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PubMed1990

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers.

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.

Base Sequence↗
PubMedDate not supplied

Cloning and expression of an amylase gene from Bacillus stearothermophilus.

In the industrial process of liquefying starch to make glucose or high fructose syrups, it is crucial that the amylase used is stable and active at about 105 degrees C at pH 6.5 or preferentially at a lower pH. The amylase from Bacillus licheniformis is well suited for this purpose but it is possible that other amylases might perform even better. Therefore, we cloned and characterized amyS encoding a heat-stable alpha-amylase from Bacillus stearothermophilus. Using a newly developed method for creating exact gene fusions by in vivo recombination, we attempted to increase expression of amyS in Bacillus subtilis. However, only by introducing the amyS gene into B. licheniformis, we obtained significantly better yields.

Bacillus↗
PubMed2006

Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography.

Formulation development is an integral step in the successful commercialization of protein-based products in both the biotechnology and pharmaceutical industries. As the number of these protein formulations increases, so does the need for innovative approaches to characterize physical and chemical product stability. In this study, the osmotic second virial coefficient (B) of a commercial amylase was evaluated by self-interaction chromatography (SIC) as an innovative approach to characterize physical protein stability. B was measured as a function of pH and several common formulation additives (cosolvents), including sodium chloride, sucrose, and sorbitol. Cosolvent- and pH-induced physical stabilization of amylase is discussed in terms of positive shifts in B. Liquid chromatographic measurements of total soluble amylase and enzymatic activity measurements correlated qualitatively with trends in B except near the pI of amylase, where physical stability was minimal.

Amylases↗
PubMed2005

Relation of Rab26 to the amylase release from rat parotid acinar cells.

Amylase secretion from rat parotid acinar cells is induced by the accumulation of cAMP in response to beta-adrenergic agonists as well as by the elevation of intracellular Ca2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised acinar cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca2+-induced release of amylase. Finally, in the early stage of beta-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon beta-adrenergic stimulation.

Adrenergic beta-Agonists↗
PubMed2005

A direct measurement of serum amylase levels produced by lung cancer.

The production of amylase by lung cancer has been previously diagnosed pathologically or immunohistochemically, or it has been confirmed by a decrease in serum levels after resection. It is possible to directly probe the continuous production of amylase by collecting samples from the inflow and outflow vessels of lung cancer. We herein describe an intraoperative measurement in which the amylase level in the pulmonary lobar vein was 3 times greater than that in the superior vena cava which was 6 times greater than the normal range. The venous level of amylase returned to a normal range after resection.

Amylases↗
PubMed2006

A highly thermostable and alkaline amylase from a Bacillus sp. PN5.

A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.

Amylases↗
PubMed2006

Comparison of the molten globule states of thermophilic and mesophilic alpha-amylases.

In recent years great interest has been generated in the process of protein folding, and the formation of intermediates during the folding process has been proven with new experimental strategies. In the present work, we have examined the molten globule state of Bacillus licheniformis alpha-amylase (BLA) by intrinsic fluorescence and circular dichroism spectra, 1-anilino naphthalene-8-sulfonate (ANS) binding and proteolytic digestion by pepsin, for comparison to its mesophilic counterpart, Bacillus amyloliquefaciens alpha-amylase (BAA). At pH 4.0, both enzymes acquire partially folded state which show characteristics of molten globule state. They unfold in such a way that their hydrophobic surfaces are exposed to a greater extent compared to the native forms. Chemical denaturation studies by guanidine hydrochloride and proteolytic digestion with pepsin show that molten globule state of BLA is more stable than from BAA. Results from gel filtration indicate that BAA has the same compactness at pH 4.0 and 7.5. However, molten globule state of BLA is less compact than its native state. The effects of polyols such as trehalose, sorbitol and glycerol on refolding of enzymes from molten globule to native state were also studied. These polyols are effective on refolding of mesophilic alpha-amylase but only slightly effect on BLA refolding. In addition, the folding pathway and stability of intermediate state of the thermophilic and the mesophilic alpha-amylases are discussed.

Bacillus↗
PubMed2006

Significant differences in the activities of alpha-amylases in the absence and presence of polyethylene glycol assayed on eight starches solubilized by two methods.

Starch is a reserve chemical source of the energy of the sun found in plants as a water-insoluble granule that differs in their chemical and physical properties, depending on the source. The granules can be solubilized by heating in water or by treatment with various reagents, such as 1M NaOH. alpha-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. We recently found that the activities of a single alpha-amylase on two different starches were significantly different. We then determined the activities of Bacillus amyloliquefaciens and porcine pancreas alpha-amylases, using eight different starches, solubilized by two methods: autoclaving at 121 degrees C and 1M NaOH at 20 degrees C. There were significant differences in the activities of both of the amylases on all eight of the starches. Previously, it had been found that polyethylene glycol (PEG) stabilized and activated the activities of both enzymes, using a soluble amylose as the substrate. Addition of PEG to the enzymes greatly increased the activities on the eight starches, but the activities still differed significantly. The different activities with the starches were hypothesized as differences in the amounts of secondary and tertiary structures that are partially retained when the different starches are solubilized; the activities on addition of PEG is hypothesized as the formation of highly active species from a series of less active forms.

