and total flavonoid content (TFC) was determined. TPC and TFC was 153 ± 38.82 mg GAE/g dw and 40.55 ± 4.409 mg QE/g dw respectively. For antioxidant potential, DPPH method was used. At concentrations of 31–1000 µg/mL MEAE showed scavenging percentage of DPPH radicals ranging from 1.618 to 13.487%. Enzymatic antioxidant activities by MEAE such as catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) showed their significant potentials with values of 3.99 ± 1.05, 1.27 ± 2.6 and 23.80 ± 0.03 U/mg protein respectively. At 50–200 µg/mL inhibition percentage of α-glucosidase and α-amylase ranged from 14.36 ± 0.099 to 19.23 ± 0.172% and 15.89 ± 1.877 to 37.19 ± 7.430% respectively. Yeast glucose uptake assay revealed that it showed glucose absorption which was directly proportional with concentration of extract and inversely proportional with the molar concentration of glucose. Maximum glucose uptake percentage by MEAE was 68.420 ± 1.752% at 3 mg/mL extract and 5 mM glucose concentration. In silico analysis, revealed that ligand molecule could bind with the α-glucosidase and α-amylase molecules. It can be concluded that A. esculentus is an important vegetable with a potent source of natural antioxidants as well as having potential antidiabetic activity thus justifying its traditional use in green therapeutics.

In vitro inhibitory effects of Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves proanthocyanidins on pancreatic α-amylase and their interaction

Chinese bayberry leaves proanthocyanidins (BLPs) belongs to the prodelphinidin category with potent EGCG unit, whose inhibition effect on α-amylase and their interaction were investigated by in vitro digestion and enzyme kinetic analysis, multi fluorescence spectroscopies (fluorescence quenching, synchronous fluorescence, and three-dimensional fluorescence), circular dichroism spectra, Fourier transform infrared spectroscopy and in silico modelling. The results revealed that BLPs was a mixed inhibitor to α-amylase with the IC50 value of 3.075 ± 0.073 μg/mL. BLPs could lead to a static fluorescence quenching of α-amylase, mainly by means of interacting with amino acids (mainly Try and Tyr residues) in one site on α-amylase molecule under the action of hydrogen bonding and/or Van der Waals force. This interaction further induced the change of secondary conformational structure, functional group structure and hydrophobicity of α-amylase, thus resulting in lowering activity. Molecular docking simulated that this binding occurred in a cavity on the surface of the α-amylase molecule, and BLPs trimer showed a relatively high binding energy. The present study provided a new insight of BLPs as an α-amylase inhibitor, which could be considered in anti-diabetic therapy.

Characterization of promising natural blue pigment from Vaccinium bracteatum thunb. leaves: Insights of the stability and the inhibition of α-amylase

A concise method of natural blue pigment from Vaccinium bracteatum thunb. leaves (VBTL) was proposed firstly in this research. The potential properties of new pigment source needed to be assessed for further application. During the shelf life and thermal-accelerated storage, the blue pigment was prone to stable in the mildly acidic system, which was observed retention of 35.85% for storage of 12 weeks in pH 4.0. The 65% of blue pigment retained after the in vitro simulated digestion. The blue pigment exhibited inhibition effect on the pancreatic α-amylase (IC50 = 2.915 mg/mL). The analysis of inhibition kinetics indicated the blue pigment was an uncompetitive inhibitor. Through fluorescence quenching and circular dichroism study, the blue pigment inhibited α-amylase activity via the structural unfolding of α-amylase molecule and changing the secondary structure. These findings provided the new evidence of the digestion resistibility of VBTL pigment and the functional food – “Wu mi”.

Investigation on the interaction between γ-cyclodextrin and α-amylase

A growing interest has been shown regarding the impact of α-amylase on the quality of fermented foods. Therefore, the interaction between γ-cyclodextrin and α-amylase was investigated in this article. The results showed that γ-cyclodextrin had an inhibitory effect on the activity of α-amylase under certain conditions, obtaining the strongest inhibitory effect when the concentration of γ-cyclodextrin was 10 mmol/L, the reaction temperature was 45 °C, pH = 5.9 and reaction time of 120 min. The results also showed important changes in the secondary structure of α-amylase induced by γ-cyclodextrin, including the transformation of α-helix, β-fold and random coil. Moreover, the addition of γ-cyclodextrin could induce the change of endogenous fluorescence intensity by changing the microenvironment of the α-amylase. According to the results of NMR, a part of the α-amylase molecule enters the cavity of the γ-cyclodextrin, and the other part forms a hydrogen bond with the outer cavity, forming a complex. The experimental results provide references for the application of γ-cyclodextrin and α-amylase.

