Showing papers on "Amylase published in 1990"

Ontogenetic Change in Digestive Enzyme Activity of Larval and Postlarval White Shrimp Penaeus setiferus (Crustacea, Decapoda, Penaeidae).

Abstract: Whole specimens of developmental stages of Penaeus setiferus (Linnaeus, 1767) were homogenized and assayed for activities of digestive enzymes. In all developmental stages, activities were present for trypsin, carboxypeptidase A and B, amylase, and non-specific esterase; none for pepsin or lipase were detected. Activities assayed with substrates for chymotrypsin and aminopeptidase are not apparently due to the presence of these enzymes in the gut. Peak activities for all enzymes occurred during late zoeal or early mysis larval stages; low activities occurred at metamorphosis. During postlarval development, amylase activity increased steadily (by a tenfold increase over five weeks), whereas most other enzyme activities were relatively constant until the fifth week of postlarval development. Although it alters enzyme activity, diet does not appear to be the primary effector of ontogenetic change in digestive enzyme activity. Instead, ontogenetic change in digestive enzyme activity may reflect either a devel...

Production of bacterial thermostable alpha-amylase by solid-state fermentation: a potential tool for achieving economy in enzyme production and starch hydrolysis.

Abstract: Publisher Summary This chapter discusses the production of bacterial thermostable α amylase by solid-state fermentation. Bacterial thermostable α-amylase (1,4-α D glucan glucanohydrolase), one of the enzymes used in saccharification of starch, randomly attacks α-D- glucosidic linkages in starch thereby leading to the production of limit dextrins. α-amylase an industrially important enzyme produced in higher quantities and represents ∼12% of the sales value of the world market for enzymes. In addition to its use in starch-processing industries, the ability of α-amylase to cause midchain random degradation of starch has been of vital importance to several other industries. This chapter analyzes various possible modes for reducing the cost of starch hydrolysis. It also establishes that the liquefaction and partial saccharification of starch by bacterial thermostable α-amylase is one of the most cost intensive unit operations, chiefly because of the cost of the enzyme. In addition, it identifies the potential of the solid-state fermentation (SSF) technique for reducing the cost of producing bacterial thermostable α-amylase and reviews the literature on its production using the SSF process.

Degradation of Native Starch Granules by Barley α-Glucosidases

Abstract: The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

Abstract: The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Physiology and enzymology of thermophilic anaerobic bacteria degrading starch

Abstract: The capability of secreting thermoactive enzymes exhibiting α-amylase and pullulanase with debraching activity, seems to be widely distributed amongst anaerobic thermophilic bacteria. Interestingly, pullulanase formed by these bacteria displays dual specificity by attacking α-1,6- as well as α-1,4-glycosidic linkages in branched glucose polymers. Unlike the enzyme system of aerobic microorganisms the majority of starch hydrolysing enzymes of anaerobic bacteria is metal indepedent and is extremely thermostable. This enzyme system is controlled by substrate induction and catabolite repression; enzyme expression is accomplished when maltose or maltose-containing carbohydrates are used as substrates. By developing a process in continuous culture we were able to greatly enhance enzyme synthesis and release by anaerobic thermophilic bacteria. An elevation in the specific activities of cell-free amylases and pullulanases could also be achieved by entrapping of bacteria in calcium alginate beads. The unique properties of extracellular enzymes of thermophilic anaerobic bacteria makes this group of organisms suitable candidates for inductrial application.

Amylase‐binding as a discriminition among oral streptococci

Abstract: The ability of 51 strains, belonging to Streptococcus sanguis, ‘S. mitior’, S. oralis and related groups, to bind salivary amylase was studied. Most strains were grouped according to their DNA-related and then compared using 14 phenotypic tests. S. mitis, ‘S. mitior’ and three relatively new groups of strains (©R’, ‘MGH’ and ‘Tufted mitior’) bound salivary amylase, while strains of S. sanguis and S. oralis did not. The ability of strains to bind amylase or not was remarkably consistent within groups and the test proved to be reproducible, rapid and easy to perform. Combination of the amylase-binding test with 6 other conventioanal physiological tests allowed the construction of a dichotomous identification key which correclty identified 95% of strains for which genetic data was available. These findigs suggest that the ability of organisms to bind salivary amylase could become a key test in identification schemes for certain oral streptococci.

