Proteinaceous Α-amylase Inhibitor Research Articles

Effects of chickpea protein fractions on α-amylase activity in digestion

This study concerns the effects of endogenous proteins on starch digestion kinetics. It investigates the effects in chickpeas of hydrolysates released from the endogenous proteins albumin, globulin and glutelin on the in vitro activity of porcine pancreatic α-amylase (PPA) and on starch digestion. Using a docking simulation, potential proteinaceous α-amylase inhibitors (α-AIs) belonging to the small peptides of albumin, globulin and glutelin are identified and their binding mechanisms with PPA are explored. It was found that cooking and pepsin hydrolysis led to severe degradation of protein fractions, accompanied by the enrichment of small (<10 kDa) peptides, among which peptides <3 kDa exhibited strong inhibitory activity (up to 50%). Most of the active α-AIs were therefore considered to be found in the fractions <3 kDa, and were further characterized by mass spectrometry combined with in silico analysis. Docking results showed that glutelin could produce the most α-AIs (45 peptides), followed by globulin (41) and albumin (12). Among these, 71 peptides were predicted to exhibit a competitive or uncompetitive type of inhibition on PPA, whereas 6 peptides performed a noncompetitive inhibition. The competitive and uncompetitive α-AIs inhibited enzyme activity mainly by binding to a flexible loop and three major catalytic residues in PPA, and the latter by interacting with the non-catalytic regions of PPA. Different inhibition types therefore can together to hinder the formation of enzyme-starch complexes, so that PPA activity is inhibited and starch digestibility could be reduced. • Chickpea proteins reduce starch digestion rate, which is nutritionally advantageous. • Cooking and pepsin hydrolysis produced albumin-, globulin- and glutelin-peptides. • Peptides<3 kDa had the highest α-amylase inhibitory activity. • 77 peptides were predicted to be α-amylase inhibitors. • Inhibitors exhibited competitive, uncompetitive and noncompetitive inhibition.

The impact of the soluble protein fraction and kernel hardness on wheat flour starch digestibility

Wheat is the staple crop for 35% of the world’s population, providing a major source of calories, mainly in the form of starch. The digestibility of wheat starch varies between different flours and products. Wheat products that are rapidly digested elicit large post-prandial glucose peaks associated with metabolic disorders. We investigated the impact of protein on starch digestion in three commercial flours with different grain hardness. A soluble extract of wheat proteins reduced starch digestion, even following gastric proteolysis. This extract was enriched in proteinaceous α-amylase inhibitors which were partially degraded during gastric proteolysis. Starch digestion kinetic analysis was carried out for flour samples pre-treated with different pepsin activities. The rate of starch digestion was altered following pepsin pre-digestion, and the extent of starch digestion increased in response to pepsin pre-digestion. We conclude that soluble proteinaceous alpha-amylase inhibitors present in wheat can escape gastric digestion and significantly contribute to reducing starch digestion in the small intestine.

High effective proteinaceous α-amylase inhibitors from grains and control release

Plant-derived α-amylase inhibitors exhibit great capacity in glycaemic control by restraining dietary starch digestion. However, natural α-amylase inhibitors with strong inhibitory activity and good stability are still scarce. In this study, the inhibitory activity and stability of proteinaceous α-amylase inhibitors derived from eleven grains were examined, and microencapsulation was carried out to protect the inhibitors in gastric fluid. We found that the highland barley protein extract (HBPE) showed the most potent α-amylase inhibitory activity (IC50 = 1.11 mg/mL). HBPE is a reversible noncompetitive α-amylase inhibitor and displayed good stability across various pH and temperatures. It showed high trypsin tolerance but low pepsin tolerance. Thus HBPE-encapsulated microspheres were prepared by the composite of sodium alginate/gellan gum with the internal ionic gelation method. The optimum encapsulation condition was determined, and the prepared microspheres displayed discrete, uniform and regular sphericities with diameters around 53 μm, and better mechanical strength with more than 81% activity retention rate. Moreover, the microspheres protected the HBPE in gastric fluid and extended HBPE release in the small intestinal fluid, revealing excellent control release of HBPE in simulated gastrointestinal digestion. Our study provides a new and effective strategy for the management of postprandial blood glucose.