Investigating lignin from Canna edulis ker residues induced activation of α-amylase: Kinetics, interaction, and molecular docking

The lignin isolated from C. edulis ker residues showed a significant activating effect on α-amylase. Further studies revealed that the isolated lignin formed a 1:1 complex with α-amylase through hydrogen bonding and quenched fluorescence of α-amylase with a static quenching procedure. Binding with lignin led to conformational and granular size changes of α-amylase. Two-dimensional nuclear Overhauser spectroscopy (2D-NOESY) spectra suggested that OH in G units and β-O-4 structure were the major binding sites of lignin on the α-amylase molecule. Molecular docking studies indicated that the binding residue on α-amylase for lignin was not the same as for chloride ions, and the major binding force was hydrogen bonding. Furthermore, the docking results also showed the structural change of lignin induced by α-amylase. Thus, this work provided a new insight into the interaction between lignin from Canna edulis ker residues and α-amylase, which may be beneficial to apply lignin in the food industry.

Encouragement of Enzyme Reaction Utilizing Heat Generation from Ferromagnetic Particles Subjected to an AC Magnetic Field.

We propose a method of activating an enzyme utilizing heat generation from ferromagnetic particles under an ac magnetic field. We immobilize α-amylase on the surface of ferromagnetic particles and analyze its activity. We find that when α-amylase/ferromagnetic particle hybrids, that is, ferromagnetic particles, on which α-amylase molecules are immobilized, are subjected to an ac magnetic field, the particles generate heat and as a result, α-amylase on the particles is heated up and activated. We next prepare a solution, in which α-amylase/ferromagnetic particle hybrids and free, nonimmobilized chitinase are dispersed, and analyze their activities. We find that when the solution is subjected to an ac magnetic field, the activity of α-amylase immobilized on the particles increases, whereas that of free chitinase hardly changes; in other words, only α-amylase immobilized on the particles is selectively activated due to heat generation from the particles.

Structure Activity Relationships of Flavonoids as Potent α-Amylase Inhibitors

The effects of three flavonoids (quercetin, luteolin, diosmetin) on alpha-amylase were examined by enzymatic kinetics and fluorescence spectroscopy. The three test flavonoids were non-competitive inhibitors of the enzyme. Addition of flavonoids led to fluorescence quenching of alpha-amylase. The quenching was initiated from the formation of a complex between the flavonoids and the enzyme, corresponding to a static quenching process. An alpha-amylase molecule provides a binding site for the test flavonoid. The main binding force was hydrophobic. The decreasing order of inhibition of alpha-amylase by flavonoids and the binding force was luteolin, diosmetin, and quercetin. It is demonstrated that hydroxylation in ring C and methylation of the hydroxyl group in ring B of flavonoids may weaken the binding affinities to alpha-amylase.

Interaction of Dihydromyricetin and α-Amylase

The interaction of dihydromyricetin (DMY) and α-amylase was investigated. The complex formed between DMY and α-amylase resulted in decreased antioxidant activity of DMY and the catalytic activity of the enzyme, as well as efficient quenching of the intrinsic fluorescence of α-amylase. An α-amylase molecule provides one binding site for a DMY molecule. These results will be useful for exploiting this compound to combat diseases efficiently.

Immobilization of a thermostable α-amylase onto reactive membranes: kinetics characterization and application to continuous starch hydrolysis