Purification and Characterization of Pea Epicotyl β-Amylase

Abstract: The most abundant β-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this β-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a β-amylase by end product analysis and by its inability to hydrolyze β-limit dextrin and to release dye from starch azure. Pea β-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a Km of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea β-amylase is competitively inhibited by its end product, maltose, with a Ki of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with α-cyclohexaamylose being a stronger inhibitor than β-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea β-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea β-amylase is the multichain type. Possible roles of pea β-amylase in cellular glucan metabolism are discussed.

Characterization of α-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

Abstract: The most abundant α-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This α-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an α-amylase by polarimetry, substrate specificity, and end product analyses. The purified α-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This α-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. β-glucan) are not substrates of the enzyme. A very low amount of this α-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or α-amylase which is integrally associated with the chloroplast.

Characterization of the α-amylase Receptor of Streptococcus gordonii NCTC 7868

Abstract: The purpose of the work described here was to investigate the mechanisms involved in the binding of salivary α-amylase to Streptococcus gordonii NCTC 7868 (Challis). Of six types of a-amylase studied, only mammalian forms of the enzyme were found to bind to S. gordonii cells. Salivary a-amylase binding was inhibited by treatment of cells with trypsin and pronase, but not with pepsin or sodium periodate. Presence of starch, dextrin, or maltoheptaose partially inhibited binding of the enzyme to S. gordonii. Both mutanolysin extracts of cells and culture supernatants contained α-amylase-binding activity, which was partially purified by Sepharose CL-6B and DEAE-ionexchange chromatography. Western blotting detected four putative receptor bands-65 kDa, 15 kDa, 12.5 kDa, and one with a very high molecular weight; the lower-molecular-weight components may be products of proteolytic degradation of the high-molecular-weight material, but their true relationship has yet to be determined. Pre-treatment of salivary α-...

Some Nutritional Properties of Starch and Dietary Fiber in Barley Genotypes Containing Different Levels of Amylose

Abstract: The nutritional properties of starch and dietary fiber (DF) were studied in barley genotypes containing different amylose contents: Waxy Campana (-8% amylose); Alva, Lina, and Glacier normal (normal varieties, 25-27% amylose); and Glacier high (-35% amylose). On an equivalent starch basis, all barley varieties showed a somewhat higher availability to aamylase than a wheat reference. Among the barley flours, starch in the waxy variety was most available to a-amylase when tested raw. With excess water (90% H2 0), the gelatinization was completed at about 80 C, as measured with differential scanning calorimetry, irrespective of amylose content. At lower moisture (50% H2 0), the temperature interval for gelatinization was considerably broadened. However, the differences in gelatinization behavior between the barley varieties were small. No

Purification of a β-Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism

Abstract: Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic β-(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the β-amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and Km for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of β-amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified β-amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated β-amylase protein.

Growth and extracellular enzyme production by strains of Bacillus species isolated from fermenting African locust bean, iru

Abstract: Adeiubigbe, E.Y. & Odunfa, S.A. 1990. Growth and extracellular enzyme production by strains of Bacillus species isolated from fermenting African locust bean, iru. Journal of Applied Bacteriology69, 662–671. Seven strains of Bacillus subtilis group, isolated from fermented African locust bean (iru), were screened for proteolytic activity. Three strains (BS2, BL2 and BP2), which were found to be highly proteolytic, were compared on the basis of growth and extracellular enzyme production in media with and without locust bean. AH had their optimum pH for growth between 7.0 and 9.0. The three strains produced varied amounts of amylase, polygalacturonase, galac-lanase and sucrase. The amounts of amylase and polygalacturonases produced by strain BS2 were significantly higher (at α= 0.5) than those of strains BL2 and BP2. None of the strains produced pectinmethylesterase in nutrient broth with or without African locust bean. Although the three strains were lipolytic on tributyrin agar plates, only trace amounts of lipase were detected titrimetrically in broth medium containing African locust bean. The three strains produced varying levels of sucrase and galactanases. Phytase activity was not detected in the broth culture of strain BS2. The presence of African locust bean in culture medium generally enhanced the production of extracellular enzymes significantly (at α= 0.05) in the three strains.

