Showing papers on "Amylase published in 1992"

Classification and measurement of nutritionally important starch fractions

Abstract: For nutritional purposes, starch in foods may be classified into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). RS may be further divided into three categories according to the reason for resistance to digestion. A method is reported for the measurement of total starch, RDS, SDS, RS and three RS fractions in starchy foods, using controlled enzymic hydrolysis with pancreatin and amyloglucosidase. The released glucose is measured by colorimetry, using a glucose oxidase kit. Values for RDS and SDS in foods obtained by the method reflect the rate of starch digestion in vivo. Values for RS are similar to the amounts of starch escaping digestion in the small intestine of ileostomates, and are a guide to the amounts of starch likely to enter the colon for fermentation. Results are given for a number of starchy foods.

Starch Hydrolysis by the Ruminal Microflora

Abstract: The effects of grain type and processing on ruminal starch digestion are well documented but poorly understood at the biochemical and molecular levels. Waxy grains have starches high in amylopectin and are more readily digested than nonwaxy grains. However, the composition of the endosperm cell matrix and the extent to which the starch granules are embedded within it also affect starch digestion rates. Continued work is needed to determine the influence of specific cell matrix proteins, protein-starch interactions and cell wall carbohydrates on starch availability. The microbial populations that metabolize starch are diverse, differing in their capacities to hydrolyze starch granules and soluble forms of starch. Surveys show that the amylases are under regulatory control in most of these organisms, but few studies have addressed the types of amylolytic enzymes produced, their regulation and the impact of other plant polymers on their synthesis. Research in these areas, coupled with the development and use of isogeneic or near-isogeneic grain cultivars with biochemically defined endosperm characteristics, will enhance our ability to identify mechanisms to manipulate ruminal starch digestion.

Process for producing ethanol

Abstract: The invention relates to a process for producing ethanol from raw materials, that contain fermentable sugars or constitu-ents which can be converted into sugars, comprising the steps of: (a) liquefaction of the raw materials in the presence of an alpha-amylase to obtain liquefied mash, (b) saccharificatiott of the liquefied mash in the presence of a glucoamylase to obtain hydro-lysed starch and sugars, (c) fermentation of the hydrolysed starch and sugars by yeast to obtain ethanol, and (d) recovering the obtained ethanol, a fungal protease being introduced to the liquefied mash during the saccharification and/or to the hydrolysed starch and sugars during the fermentation. The invention relates also to a composition containing a glucoamylase and an acid fungal protease.

Functional changes in salivary glands of autoimmune disease-prone NOD mice.

Abstract: Lymphocytic infiltration of the salivary glands in autoimmune diseases results in the human condition known as xerostomia. To date, an animal model for the autoimmune development of salivary gland dysfunction has yet to be described. With the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain, salivary flow rates and total saliva protein concentration in both male and female mice showed a progressive decline in the nondiabetic and diabetic states. Submandibular gland weight decreased from control mice with the progression to onset of diabetes in both sexes, whereas the weight of the parotid gland remained unchanged. The level of saliva amylase activity, when measured relative to unit volume, decreased in nondiabetic males but increased upon onset of diabetes to control values. When expressed relative to protein concentration in saliva, amylase activity was depressed for both sets of NOD mice but was higher upon diabetes onset than in the nondiabetic animals. In females a similar pattern was observed except that amylase activity expressed relative to unit volume was not significantly depressed in either set of NOD mice. The same observations were made for glandular amylase activity. The level of epidermal growth factor (a product of the ductal cells of the submandibular gland) was reduced over 500- and 18-fold for male and female diabetic mice, respectively. Sodium dodecyl sulfate polyacrylamide gels of total saliva showed changes in mobility as well as concentration of several proteins in the NOD mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Starch digestion and absorption in nonruminants.