Effect of α-amylase inhibitor from Withania somnifera on growth and development of Callosobruchus chinensis and in silico studies on its interactions with insect amylase

Pulse beetle (Callosobruchus chinensis) is a cosmopolitan stored grain pest of legumes responsible for significant losses in them during storage. A proteinaceous α-amylase inhibitor, isolated and purified from Withania somnifera (WSAI), affected various growth and developmental parameters of Callosobruchus chinensis. Whereas female fecundity, adult emergence and % seed damaged were found to be reduced, mean development period and feeding deterrence index increased as a function of WSAI treatment. Almost 59% relative amylase activity was observed at 50 µg/mL of WSAI and confirmed by starch activity gel assay. Longevity of survived insects of treated sets was reduced by 2 days compared to control sets. Possible homology modelled structures for WSAI and C. chinensis proteins were proposed. The In-silico protein-protein docking studies revealed the hydrogen bonding network, binding energy (−16.5 Kcal) and amino acids involved in forming interactions which will be helpful in understanding the mechanism of inhibition in future.

The role of thermostable proteinaceous α-amylase inhibitors in slowing starch digestion in pasta

Starch in pasta is slowly digested, which is nutritionally advantageous because it reduces post-prandial glycemia, a risk factor for type 2 diabetes. We examine the role of proteinaceous amylase inhibitors, providing insights into how wheat proteins slow starch digestion. Gluten samples were extracted in the laboratory and also commercially obtained, and incubated in water at 37 °C and 100 °C. The centrifuged supernatant and gluten pellet were incubated with porcine α-amylase, and enzymatic activity measurement revealed that the supernatant from gluten heated to 100 °C retained the capacity to significantly reduce amylolytic activity. Confocal microscopy showed that the gluten pellet had become porous following heat treatment. These results are consistent with an increased concentration of soluble proteins after cooking. Proteins in the soluble fraction and gluten pellet were identified with mass spectrometry proteomics, which screened a limited number of endogenous double-headed α-amylase/trypsin inhibitors (α-AI(s)), found to be enriched in the supernatant after cooking. Endogenous α-AI(s) play a role in slowing starch digestion by interacting with α-amylase as it penetrates the gluten network that entraps the starch, resulting in an immobilization of the enzyme onto the gluten network, so that the digestion of starch entrapped by the gluten network is inhibited.

A cupin domain is involved in α-amylase inhibitory activity

Proteinaceous α-amylase inhibitors have specialized activities that make some strong inhibition of α-amylases. New α-amylase inhibitors continue to be discovered so far. A proteinaceous α-amylase inhibitor CL-AI was isolated and identified from chickpea seeds. CL-AI, encoded by Q9SMJ4, was a storage legumin precursor containing one α-chain and one β-chain, and each chain possessed a same conserved cupin domain. Amino acid mutation and deficiency of cupin domain would lead to loss of α-amylase inhibitory activity, indicating that it was essential for inhibitory activity. CL-AI(α + β) in its single stranded state in vivo had inhibitory activity. After it was processed into one α-chain and one β-chain, the two chains were connected to each other via disulfide bond, which would cover the cupin domains and lead to the loss of inhibitory activity. The CL-AI(α + β), α-chain and β-chain could inhibit various α-amylases and delay the seed germination of wheat, rice and maize as well as the growth and development of potato beetle larva. Two cupin proteins, Glycinin G1 in soybean and Glutelinin in rice were also found to have inhibitory activity. Our results indicated that the cupin domain is involved in α-amylase inhibitory activity and the proteins with a cupin domain may be a new kind of proteinaceous α-amylase inhibitor.

Isolation, Identification and Characterization of a New Type of Lectin with α-Amylase Inhibitory Activity in Chickpea (Cicer arietinum L.)