Structure and biosynthesis of the xylose‐containing carbohydrate moiety of rice α‐amylase

Abstract: Suspension-cultured cells of rice secrete alpha-amylase into the culture medium. It has been shown that the mature form of the alpha-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete alpha-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, alpha-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.

Regulation of amylase gene expression in diabetic mice is mediated by a cis-acting upstream element close to the pancreas-specific enhancer.

Abstract: We localized the cis-acting sequences that mediate the regulation of a pancreatic amylase gene, Amy-2.2, in diabetic mice. We constructed three hybrid genes containing sequences from the 5'-flanking region of the amylase gene upstream of the heterologous elastase promoter linked to the CAT structural gene. These constructs were transferred to the germ line of transgenic mice by microinjection of fertilized eggs. The amylase sequences had two effects on expression of the elastase promoter: Basal expression was increased, and expression in diabetic animals was reduced to approximately 2% of basal levels. A 30-bp amylase fragment was sufficient to transfer both of these regulatory functions to the elastase promoter. Sequences within this 30-bp fragment are included in the binding site for the pancreatic nuclear protein PTF1. The close association of the PTF1-binding site and the regulatory functions is consistent with a mechanism based on interference with activation by PTF1 in diabetic pancreas. PTF1-binding activity is not reduced in diabetic pancreas. The data presented here demonstrate that the 5'-flanking region of the pancreatic amylase gene contains a novel insulin-dependent element (IDE) that mediates the loss of expression in diabetic animals.

Characterization of an endo-Acting Amylopullulanase from Thermoanaerobacter Strain B6A.

Abstract: A thermoanaerobe (Thermoanaerobacter sp.) grown in TYE-starch (0.5%) medium at 60°C produced both extra- and intracellular pullulanase (1.90 U/ml) and amylase (1.19 U/ml) activities. Both activities were produced at high levels on a variety of carbon sources. The temperature and pH optima for both pullulanase and amylase activities were 75°C and pH 5.0, respectively. Both the enzyme activities were stable up to 70°C (without substrate) and at pH 4.5 to 5.0. The half-lives of both enzyme activities were 5 h at 70°C and 45 min at 75°C. The enzyme activities did not show any metal ion activity, and both activities were inhibited by β- and γ-cyclodextrins but not by α-cyclodextrin. A single amylolytic pullulanase responsible for both activities was purified to homogeneity by DEAE-Sepharose CL-6B column chromatography, gel filtration using high-pressure liquid chromatography, and pullulan-Sepharose affinity chromatography. It was a 450,000-molecular-weight glycoprotein composed of two equivalent subunits. The pullulanase cleaved pullulan in α1,6 linkages and produced multiple saccharides from cleavage of α-1,4 linkages in starch. The Kms for pullulan and soluble starch were 0.43 and 0.37 mg/ml, respectively.

Immunohistochemical localization of the glucocorticoid receptor in pancreatic β-cells of the rat

Abstract: We used a monoclonal antibody against an epitope located in the N-terminal moiety of the rat glucocorticoid receptor to identify the glucocorticoid receptor-containing cells in the rat pancreas. Monospecific polyclonal antisera against insulin, glucagon, somatostatin, and amylase were applied to serial sections in colocalization studies to identify the respective endocrine and exocrine cells. Glucocorticoid receptor immunoreactivity was exclusively present in nuclei and cytoplasm of the beta-cells of pancreatic islets. Western blots using the glucocorticoid receptor antibody resulted in identical 94K immunoreactive proteins in both liver and pancreas. After adrenalectomy, the glucocorticoid receptor immunoreactivity of beta-cells decreased significantly. A computer-assisted method of semiquantitative evaluation of the glucocorticoid receptor immunoreactivity demonstrated a significant decrease in the staining intensity of the beta-cells by 23.5% and in that of insulin antibodies by 10.4%, while amylase immunoreactivity was only slightly decreased. Serum levels of corticosterone determined by RIA decreased from 225 micrograms/ml in sham-operated animals to 55 micrograms/ml in animals 14 days after adrenalectomy, while the tissue content of amylase decreased by 45%. The immunohistochemical findings give circumstantial evidence of the presence of glucocorticoid receptor in beta-cells. We interpret our data as indicating an indirect effect of glucocorticoids on amylase synthesis via a glucocorticoid-insulin-exocrine cell pathway.