Physical and catalytic properties of alpha-amylase from Tenebrio molitor L. larvae.

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an alpha-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect alpha-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch.     

Interaction of human alpha-amylases with inhibitors from wheat flour

The interaction of four purified alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic alpha-amylases was investigated. The inhibitory activity of the four proteins towards salivary alpha-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic alpha-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic alpha-amylase and inhibitor 4 was observed, though complex formation between pancreatic alpha-amylase and the other inhibitors was not clearly demonstrated.     

Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean)

Herein, we report the purification and biochemical characterization of a novel bi-functional protein proteinase/amylase inhibitor from the dietary leguminous pulse Phaseolus aureus Roxb. (Vigna radiata L.) by means of acetic acid precipitation, salt fractionation, ion-exchange chromatography (DEAE-cellulose) and affinity chromatography on trypsin-sepharose column. P. aureus inhibitor is a bi-functional inhibitor since it exhibits inhibitory activity towards trypsin-like and alpha-chymotrypsin-like serine proteinases as well as against alpha-amylases. It is a helix-rich protein (Mr 13,600) containing approximately eight tyrosines, one tryptophan and two cystines. N-terminal sequence alignment reveals no homology to other proteinase inhibitors reported from Phaseolus sp. thereby confirming that it is a novel inhibitor. Inhibitory activity measurements show that the inhibitor is quite stable even at extremely high temperatures and is only slightly affected by pH changes. Circular dichroism (CD) conformational studies revealed some changes in its near- as well as far-ultraviolet spectrum at extremes of pH and temperature. Treatments with trypsin for varying time periods did not alter its proteolytic inhibitory activity but caused some reduction in its amylase inhibitory activity.     

New alpha-amylase and trypsin inhibitors among the CM-proteins of barley (Hordeum vulgare)

Barley CM-proteins are a group of at least five salt-soluble components (CMa-e) that can be selectively extracted from endosperm with chloroform/methanol mixtures. N-terminal sequences of proteins CMa, CMb and CMc have been determined and found to be homologous to those previously determined for CMd and CMe, an observation which confirms that their structural genes are members of a dispersed multi-gene family. The purified CM-proteins were tested against trypsin and against alpha-amylases from saliva, pancreas, Aspergillus oryzae, Tenebrio molitor and barley. Besides CMe, which was known to be a trypsin inhibitor, CMc also showed antitrypsin activity, whereas CMa was specifically active against the alpha-amylase from T. molitor and no inhibitory activity was found for proteins CMb and CMd. The evolutionary implications of these findings are discussed.     

Integrity of proteins in human saliva after sterilization by gamma irradiation

Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10(-6) was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form.     

Starch degradation during germination of Civer arietinum L. seeds.

The variations in starch and soluble sugar content, in phosphorylase and amylase activities in cotyledons of germinating seeds of Cicer arietinum L. are determined. Results from various experiments prove that the alpha-amylases are chiefly responsible for amylase activity. Phosphorylase plays an important r?le during the first two days of germination, but it is relegated to a secondary position as the amylase activity increases. Disc electrophoresis on polyacrylamide gel shows the existence of a phosphorylase throughout germination, and detects two alpha-amylases after 48 and 96 h germination respectively. The increase in alpha-amylase activity during germination is due to de novo synthesis of the two isoenzymes, since both are inhibited by cycloheximide and actinomyces D. This de novo synthesis depends on some embryo produced factor, unreplaceable either by giberellic acid or by kinetin.     

Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch

Background

The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs) in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored.

Principal Findings

Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual''s amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning.

Conclusions

By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status.     

Identification of albumin 0.19 in wheat and other cereal proteins

It was found that albumin 0.19, being an inhibitor of alpha-amylases from human and insect saliva, is not specific for T. aestivum and is also revealed immunochemically by means of antiserum in the proteins of T. durum. Using rocket immunoelectrophoresis, it was shown that the albumin component specific for T. aestivum is not identical to albumin 0.19. The levels of albumin 0.19 in the proteins of wheat, aegilops. secale, agropyron and barley grains were studied.     