Abstract: Starch digestion and absorption is augmented appreciably by physical processing of grain or legume and by heating to 100 degrees C for several minutes before its ingestion. Starch, a polysaccharide composed of alpha 1,4-linked glucose units (amylose) and alpha 1,4-1,6-linked branched structure (amylopectin), is cleaved in the duodenal cavity by secreted pancreatic alpha-amylase to a disaccharide (maltose), trisaccharide (maltotriose), and branched alpha-dextrins. These final oligosaccharides are hydrolyzed efficiently by complimentary action of three integral brush border enzymes at the intestinal surface: glucoamylase (maltase-glucoamylase, amyloglucosidase), sucrase (maltase-sucrase) and alpha-dextrinase (isomaltase). The final monosaccharide glucose product is then cotransported into the enterocyte along with Na+ by a specific brush border 75-kDa transport protein in the rate-limiting step for overall starch assimilation. By virtue of this sequential luminal and membrane digestion followed by glucose transport, starch is assimilated in a very efficient manner in nonruminants.

Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36.

Abstract: A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.

A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

Abstract: In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.

Formation of salivary-mucosal pellicle: the role of transglutaminase

Abstract: The present investigation was carried out to identify salivary components of mucosal pellicles in vivo and explore further the mechanism of interaction between salivary molecules and buccal epithelial cells. By using specific antisera and immunoprotein blotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, salivary cystatins and proline-rich proteins were detected within mucosal pellicle in vivo. In addition, the data indicated that the mucins and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. The role of buccal epithelial cell transglutaminase in these interactions was further studied by utilizing purified iodinated amylase, neutral cystatin SN and acidic proline-rich proteins 1 and 3 (APRP1 and 3). After incubation with buccal epithelial cells in vitro 125I-labelled APRPs appeared to undergo a greater degree of cross-linking than 125I-labelled cystatin SN, as determined by SDS/PAGE/autoradiography. Amylase did not appear to be cross-linked at all. Recovery of 125I-labelled APRPs and 125I-labelled cystatin SN with epithelial cell envelopes after repeated extraction suggested that both molecules were cross-linked to envelope proteins, but that 125I-labelled APRPs were cross-linked to a greater degree than 125I-labelled cystatin SN. Cross-linking in buccal epithelial cell preparations was inhibited by an excess of methylamine hydrochloride, a transglutaminase substrate. In a further assessment of amylase, cystatin and APRPs as transglutaminase substrates, only APRP3 and a partially purified preparation of APRPs acted as an amine acceptor for the cross-linking of [14C]methylamine by purified transglutaminase, as determined by SDS/PAGE/fluorography. This reaction was completely inhibited by excess EDTA. The combined data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to buccal epithelial cell surfaces, and that, within this group, selected components are enzymically cross-linked by an epithelial transglutaminase and/or proteolytically cleaved into smaller fragments.

Process for making amylase resistant starch from high amylose starch

Abstract: A process to increase the amount of resistant starch in a starch product to at least about 15% resistant starch using a high amylose starch, such as HYLON V or HYLON VII, as the starting starch, consists essentially of the steps of gelatinizing a starch slurry, enzymatically debranching the starch, and isolating the starch product by extrusion or drying. A further increase is obtained by the addition of an inorganic salt to the debranched starch before isolation.

Powdered automatic dishwashing composition containing enzymes

Abstract: A phosphate-free powdered dishwashing composition containing a mixture of a protease enzyme and an amylase enzymes have been found to be very useful in the cleaning of dishware. The compositions contain nonionic surfactants and a alkali metal silicate and bleaching agent.

Polymer compatibility and biodegradation of Starch-poly(ethylene-co-acrylic acid)-polyethylene blends

Abstract: X-ray diffraction, CP/MAS C-13 NMR, DSC, FTIR and fluorescence microscopy have been used to study the structure, compatibility, and morphology of films made from starch, poly(ethylene-co-acrylic acid) (EAA), and polyethylene (PE) before and after exposure to a mixture of highly amylolytic bacteria. The components of starch, amylose and amylopectin, interact with EAA via the formation of V-type inclusion complexes and hydrogen bonds. PE appears to be immiscible with the starch–EAA complex, with each forming sheetlike domains. The amylopectin in the films is susceptible to digestion by the bacterial consortium while the crystalline EAA–amylose complex is resistant. Digestion begins at the film surface and then proceeds inwards with sheetlike areas of starch removed. The good compatibility between starch and EAA as well as migration of EAA to the film surface explains the resistance of such films to digestion by conventional amylases.