Animals↗
PubMed2006

Characterization of a partial alpha-amylase clone from red porgy (Pagrus pagrus): expression during larval development.

A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.

Age Factors↗
PubMed2004

Pharmacological characterization of rebamipide: its cholecystokinin CCK1 receptor binding profile and effects on Ca2+ mobilization and amylase release in rat pancreatic acinar cells.

We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 microM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+ -mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.

Alanine↗
PubMed2004

Comparative studies on trifluoroethanol (TFE) state of a thermophilic alpha-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes.

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial alpha-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the alpha-amylases, a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the alpha-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease.

Anilino Naphthalenesulfonates↗
PubMed2004

Lack of nitric oxide-mediated regulation of amylase secretion stimulated by VIP in parotid glands of NOD mice.

The non-obese diabetic (NOD) mouse is chosen among other experimental models to study autoimmune sialadenitis resembling Sjögren's syndrome (SS), because of its unique characteristic of developing salivary dysfunction. Based on the deep loss of nitric oxide synthase (NOS) activity in parotid glands of NOD mice observed from early stages of disease and the inhibitory effect of nitric oxide (NO) donors on amylase secretion in normal salivary glands, our goal was to investigate whether parotid glands from NOD mice lacking NOS activity presented this regulatory mechanism of amylase secretion. We found that parotid glands from NOD mice lack nitric oxide-mediated regulation of amylase secretion in response to VIP stimulation. The lack of regulation might be assigned to the loss of NOS activity as derived from the results with NOS inhibitors and increasing concentrations of VIP. These functional differences observed in NOD vs. BALB/c parotid glands occur in the absence of immune infiltrates in exocrine tissue, and it is not related to cAMP accumulation. NO-mediated regulation of amylase secretion was not observed in BALB/c submandibular glands to the same extent as described in parotid glands and was absent in submandibular glands of NOD mice.

Amylases↗
PubMed2006

Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay.

BACKGROUND: Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with direct on-site demonstration of a bioallergen exposure hazard. OBJECTIVE: In a field study, we evaluated a recently developed LFIA for fungal alpha-amylase, an important bakery allergen. METHODS: Airborne and surface dust (wipe) samples and samples from flours and baking additives used at the workplace were collected in 5 industrial bakeries and tested in the LFIA for fungal amylase. For comparison, amylase was measured in sample eluates with the reference EIA method. RESULTS: Sensitivity of the LFIA was 1 to 10 ng/mL, and of EIA, approximately 25 pg/mL. In LFIA, most flour samples, 84% of wipe samples, 26% of personal airborne dust, and none of the 26 ambient air dust samples produced a visible reaction. Wipe samples from dough-making areas and flour samples gave the strongest reactions. All extracts with >5 ng allergen per milliliter showed a positive LFIA reaction. CONCLUSION: The LFIA for fungal amylase is an easy and rapid method to demonstrate the allergen directly at the worksite in less than 10 to 20 minutes. Similar LFIA methods may be used for other occupational allergens in other work environments. CLINICAL IMPLICATIONS: Lateral flow immunoassays for occupational allergens may be of great value in occupational hygiene surveys to demonstrate directly to workers and supervisors the hazards of work-related bioallergen exposure.

Air Microbiology↗
PubMed2006

Purification by expanded bed adsorption and characterization of an alpha-amylases FORILASE NTL from A. niger.

In this work the purification and biochemistry characterization of alpha-amylases from Aspergillus niger (FORILASE NTL) were studied. The effects of expansion degree of resin bed on enzyme purification by expanded bed adsorption (EBA) have also been studied. Residence time distributions (RTD) studies were done to achieve the optimal conditions of the amylases recovery on ion-exchange resin, and glucose solution was used as a new tracer. Results showed that height equivalent of the theoretical plates (HETP), axial dispersion and the Prandt number increased with bed height, bed voidage and linear velocity. The adsorption capacity of alpha-amylases, on the resin, increased with bed height and the best condition was at four-expansion degree. alpha-Amylase characterization showed that this enzyme has high affinity with soluble starch, good hydrolysis potential and molecular weight of 116 kDa.

Adsorption↗
PubMed2005

In vitro analysis of the digestive enzymes amylase and alpha-glucosidase in the midguts of Locusta migratoria L. in response to the myosuppressin, SchistoFLRFamide.