Plant lectins are a group of highly diverse proteins that possess at least one non-catalytic domain that binds reversibly to a specific mono- or oligosaccharide. So far, only seven members in the lectin-arcelin-αAI1 supergene family in legume lectins have been reported to have inhibitory activity of α-amylases. A proteinaceous α-amylase inhibitor was isolated and purified using Ammonium sulfate precipitation (ASP), Ion exchange chromatography (IEC) and Reversed phase liquid chromatography (RPLC) from the mature seeds of chickpea. Identification by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) indicated that the purified proteinaceous α-amylase inhibitor was a chickpea lectin CAL in GenBank (accession No. AGL46982.1). CAL had 227 aa containing a hemopexin- like repeats domain and was a cytoplasm protein. It had very low (<17%) identity with seven members in the lectin-arcelin-aAI1 supergene family in legumes that have α-amylase inhibitory activity. The purified CAL derived from prokaryotic expression was confirmed to have inhibitory activity against various α-amylases. The inhibitory activity of CAL against various α-amylases was severely affected by temperature, pH, incubation time, substrate concentration and CAL protein concentration. Feeding CAL reduced the weight of potato beetle larvae by 27.21% (P<0.05) and survival rate by 6.67% (P>0.05). Our results indicated that CAL is a new type of lectin with inhibitory activity against α-amylases in legume lectins, which can be used as a candidate in genetic engineering for breeding for pest resistance.

Isolation, purification and characterization of proteinaceous fungal α-amylase inhibitor from rhizome of Cheilocostus speciosus (J.Koenig) C.D.Specht

As the aim of this present study, a proteinaceous α-amylase inhibitor has been isolated from the rhizome of Cheilocostus specious (C. speciosus) and was purified using DEAE cellulose anion exchange chromatography followed by gel filtration using Sephacryl-S-200 column. The purity and molecular mass of the purified inhibitor was determined by SDS-PAGE and LC-MS respectively. The molecular mass of the purified inhibitor was determined to be 31.18kDa. Protein-protein docking was also carried out as molecular model. Model validation methods such as Ramachandran plot and Z-score plot were adopted to validate the structural description (sequence analysis) of proteins. The inhibitory activity was confirmed using spectrophotometric and reverse zymogram analyses. This 31.18kDa protein from C. speciosus inhibited the activity of fungal α-amylase by 71% at the level of ion exchange chromatography and 96% after gel filtration. The inhibition activity of the α-amylase inhibitor was stable and high at optimum pH6 (52.2%) and temperatures of 30–40°C (72.2%). Thus it was suggested that the main responsible for the versatile biological and pharmacological activities of C. speciosus is due to its primary metabolites (proteins) only.

Bioprospecting of bacillus pumilus ngp-2 for the production Of proteinaceous α-amylase inhibitor

Bioprospecting of bacillus pumilus ngp-2 for the production Of proteinaceous α-amylase inhibitor

Isolation of proteinaceous α-AMYLASE inhibitor from Bacillus pumilus NGP-1

Isolation of proteinaceous α-AMYLASE inhibitor from Bacillus pumilus NGP-1

Isolation and characterization of α-amylase inhibitor from Leucas aspera (Willd) Link: α-amylase assay combined with FPLC chromatography for expedited identification

The aim of the present study was to isolate and characterize a proteinaceous α-amylase inhibitor from the whole plant extract of Leucas aspera (Willd) Link. The proteins were further purified by fast and reliable ion-exchange chromatography. A ~ 28 kDa protein from L. aspera inhibited the activity of fungal α-amylase by 90% at 80:1 (inhibitor:enzyme) ratio. The inhibition activity was examined in various α-amylases and its enhanced inhibition activity was witnessed. The activity of the inhibitor on α-amylase was stable and high at pH 6–7 and at temperatures of 30–50 °C. The high-resolution α-amylase inhibition assay/FPLC-MS-SPE platform allowed identification of 28 kDa protein with high purification fold as the α-amylase inhibitor in L. aspera and peptides were matched with highest score of alpha-amylase/trypsin inhibitor of Zea mays. In conclusion, results here obtained suggested that the primary metabolites (proteins) in L. aspera are mainly responsible for its versatile biological and pharmacological activities.

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785.

A maltotriose-forming amylase (G3Amy) from Kitasatospora sp. MK-1785 was successfully isolated from a soil sample by inhibiting typical extracellular α-amylases using a proteinaceous α-amylase inhibitor. G3Amy was purified from the MK-1785 culture supernatant and characterized. G3Amy produced maltotriose as the principal product from starch and was categorized as an exo-α-amylase. G3Amy could also transfer maltotriose to phenolic and alcoholic compounds. Therefore, G3Amy can be useful for not only maltotriose manufacture but also maltooligosaccharide-glycoside synthesis. Further, the G3Amy gene was cloned and expressed in Escherichia coli cells. Analysis of its deduced amino acid sequence revealed that G3Amy consisted of an N-terminal GH13 catalytic domain and two C-terminal repeat starch-binding domains belonging to CBM20. It is suggested that natural G3Amy was subjected to proteolysis at N-terminal region of the anterior CBM20 in the C-terminal region. As with natural G3Amy, recombinant G3Amy could produce and transfer maltotriose from starch.

