Flow-injection-type biosensor system for salivary amylase activity

The authors aim to establish a method that can quantitatively evaluate vital reactions to stress. We have been examining the correlation between stress and salivary amylase activity in order to verify its validity as a stress index. In order to quantify human stress, which changes over time, the relationship between stress and salivary amylase activity must be verified by fast and repeated analysis of salivary amylase activity. Standard biosensors are designed such that the enzyme immobilized on an electrode (enzyme electrode) and the substrate-dependent activity is measured. The reverse approach of measuring the alpha-amylase-dependent activity was adopted. We fabricated an amylase activity analytical system. Maltopentaose was selected as a substrate for alpha-amylase and a flow-injection-type device was used to supply maltopentaose continuously. alpha-Glucosidase, having relatively low enzyme activity, was immobilized on a pre-activated membrane so that it could be enclosed in a pre-column, Glucose oxidase, having higher enzyme activity, was immobilized on a working electrode so that it could function as an amperometric biosensor. A saliva-collecting device was fabricated to make saliva pretreatment unnecessary. As a result, an amylase activity analytical system was fabricated that enabled us to measure salivary amylase activity from 0 to 30 kU/l, with an R(2) value of 0.97. The time-course changes in the salivary amylase activities for 1 week were 5.1%, and the initial sensitivity remained nearly constant. Through this study, we were able to verify the possible development of the amylase activity analytical system.     

The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes

Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively.     

Structural relationship between the enzymatic and streptococcal binding sites of human salivary alpha-amylase

Previous studies have demonstrated that human salivary alpha-amylase specifically binds to the oral bacterium Streptococcus gordonii. This interaction is inhibited by substrates such as starch and maltotriose suggesting that bacterial binding may involve the enzymatic site of amylase. Experiments were performed to determine if amylase bound to the bacterial surface possessed enzymatic activity. It was found that over one-half of the bound amylase was enzymatically active. In addition, bacterial-bound amylase hydrolyzed starch to glucose which was then metabolized to lactic acid by the bacteria. In further studies, the role of amylase's histidine residues in streptococcal binding and enzymatic function was assessed after their selective modification with diethyl pyrocarbonate. DEP-modified amylase showed a marked reduction in both enzymatic and streptococcal binding activities. These effects were diminished when DEP modification occurred in the presence of maltotriose. DEP-modified amylase had a significantly altered secondary structure when compared with native enzyme or amylase modified in the presence of maltotriose. Collectively, these results suggest that human salivary alpha-amylase may possess multiple sites for bacterial binding and enzymatic activity which share structural similarities.     

Targeted disruption of the alpha-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.     

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.     

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.     

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.     

The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636

AIMS: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA.METHODS AND RESULTS: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases.CONCLUSIONS: AmyB has been isolated, characterized and then compared to AmyA.SIGNIFICANCE AND IMPACT OF THE STUDY: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.     

Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.     

Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

Identification of Kernel Proteins Associated with the Resistance to Fusarium Head Blight in Winter Wheat (Triticum aestivum L.)

Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.     

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.     

Spatio-temporal profiling and degradation of alpha-amylase isozymes during barley seed germination

Ten genes from two multigene families encode barley alpha-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the alpha-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 alpha-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding alpha-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both alpha-amylase 2 entries co-migrated in five full-length and one fragment spot. The alpha-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that alpha-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (betaalpha)(8)-barrel between beta-strand 3 and alpha-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other alpha-amylase 2 (gi|166985). The 2D gels of dissected tissues showed alpha-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length alpha-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.     

Alpha-amylases of the coffee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors

The adult coffee berry borer (Hypothenemus hampei Ferrari [Coleoptera: Scolytidae]), a major insect pest of coffee, has two major digestive alpha-amylases that can be separated by isoelectric focusing. The alpha-amylase activity has a broad pH optimum between 4.0 and 7.0. Using pH indicators, the pH of the midgut was determined to be between 4.5 and 5.2. At pH 5.0, the coffee berry borer alpha-amylase activity is inhibited substantially (80%) by relatively low levels of the amylase inhibitor (alphaAI-1) from the common bean, Phaseolus vulgaris L., and much less so by the amylase inhibitor from Amaranthus. We used an in-gel zymogram assay to demonstrate that seed extracts can be screened to find suitable inhibitors of amylases. The prospect of using the genes that encode these inhibitors to make coffee resistant to the coffee berry borer via genetic engineering is discussed.     

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.     

Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.     

Determination of the primary structure of Paim II, an alpha-amylase inhibitor from Streptomyces coruchorushii, by high-performance tandem mass spectrometry

This study indicates one of the advantages of tandem mass spectrometry; the primary structures of proteins with little structural difference can be determined by using tandem mass spectrometry without prior purification of each component. The primary structure of Paim II, a protein alpha-amylase inhibitor from Streptomyces coruchorushii, was determined by using tandem mass spectrometry. Paim II consists of two component proteins with ragged N-terminus, and was sequenced on the basis of the structure of Paim I, an analogous alpha-amylase inhibitor from the same natural origin.     

Hand-held monitor of sympathetic nervous system using salivary amylase activity and its validation by driver fatigue assessment

In order to realize a hand-held monitor of the sympathetic nervous system, we fabricated a completely automated analytical system for salivary amylase activity using a dry-chemistry system. This was made possible by the fabrication of a disposable test-strip equipped with built-in collecting and reagent papers and an automatic saliva transfer device. In order to cancel out the effects of variations in environmental temperature and pH of saliva, temperature- and pH-adjusted equations were experimentally determined, and each theoretical value was input into the memory of the hand-held monitor. Within a range of salivary amylase activity between 10 and 140 kU/l, the calibration curve for the hand-held monitor showed a coefficient with R(2)=0.97. Accordingly, it was demonstrated that the hand-held monitor enabled a user to automatically measure the salivary amylase activity with high accuracy with only 30 microl sample of saliva within a minute from collection to completion of the measurement. In order to make individual variations of salivary amylase activity negligible during driver fatigue assessment, a normalized equation was proposed. The normalized salivary amylase activity correlated with the mental and physical fatigue states. Thus, this study demonstrated that an excellent hand-held monitor with an algorithm for normalization of individuals' differences in salivary amylase activity, which could be easily and quickly used for evaluating the activity of the sympathetic nervous system at any time. Furthermore, it is suggested that the salivary amylase activity might be used as a better index for psychological research.     

Ontogenesis of alpha-amylase in rat parotid gland during postnatal development

Changes in alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) of parotid gland were investigated during postnatal development of the rat. Modifications in amylase activity after birth allow the distinction of three stages which can be correlated with the morphologic development of the parotid gland. Significant sexual differences in the evolution of alpha-amylase activity were found. During the first stage (from birth to the 20th day) there is a higher increase in females, while males have a more pronounced increment in the second stage (from the 20th to the 30th day). By means of gel electrophoresis of parotid extracts, four molecular forms of amylase can be separated. The slowest migrating band (Form 1) is not detected at the initial stage.