We have investigated the effect of the locust myosuppressin, SchistoFLRFamide, on the activity of amylase and alpha-glucosidase in the midgut of 2-week old male locusts. Total enzyme activity in the lumen contents and tissue extracts of midguts responds to SchistoFLRFamide in a dose-dependent manner that appears to vary with the feeding state of the locust and duration of exposure to the peptide. Starvation for 24h prior to assessment alters the distribution of enzyme activity between the midgut lumen contents and tissue extracts in response to SchistoFLRFamide when compared with fed locusts. Duration of exposure to SchistoFLRFamide also alters the distribution of total amylase and alpha-glucosidase activity; as duration of exposure increases, lower concentrations of SchistoFLRFamide increase total enzyme activity in the lumen contents while decreasing total enzyme activity in the tissue extracts. We suggest that the minimum amino acid sequence in SchistoFLRFamide necessary to increase both amylase and alpha-glucosidase activity is DHVFLRFamide. We have determined that two other peptides endogenous to the locust, AFIRFamide and GQERNFLRFamide, increase amylase and alpha-glucosidase activity in midgut lumen contents.

Amylases↗
PubMed2006

An alpha-amylase inhibitor gene from Phaseolus coccineus encodes a protein with potential for control of coffee berry borer (Hypothenemus hampei).

Plant alpha-amylase inhibitors are proteins found in several plants, and play a key role in natural defenses. In this study, a gene encoding an alpha-amylase inhibitor, named alphaAI-Pc1, was isolated from cotyledons of Phaseolus coccineus. This inhibitor has an enhanced primary structure to P. vulgaris alpha-amylase inhibitors (alpha-AI1 and alpha-AI2). The alphaAI-Pc1 gene, constructed with the PHA-L phytohemaglutinin promoter, was introduced into tobacco plants, with its expression in regenerated (T0) and progeny (T1) transformant plants monitored by PCR amplification, enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis, respectively. Seed protein extracts from selected transformants reacted positively with a polyclonal antibody raised against alphaAI-1, while no reaction was observed with untransformed tobacco plants. Immunological assays showed that the alphaAI-Pc1 gene product represented up to 0.05% of total soluble proteins in T0 plants seeds. Furthermore, recombinant alphaAI-Pc1 expressed in tobacco plants was able to inhibit 65% of digestive H. hampei alpha-amylases. The data herein suggest that the protein encoded by the alphaAI-Pc1 gene has potential to be introduced into coffee plants in order to increase their resistance to the coffee berry borer.

Amino Acid Sequence↗
PubMed2005

Isolation and characterization of an alpha-amylase gene in cassava (Manihot esculenta).

The roots of cassava plants (Manihot esculenta Crantz) accumulate starch as their major form of carbohydrate reserve. Starch accumulation and properties are determined by a balance between starch biosynthesis and degradation processes. Alpha-amylases (EC 3.2.1.1) are alpha-1,4 endoglycolytic enzymes, responsible for the mobilization of stored carbohydrate reserves by initiating the degradation process. Alpha-amylase genes have been shown to be differentially expressed at various developmental stages and environmental conditions through the action of plant hormones such as abscisic acid (ABA) and gibberellic acid (GA). In this study, we isolated an alpha-amylase gene from cassava tuberous roots (designated as MEamy2, GenBank accession number DQ011041). The deduced product of MEamy2 is 407 amino acid residues in length, with a calculated molecular mass of 46.7 kDa and an isoelectric point of 8.66. Southern blot analysis showed that the MEamy2 is present as a single copy in cassava genome. It shares the highest homology with AMY8 from apple fruit. The predicted structural model of MEamy2 contains three domains, active sites and starch-binding domain that are common with other plant alpha-amylases. RT-PCR analysis showed that the MEamy2 gene expression was induced in cassava roots within 2 hours after treatment with GA, but not ABA.

Amino Acid Sequence↗
PubMed2001

Phlebotomus papatasi and Leishmania major parasites express alpha-amylase and alpha-glucosidase.

Alpha-amylase and alpha-glucosidase activities were found in homogenates of young, unfed male and female Phlebotomus papatasi and in gut and salivary gland preparations. A significant increase in both enzyme activities in females and of alpha-amylase in males was recorded for flies that had fed overnight on a plant (Capparis spinosa). After plant feeding, alpha-amylase activity was relatively lower in female salivary glands and higher in guts, while in the males the activity in the salivary glands had increased. Alpha-glucosidase activity increased in guts of both sexes and in the salivary glands of the females. In addition, alpha-amylase activity was found in preparations of Leishmania major and L. infantum promastigotes, but not in those of L. donovani or L. tropica. Alpha-glucosidase activity was present in promastigote preparations of L. major, L. infantum, L. donovani, L. braziliensis, Crithidia fasciculata and Herpetomonas muscarum. It was lacking in similar preparations of L. tropica, Sauroleishmania agamae or Leptomonas seymouri. The growth rate of L. major promastigotes in medium supplemented with starch or with glucose was similar and it was significantly higher than in glucose poor medium. In this study, we demonstrate that P. papatasi and L. major possess the enzymes for hydrolyzing starch grains that are included in the plant tissue-diet of the sand flies.

Animals↗