Proteinaceous α-amylase inhibitors of Setaria italica Linn (Co-6) and its effect on α-amylase from human saliva and Bacillus sp

The specific α-amylase inhibitory activity of Setaria italica Linn (Co-6) was determined by using heat-treated and ammonium sulphate precipitated fractions of α-amylase isolated from Bacillus sp. The α-amylase inhibitor from S. italica Linn (Co-6) was precipitated with 1.0 M fraction of ammonium sulphate and used for further purification and characterization. To overcome the endogenous α-amylase activity, the extract was subjected to heat treatment at 60oC. At temperature above 85o C, complete denaturation of α-amylase inhibitory activity of the millet extract was evidenced. The results of the study suggest the use of S. italica Linn (Co-6) flour, which exhibits significant of α-amylase inhibitory activity, as a dose dependent diet for Non-Insulin dependent diabetes mellitus.

Isolation and characterization of a proteinaceous α-amylase inhibitor AAI-CC5 from Streptomyces sp. CC5, and its gene cloning and expression.

An α-amylase inhibitor producing Streptomyces sp. strain CC5 was isolated from soil. A proteinaceous α-amylase inhibitor AAI-CC5 was purified from strain CC5. AAI-CC5 specifically inhibited mammalian α-amylases. The molecular weight of the inhibitor was determined to be 8,212 Da by MALDI-TOF Mass Spectrum. The N-terminal 15 amino acid residues of the purified AAI-CC5 were DTGSPAPECVEYFQS, which is dissimilar to other reported proteinaceous α-amylase inhibitors. AAI-CC5 is a pH insensitive and heat-stable protein, and cannot be hydrolysed by trypsin. AAI-CC5 was cloned and expressed in Escherichia coli BL21 (DE3) with a hexa-histidine tag on the C terminal. AAI-CC5 shared 82 % identity with Parvulustat. The recombinant α-amylase inhibitor was purified to homogeneity by one-step affinity chromatography using Ni(2+)-NTA resin with molecular mass of 9,404 Da. Steady state kinetics studies of α-amylase and the inhibitor revealed an irreversible, non-competitive inhibition mechanism with IC50 and Ki value of 6.43 ×1 10(-11) and 4.45 × 10(-11) M respectively. These results suggest this novel α-amylase inhibitor possessed powerful inhibitory activity for α-amylase, and it may be a candidate in research of diabetes therapy and obesity treatment.

Discovery and characterization of pseudocyclic cystine-knot α-amylase inhibitors with high resistance to heat and proteolytic degradation.

Obesity and type 2 diabetes are chronic metabolic diseases, and those affected could benefit from the use of α-amylase inhibitors to manage starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated from an Apocynaceae plant show promise for further development as orally active α-amylase inhibitors. These linear peptides retain the stability known for cystine-knot peptides in the presence of harsh treatment. They are resistant to heat treatment and endopeptidase and exopeptidase degradation, which is characteristic of cyclic cystine-knot peptides. Our NMR and crystallography analysis also showed that wrightides, which are currently the smallest proteinaceous α-amylase inhibitors reported, contain the backbone-twisting cis-proline, which is preceded by a nonaromatic residue rather than a conventional aromatic residue. The modeled structure and a molecular dynamics study of Wr-AI1 in complex with yellow mealworm α-amylase suggested that, despite having a similar structure and cystine-knot fold, the knottin-type α-amylase inhibitors may bind to insect α-amylase via a different set of interactions. Finally, we showed that the precursors of pseudocyclic cystine-knot α-amylase inhibitors and their biosynthesis in plants follow a secretory protein synthesis pathway. Together, our findings provide insights for the use of the pseudocyclic α-amylase inhibitors as useful leads for the development of orally active peptidyl bioactives, as well as an alternative scaffold for cyclic peptides for engineering metabolically stable human α-amylase inhibitors.

