Proteinaceous Alpha-amylase Inhibitor Research Articles

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.

A Proteinaceous Alpha-Amylase Inhibitor from Moringa Oleifera Leaf Extract: Purification, Characterization, and Insecticide Effects against C. maculates Insect Larvae.

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the Kmax and Vmax of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.

Identification of a Proteinaceous Alpha Amylase Inhibitor from a Medicinal Herb Oxalis corniculata L. (Oxalidaceae)

Health management through traditional medicine is a promising approach worldwide as it represents a multifaceted approach to health care than conventional medicine. The heightened importance to validate the efficacy and standards of traditional herbal medicine is the thrust area of present day research. The present study is one such attempt to evaluate the alpha amylase inhibitory potential and identify the candidate inhibitor from Oxalis corniculata L, a potent indigenous medicinal plant. Sequential solvent extraction of the leaves of O. corniculata was performed in previous work and the aqueous extract, showing maximum inhibition against porcine pancreatic alpha amylase against starch as substrate (IC50 value 68.08+0.06), was selected for purification using ammonium sulphate precipitation. The pellet obtained at 40%-80% precipitation was further subjected to ion exchange chromatography on DEAE-cellulose and gel filtration chromatography using Sephadex G-100. The molecular weight of the proposed amylase inhibitor named AI-1 was estimated to be about 30 kda on SDS-PAGE. Temperature sensitivity and pH stability of the inhibitor was studied. The AI-1 protein was stable up to 400 C and was totally destroyed beyond 700 C and showed maximum activity at pH 6. Further characterization of the AI-1 protein and its sequence determination will help in discovering a novel proteinaceous alpha amylase inhibitor from O. corniculata. Research of this kind will help to usher the identification of active bioconstituents from plants with great medicinal activities.

Full‐fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3‐D analytical technique: Identification of new heterodimeric aggregation states

Wheat proteinaceous alpha-amylase inhibitors (alpha-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive alpha-AIs that share sequence homology, but differ in their specificity against alpha-amylases from different species and for their aggregation states. Wheat alpha-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive alpha-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of alpha-AIs (resistant to T > 70 degrees C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the alpha-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric alpha-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric alpha-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat alpha-AIs.

Molecular cloning and expression of proteinaceous alpha-amylase inhibitor gene from Streptomyces nitrosporeus in Escherichia coli.

The proteinaceous alpha-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous alpha-amylase inhibitors.

PURIFICATION AND CHARACTERIZATION OF ESCULENTAMIN, A PROTEINACEOUS ALPHA-AMYLASE INHIBITOR FROM THE TARO ROOT, COLOCASIA ESCULENTA

A proteinaceous alpha-amylase inhibitor from the cormel of taro (Colocasia esculenta) was purified by extraction, heat treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-100, BioGel P-30, and DE-53 cellulose. This protein (aptly named esculentamin) was shown to be an acidic (pI = 4.4) glycoprotein with a carbohydrate content of 3.64% and a molecular weight of 11,800 daltons. Esculentamin activity was essentially stable to boiling for 3 h (pH 7.0), to a pH range of 2.0 to 12.0 (at 4°C), and to 6 M guanidine hydrochloride and 8 M Urea. Amino acid analysis indicated a predominance of acidic over basic amino acids, while side-by-side neutral and alkaline absorption spectra strongly suggested a lack of tryptophan. “Bifunctional” inhibitory activity towards both α-amylase and proteases was not observed with esculentamin (i. e., it did not inhibit the several proteases tested, viz. trypsin and subtilisin).

Nucleotide sequence and expression of a Streptomyces griseosporeus proteinaceous alpha-amylase inhibitor (HaimII) gene.

The coding region of the alpha-amylase inhibitor (HaimII) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimII gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimII protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimII protein. The pre-HaimII protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimII protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimII gene.

Purification of the proteinaceous alpha-amylase inhibitor from Phaseolus vulgaris by affinity chromatography with immobilized enzyme or antibody.

Abstract A protein inhibiting salivary and pancreatic a-amylase of mammalian origin is contained in dry seeds of beans (Phaseolus vulgaris). Starting from a crude bean extract, the amylase inhibitor may be purified about 30fold in one step to apparent homogeneity by chromatography on matrix-bound salivary amylase. Compared with protein obtained by a conventional purification procedure and in similar yield, the amylase inhibitor obtained by affinity chromatography had the same specific activity (4.5 (akat inhibitor units/mg protein). A one step purification from crude extracts to homogenous inhibitor with the same specific activity was achieved by immuno-affinity chromatography on immobilized rabbit antibody raised against pure amylase inhibitor. The yield was 60 % that of a conventional purification. Criteria of purity of the inhibitor protein were thin-layer electrofocussing and immuno-electrophoresis.