Study of the inhibition of alpha-amylase by the benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine

Inhibition of porcine pancreas and human saliva alpha-amylase (EC 3.2.1.1) by sanguinarine and chelerythrine was studied. The inhibition of alpha-amylase was assayed using a biosensor method which utilises a flow system equipped with a peroxide electrode. 250 microM sanguinarine and 250 microM chelerythrine cause complete inhibition of 1.9 nkat alpha-amylase from porcine pancreas. The same concentration of sanguinarine and chelerythrine caused 23.9% and 7.5% inhibition, respectively, of 1.9 nkat alpha-amylase from human saliva. Mixed type and partially reversible inhibition was found for both alpha-amylases treated with either alkaloid.     

Cloning and nucleotide sequence of the maltopentaose-forming amylase gene from Pseudomonas sp. KO-8940.

The gene coding for the maltopentaose-(G5)-forming amylase of Pseudomonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides were sequenced. It was expected that a long open reading frame composed of 1,842-bp that encoded 614 amino acid residues for secretory precursor polypeptide including the typical signal sequence with an NH2-terminal was the gene. An extract of Escherichia coli carrying the cloned G5-forming amylase gene had amylolytic activity with which produced only G5 from starch, the same as that of the donor strain enzyme. In the deduced primary structure of this enzyme, the four conserved regions of many alpha-amylases were found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases.     

Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195.

Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.     

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.     

Glycosylated salivary alpha-amylases are capable of maltotriose hydrolysis and glucose formation

The physiological and/or clinical significance of sugar chains in human salivary alpha-amylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated alpha-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G3) on starch hydrolysis of the amylase fraction, suggests that starch and G3 can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G3 hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G3, G4, G5 and G7, as substrates, the substrate-specificities and G3/G5 ratio of amylase activities in the four fractions were examined. Maltopentaose, G5, is routinely used as a substrate for alpha-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G5. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G3 hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G4, G5 and G7. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.     

Circular dichroism of mammalian follitropins and the effects of treatment with N-bromosuccinimide

The circular dichroism of the tryptophan containing glycoprotein hormone, follitropin, displays bands in the near ultraviolet which are absent in homologous, tryptophan-free hormones. In the far ultraviolet, the dichroism is very similar to the other glycoprotein hormones with little or no indication of α-helix. The single tryptophan of follitropin is in a domain of the β subunit sequences of these hormones which is highly conserved from hormone to hormone. Without prior dissociation of the follitropin into subunits, no change is seen in circular dichroism, absorption at 280 nm, fluorescence emission or hormonal activity after treatment with N-bromosuccinimide. In contrast, these properties change when intact human lutropin is studied; its tryptophan residue is a position different than in follitropin. These results support the proposal that the domain containing the tryptophan in follitropin is in or near a region of subunit-subunit contact in the glycoprotein hormones.     

Complete amino acid sequence of an alpha-amylase inhibitor in wheat kernel (0.19-inhibitor)

Amino acid composition of the 0.19-inhibitor from wheat kernel is very similar to that of the 0.53-inhibitor, but a marked difference in inhibitory activity towards human salivary and pancreatic alpha-amylases was detected between the two inhibitors. Elucidation of the primary structure of the 0.19-inhibitor and structural comparison with the 0.53-inhibitor is essential to understand not only the mechanism of the selective inhibitory behaviors but also evolutional relationship of these inhibitors. The complete amino acid sequence of the 0.19-inhibitor was determined after cleaving the protein with cyanogen bromide and trypsin. As in the case for the 0.53-inhibitor, the 0.19-inhibitor is composed of two identical subunits with 124 amino acid residues. Comparison of the sequence of the 0.53- and 0.19-inhibitor shows very high sequence homology with amino acid substitutions at seven positions.     

Substrate-inhibitor interactions in the kinetics of alpha-amylase inhibition by ragi alpha-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments

The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.     