Histopathologic correlates of serum amylase activity in acute experimental pancreatitis

Abstract: The association of serum amylase activity with the extent of pancreatic injury in acute pancreatitis is unclear. To clarify this relationship, we induced acute pancreatitis ranging from mild to lethal in 118 Sprague-Dawley rats (350–450 g). This was achieved by controlled intraductal infusion of low- or high-dose bile salt, with or without enterokinase, followed by intravenous cerulein or saline for 6 hr. Serum amylase was measured at baseline and 6 hr. Pancreatic histopathology was evaluated by two blinded pathologists employing total surface scoring (N=118) and morphometric 20-field documentation (N =22). Serum amylase correlated best with edema (r=0.61) and fat necrosis (r=0.58), less well with acinar necrosis (r=0.53) and inflammation (r=0.50), and poorly with hemorrhage (r=0.33) and perivascular infiltrate (r=0.31). Inasmuch as edema and fat necrosis are not important determinants of severity, these observations could explain the poor prognostic value of serum amylase activity in patients with acute pancreatitis.

In‐vivo and in‐vitro digestibility of starch in autoclaved pea and potato products

Abstract: The digestibility of starch in homogenized/autoclaved pea plus potato products was studied in vitro and in vivo. The products were a canned infant puree based on peas and potatoes and products prepared in the laboratory by repeated autoclaving and cooling of either homogenized potatoes or homogenized peas. Small-intestinal digestibility was evaluated through balance experiments in rats treated with an antibiotic (Nebacitin) to supress microbial activity in the hind gut. Parallel experiments in normal rats were performed to study the fermentability of undigested starch. The small-intestinal digestibility was 93, 82 and 70% of tolal starch in the potato product, infant puree and pea product, respectively. Consequently, significant amounts of starch left the small intestine undigested, particularly with pea-based products. The major portion of the undigested starch consisted of a fraction which resisted amylases in vitro unless solubilized in alkali, ie retrograded amylose. The fermentability of starch reaching the hind gut was high, about 90%. In-vitro digestibility figures varied depending on the method used and were in the ranges 91–93, 76–86 and 71–77% in the potato product, infant puree and pea product, respectively. One of the methods allowed simultaneous and accurate determination of the in-vivo resistant retrograded amylose fraction.

A comparative study of allergenic and potentially allergenic enzymes from Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei

Abstract: The presence of the enzymatically active allergens equivalent toDer p I (cysteine protease),Der p III (serine protease) and amylase in extracts ofDermatophagoides pteronyssinus, D. farinae andEuroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived fromD. pteronyssinus andD. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared fromD. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.

Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus.

Abstract: A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of Chloroflexus aurantiacus J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55 degrees C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71 degrees C, respectively. Metal ions such as Hg, Zn, Cu, Mn, and Ni strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.

Method of purification of amylase by precipitation with a metal halide and 4-hydroxybenzic acid or a derivative thereof

Abstract: The present invention relates to a method for the preparation of a purified amylase recovered from a fermentation broth, by the addition of a precipitation agent which is a halide salt, most preferably sodium or potassium chloride. According to a preferred embodiment, a further precipitation agent is added which is an organic compound, most preferably an alkyl ester of 4-hydroxybenzoic acid. The present invention also relates to the purified amylases so obtained and to the uses of these purified amylases.

Amylase synthesis in Aspergillus flavus and Aspergillus niger grown on cassava peel

Abstract: Aspergillus flavus andAspergillus niger produce extracellular amylase into the culture medium when grown on basal medium containing 2% (w/v) soluble starch or cassava peel as the sole carbon source. On soluble tarch the highest amylase activities were 1.6 and 5.2 mg of starch hydrolyzed/min per mg protein forA. flavus andA. niger, respectively. When grown on cassava peel, the highest amylase activity in the culture filtrate ofA. flavus was 170-times higher than that on soluble starch, while that ofA. niger was 16-times higher. The mycelial dry weight for both organisms was not significantly affected by the carbon sources. Maximum enzyme activity was obtained at the growth temperature of 29.0±1°C and pH 7 for both organisms. It is concluded that cassava peel might be a better substrate for the production of amylase byA. flavus andA. niger than commercial soluble starch.