Epoxy groups containing porous membranes were prepared by UV-initiated photopolymerisation of hydroxyethylmethacrylate (HEMA) and glycidyl methacrylate (GMA). Epoxy supports could provide multipoint covalent attachment of enzymes, therefore, to stabilize their three-dimensional structure. α-Amylase was immobilized onto the poly(HEMA-GMA-1-3) membranes by means of the amide linkage formation between the amino groups of α-amylase and the epoxy groups of the support. The α-amylase immobilization capacity of the membranes was increased as the GMA ratio increased in the membrane structure. The retained activity of the immobilized α-amylase was 76% with poly(HEMA-GMA-2) membrane. The decrease in activity of the immobilized α-amylase could be considered to be due to reduced conformational flexibility of the immobilized α-amylase molecules for binding its large substrate, starch, as a result of the covalent immobilization. The immobilized α-amylase has more resistance to temperature inactivation than that of the free form. The optimum pH value of α-amylase was not affected by the immobilization reaction, but the pH profile was broadened for the immobilized enzyme. Kinetic parameters were determined for immobilized α-amylase as well as for the free enzyme. The values of the Michaelis constant K m of α-amylase, were significantly larger (ca. 2.3 times) upon immobilization, indicating decreased affinity of the enzyme for its substrate, whereas V max was smaller for immobilized α-amylase. In a 120 h continuous operation at 35 °C, only 4% of immobilized α-amylase activity was lost. The operational inactivation rate constant ( k opi) of the immobilized α-amylase with 2% starch was 8.06×10 −6 min −1.

Sucrose-Controlled Transport and Turnover of -Amylase in Rice (Oryza sativa L.) Cells

We studied the sucrose-controlled intracellular transport and turnover of α-amylase molecules in suspension-cultured cells of rice (Oryza sativa L.) employing pulse labeling techniques with [35S] amino acids. The secretion of two classes of α-amylase isoforms, α-amylase I-1 (encoded by RAmy1A) and II-4 (encoded by RAmy3D) was differentially controlled by sucrose. In rice cells labeled with [35S] amino acids under different sucrose-supplemented conditions, sucrose preferentially prevented the extracellular liberation of [35S]-labeled α-amylase II-4 molecules from rice cells at around 2 mM, whereas the de novo protein synthesis still occurred at this concentration. Pulse-chase experiments showed that sucrose regulates the intracellular transport of [35S]α-amylase II-4 molecules and stimulates the protein turnover. However, cycloheximide, a protein synthesis inhibitor, was induced the extracellular liberation and reduced the turnover of [35S]α-amylase II-4 molecules in the presence of sucrose. These results strongly suggested that newly synthesized sucrose-induced proteins are involved in the posttranslational regulation on sucrose-controlled α-amylase secretion in rice cells.

Sugar-Controlled Ca2+ Uptake and -Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberation of α-amylase molecules in suspension-cultured cells of rice (Oryza sativa L.) required Ca2+, although the level of translatable α-amylase mRNA was not affected in the presence of Ca2+. Sugar depletion markedly stimulated Ca2+ uptake by rice cells and sucrose supplementation reduced it. Immunohistochemical and electron probe microanalyzer studies indicated an apparent resemblance between the distribution pattern of Ca2+ and that of α-amylase molecules induced in the sugar-depleted cells. Ca2+ uptake was reduced by sucrose, maltose, fructose, and glucose similarly at more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose (10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effective Ca2+ channel blocker, La3+ significantly inhibited the Ca2+ uptake and the synthesis and extracellular liberation of α-amylase molecules in the absence of sucrose, while a general P-type ATPase inhibitor, vanadate greatly stimulated both in the presence of sucrose. We concluded that, by controlling the Ca2+ uptake, metabolic sugars regulate the protein synthesis and posttranslational secretory processes of α-amylase molecules in rice cells.

Enzyme-based bioartificial polymeric materials: the α-amylase-poly(vinyl alcohol) system

The possibility of realizing bioartificial polymeric materials from blends of the enzyme α-amylase (EC 3.2.1.1) and poly(vinyl alcohol), in the form of film and hydrogel, was investigated. The interactions at molecular level between the enzyme and the synthetic polymer have been studied by calorimetry, X-ray diffractometry and morphological analysis. A certain degree of interaction through which the α-amylase molecules influence the crystallinity of the poly(vinyl alcohol) was observed. The thermal stability of the enzyme in the blends was enhanced, while its biological activity did not vary significantly. In addition, hydrogels prepared by a freeze–thawing method can be considered suitable for manufacturing delivery systems capable of releasing enzymes which maintain their full biological activity. ©1997 SCI

Electron microscopic investigation of the diffusion of Bacillus licheniformis α-amylase into corn starch granules

A method for the direct electron microscopic observation of amylases in interaction with starch granules is presented. The technique involves immuno-gold labeling of the enzymes and cross-sectioning of hydrated starch granules. This approach allows the analysis of the internal degradation of starch with a concomitant visualization of enzymes at the sites of hydrolysis. The visualization of enzymes at the surface, inside the channel and inside the core of the degraded granules shows that the α-amylase molecules first proceed from the surface toward the center (centripetal hydrolysis). Then the core is completely degraded from within by erosion of its periphery (centrifugal hydrolysis). In the first case (centripetal hydrolysis), the enzymes act by progressing along the polysaccharide chains. By contrast, the centrifugal hydrolysis leads to even erosion, indicative of a more diffusive motion of the enzymes.