Structural basis for the inhibition of mammalian and insect alpha-amylases by plant protein inhibitors

Alpha-amylases are ubiquitous proteins which play an important role in the carbohydrate metabolism of microorganisms, animals and plants. Living organisms use protein inhibitors as a major tool to regulate the glycolytic activity of alpha-amylases. Most of the inhibitors for which three-dimensional (3-D) structures are available are directed against mammalian and insect alpha-amylases, interacting with the active sites in a substrate-like manner. In this review, we discuss the detailed inhibitory mechanism of these enzymes in light of the recent determination of the 3-D structures of pig pancreatic, human pancreatic, and yellow mealworm alpha-amylases in complex with plant protein inhibitors. In most cases, the mechanism of inhibition occurs through the direct blockage of the active center at several subsites of the enzyme. Inhibitors exhibiting "dual" activity against mammalian and insect alpha-amylases establish contacts of the same type in alternative ways.     

Interaction of wheat monomeric and dimeric protein inhibitors with alpha-amylase from yellow mealworm (Tenebrio molitor L. larva).

The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed 'inhibitor 0.19') and one monomeric (termed 'inhibitor 0.28'), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase--inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase--inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase--inhibitor-0.28 complex were observed. Dissociation constants of the amylase--inhibitor-0.19 and amylase--inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed.     

Cloning, nucleotide sequence, and enzymatic characterization of an alpha-amylase from the ruminal bacterium Butyrivibrio fibrisolvens H17c.

A Butyrivibrio fibrisolvens amylase gene was cloned and expressed by using its own promoter on the recombinant plasmid pBAMY100 in Escherichia coli. The amylase gene consisted of an open reading frame of 2,931 bp encoding a protein of 976 amino acids with a calculated Mr of 106,964. In E. coli(pBAMY100), more than 86% of the active amylase was located in the periplasm, and TnphoA fusion experiments showed that the enzyme had a functional signal peptide. The B. fibrisolvens amylase is a calcium metalloenzyme, and three conserved putative calcium-binding residues were identified. The amylase showed high sequence homology with other alpha-amylases in the three highly conserved regions which constitute the active centers. These and other conserved regions were located in the N-terminal half, and no similarity with any other amylase was detected in the remainder of the protein. Deletion of approximately 40% of the C-terminal portion of the amylase did not result in loss of amylolytic activity. The B. fibrisolvens amylase was identified as an endo-alpha-amylase by hydrolysis of the Phadebas amylase substrate, hydrolysis of gamma-cyclodextrin to maltotriose, maltose, and glucose and the characteristic shape of the blue value and reducing sugar curves. Maltotriose was the major initial hydrolysis product from starch, although extended incubation resulted in its hydrolysis to maltose and glucose.     

Purification, biochemical characterization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming alpha-amylase from an endophytic ascomycete Fusicoccum sp. BCC4124

An endophytic fungus, Fusicoccum sp. BCC4124, showed strong amylolytic activity when cultivated on multi-enzyme induction enriched medium and agro-industry substrates. alpha-Amylase and alpha-glucosidase activities were highly induced in the presence of maltose and starch. The purified target alpha-amylase, Amy-FC1, showed strong hydrolytic activity on soluble starch (kcat/Km=6.47 x 10(3) min(-1)(ml/mg)) and selective activity on gamma- and beta-cyclodextrins, but not on alpha-cyclodextrin. The enzyme worked optimally at 70 degrees C in a neutral pH range with t(1/2) of 240 min in the presence of Ca(2+) and starch. Maltose, matotriose, and maltotetraose were the major products from starch hydrolysis but prolonged reaction led to the production of glucose, maltose, and maltotriose from starch, cyclodextrins, and maltooligosaccharides (G3-G7). The amylase showed remarkable glucose tolerance up to 1 M, but was more sensitive to inhibition by maltose. The deduced protein primary structure from the putative gene revealed that the enzyme shared moderate homology between alpha-amylases from Aspergilli and Lipomyces sp. This thermotolerant, glucose tolerant maltooligosaccharide-forming alpha-amylase is potent for biotechnological application.