Digestive α-amylase of Bacterocera oleae Gmelin (Diptera: Tephritidae): Biochemical characterization and effect of proteinaceous inhibitor

Digestive α-amylase of Bacterocera oleae larvae was characterized and treated by an inhibitor to gain a better understanding of the degradation of nutritional molecules as a potential target for controlling the pest. Presence of α-amylase was confirmed in the gut of olive fruit fly through the use of a negative control in dinitrosalicylic acid procedure. An optimal pH of 5 was found for amylolytic activity in the gut. The enzyme had optimal activity in a broad range of temperatures 20–45°C. Among used cations and specific inhibitors, Ca2+, phenylmethylsulphonyl fluoride (PMSF) and ethylene glycol-bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA) had statistical differences on amylolytic activity indicating the presence of amino acid triad and Ca2+ in active site of the enzyme. A proteinaceous α-amylase inhibitor was extracted from Polygonum persicaria, a medicinal plant, that widely grows in North of Iran. IC50 value of PPAI was 0.062mg/ml (i.e. 0.062mg/ml of extracted inhibitor inhibited 50% of amylolytic activity in the gut of B. oleae larvae) and was temperature and pH dependent. The use of enzyme inhibitors from different plant sources may serve as an important pest control strategy via plant breeding programs. Identification of genes responsible for these inhibitor proteins could be a first step to provide a resistant variety of olive.

Novel isoforms of proteinaceous α-amylase inhibitor (α-AI) from seed extract of Albizia lebbeck

Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-αAI showed two polypeptide bands of ~35.8 and ~32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 °C. The finding and information obtained in the present investigation about novel isoforms of α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.

Inhibitory activity of proteinaceous α-amylase inhibitors from Triticale seeds against Eurygaster integriceps salivary α-amylases: Interaction of the inhibitors and the insect digestive enzymes

In many countries, the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is one of the most important insect pests of wheat. Chemical control of E. integriceps is not efficient making it essential to find other control methods. Plant α-amylase inhibitors expressed as transgenes have emerged as a promising way of controlling insect pests and several crop species expressing transgenic amylase inhibitors have been shown to be resistant to certain insect pest. Here we report inhibition of salivary α-amylases of E. integriceps by semi-purified proteinaceous α-amylase inhibitors from Triticale (TAI). A dose dependent trend of inhibition of the enzyme was observed using TAI with an I50 value of 0.79μg of TAI. At the highest concentration of the inhibitor used here, we found 87% inhibition of amylase activity. The inhibitory activity was maximal at pH at 5.0, which is in accordance with the pH optimum of the salivary gland enzymes. Kinetic studies of enzyme-inhibitor interaction revealed a kind of partial mixed inhibition with an apparent inhibitory constant value (Ki[app]) of 1.1μg of TAI against salivary α-amylase in the assay medium. In gel inhibition assays showed that all isoforms of salivary glands α-amylase were sensitive to the TAI, although to different degrees. Moreover, the effectiveness of TAI was retained in different stages of pre-oral and oral digestion of E. integriceps, although some inhibitory activity was lost, possibly by the action of the insect proteases. These findings indicate that TAI shows promise for use in the management of this pest. To the best of our knowledge, this is the first study of inhibitor-insect digestive enzymes interaction, based on the insect feeding strategy and digestion process.

【総説-糖質関連酵素化学シンポジウム-】 タンパク性α-アミラーゼ阻害剤を生産する放線菌の澱粉資化戦略

【総説-糖質関連酵素化学シンポジウム-】 タンパク性α-アミラーゼ阻害剤を生産する放線菌の澱粉資化戦略

Biophysical characterization of α-amylase inhibitor Parvulustat (Z-2685) and comparison with Tendamistat (HOE-467)

Parvulustat is a small, highly active proteinaceous α-amylase inhibitor whose high-resolution NMR structure was recently solved in Frankfurt. Here, we present its biochemical and biophysical characterization. Several spectroscopic methods such as UV, fluorescence and CD were utilized to extract conformational changes upon modification of pH, temperature and chemical denaturant. Parvulustat revealed native like behavior over a wide range of denaturizing agents as reflected in terms of activity and thermodynamic data. In addition, spectroscopic and thermodynamic properties of Parvulustat were compared to the well-characterized Tendamistat. Despite the overall structural similarity, the thermodynamic stability of the two proteins is different. Our analysis led to the conclusion that Parvulustat is even more stable than Tendamistat. Furthermore, investigations on three C-terminally truncated Parvulustat derivatives indicate that the higher stability is caused by the long flexible C-terminus.