タンパク質工学によるα‐アミラーゼの糖転移酵素化

The 210 th lysine residue in Saccharomycopsis α-amylase (Sfamy) molecule was replaced by arginine and asparagine residues. The resulting K210R and K210N enzymes cleave mainly the first glycosidic bond from the reducinng end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond. We changed successfully the major cleavage point in the hydrolysis reaction of G4. We estimated the 8th subsite affinities of the mutant enzymes and compared them with that of the native enzyme. These facts suggest that the K210 residue composes the 8th subsite, one of the major subsites, and that a positively charged s-amino residue is necessary for the 8th subsite affinity. The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite. The 84th tryptophan residue was replaced by leucine residues. The resulting W84L enzyme showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. These large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the W84 residue located at subsite 5 plays an important role in the addition of a water molecule to a carbonium ion intermediate and/or in the liberation of the hydrolysis product from the substrate binding pocket. The doubly mutated enzymes, W84LK210 N, are expected to form the transglycosylation products different in size from those produced by the single mutant, W84L enzyme.

Intestinal cell types in the freshwater snail planorbarius corneus: Histochemical, immunocytochemical and ultrastructural observations

The intestinal cell types of the pulmonate gastropod Planorbarius corneus were studied using histochemical, immunocytochemical and ultrastructural procedures. Supporting cells, mucous cells and gland cells were observed in the intestine epithelium. The supporting cells seem to be involved in absorption process, showing endocytosis vesicles, evidence of lysosomal activity and storage of reserve materials. The presence of epithelial cells specialized in secretion indicates that digestive enzymes may be produced. Indeed, immunoreactive α-amylase molecules have been demonstrated. Some gland cells showed the ultrastructural features of invertebrate and vertebrate enteroendocrine cells and were also positive to vertebrate anti-insulin, anti-gastrin and anti-serotonin antibodies.

An increase in the transglycosylation activity of Saccharomycopsis α-amylase altered by site-directed mutagenesis

The 84th tryptophan residue in Saccharomycopsis α-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G 7), for example, maltooligosaccharide products larger than maltodecaose (G 10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.

Preferential Secretion of R-Type α-Amylase Molecules in Rice Seed Scutellum at High Temperatures

Exposure of fresh scutella excised from 4-day-old rice seedlings to higher temperatures, (40-42 degrees C), drastically reduced the biosynthesis of alpha-amylase as determined by the incorporation of [(35)S]methionine into the immunoprecipitable product. However, the intracellular transport and extracellular secretion of the enzyme molecules were enhanced at high temperatures, indicating that the biosynthesis and secretion of alpha-amylase are distinguishable in their temperature dependency. At the higher temperature regime (>==40 degrees C), the complex-type alpha-amylase isoform, resistant to hydrolytic digestion by endo-beta-N-acetylglucosaminidase H (Endo-beta-H) was predominantly secreted, whereas at lower temperatures (<==15 degrees C), the isoform susceptible to Endo-beta-H attack was the major molecular form secreted.

Degradation of α-amylase fromAspergillus oryzae with firmly bound proteinases at pH 3.0

Incubation of highly purified α-amylase fromAspergillus oryzae (EC 3.2.1.1) with 0.01M acetate buffer, pH 3.0, resulted in degradation of the α-amylase. The molecular weight values of degradation products were 42 K, 37 K, and 28 K. Incubation of the purified α-amylase in 0.02m phosphate buffer, pH 7.5, at 30°C for 17 h, however, resulted in no degradation of the α-amylase molecule. Incubation of the purified α-amylase with proangiotensin at pH 3.0 for 24 h resulted in cleavage of Tyr4-Ile5, His6-Pro7, Pro7-Phe8, Phe8-His9, and His9-Leu10. Thus, it appears that proteolytic activities firmly bound to α-amylase are identical withAspergillus aspartic proteinase (EC 3.4.23.6) andAspergillus acid carboxypeptidase (EC 3.4.16.1).