Showing papers on "Amylase published in 1998"
TL;DR: The levels of particle degradation and starch digestion before swallowing were compared for bread and spaghetti, and it was clear that starch hydrolysis depended on the initial structure of the food, as in the breakdown of solid food.
Abstract: Chemical and physical transformations of solid food begin in the mouth, but the oral phase of digestion has rarely been studied. In the present study, twelve healthy volunteers masticated mouthfuls of either bread or spaghetti for a physiologically-determined time, and the levels of particle degradation and starch digestion before swallowing were compared for each food. The amounts of saliva moistening bread and spaghetti before swallowing were, respectively, 220 (SEM 12) v. 39 (SEM 6) g/kg fresh matter. Particle size reduction also differed since bread particles were highly degraded, showing a loss of structure, whereas spaghetti retained its physical structure, with rough and incomplete reduction of particle size. Starch hydrolysis was twice as high for bread as for spaghetti, mainly because of the release of high-molecular-mass α-glucans. The production of oligosaccharides was similar after mastication of the two foods, respectively 125 (SEM 8) and 92 (SEM 7) g/kg total starch. Starch hydrolysis, which clearly began in the mouth, depended on the initial structure of the food, as in the breakdown of solid food. These significant physical and chemical degradations of solid foods during oral digestion may influence the entire digestive process.
192 citations
TL;DR: Activity staining revealed that two amylases with molecular masses of 150 and 42 kDa were produced when maltose, soluble starch or amylose was used as carbon source for growth, whereas only the 150 kDa protein was detected in the medium containing water‐ insoluble carbon sources.
Abstract: The optimum temperature and initial medium pH for amylase production by Bacillus sp. TS-23 were 55 degrees C and 8.5 respectively. Maximum amylase activity was obtained in a medium containing peptone and soluble starch as nitrogen and carbon sources. Activity staining revealed that two amylases with molecular masses of 150 and 42 kDa were produced when maltose, soluble starch or amylose was used as carbon source for growth, whereas only the 150 kDa protein was detected in the medium containing water-insoluble carbon sources. A raw-starch-degrading amylase was purified from culture supernatant of Bacillus sp. TS-23. The molecular mass of the purified amylase was estimated at 42 kDa by electrophoresis. The enzyme had a pI of 4. 2. The optimal pH and temperature for activity were 9.0 and 70 degrees C respectively. The thermoactivity of the purified enzyme was enhanced in the presence of 5 mM Ca2+; under this condition, enzyme activity could be measured at a temperature of 90 degrees C. The enzyme was strongly inhibited by Hg2+, Pb2+, Zn2+, Cu2+ and EDTA, but less affected by Ni2+ and Cd2+. The enzyme preferentially hydrolysed high-molecular-mass substrates with an alpha-1, 4-glucosidic bond except glycogen. The raw starches were partly degraded by the purified amylase to yield predominantly oligosaccharides with degrees of polymerization 3, 4 and 5.
192 citations
TL;DR: This study shows for the first time that the molecular mechanisms which control the dietary adaptation of trypsin and amylase are independently regulated, age-dependent and influenced by the composition and the quantity of the diet.
Abstract: Specific activities and mRNA levels of trypsin and amylase were studied in sea bass larvae. From day 20 to day 40, Dicentrarchus labrax were fed two rations of one day old Artemia: satiation (LP) and one-eighth of the satiation ration (LP/8) or two isoenergetic compound diets that varied in protein (30 and 60%) and carbohydrate (37 and 7%) content (FP30 and FP60 respectively). Trypsin mRNA levels and specific activities were mainly influenced by the nature of dietary protein and the Artemia ration. By using fish meal as protein source, dietary protein concentration did not affect either mRNA level nor specific activity of trypsin. These results suggested that the trypsin synthesis was not affected at a transcriptional level by the protein ration, i.e., Artemia ration. Decrease in amylase mRNA observed from day 29 in the four dietary groups suggested that this decrease in amylase expression is genetically programmed during sea bass larvae development. Nevertheless, the composition and the quantity of the diet influenced the amylase specific activities revealing primarily translational regulation of amylase. This study shows for the first time that the molecular mechanisms which control the dietary adaptation of trypsin and amylase are independently regulated, age-dependent and influenced by the composition and the quantity of the diet.
191 citations
TL;DR: The hypothesis that tea consumption can be effective in reducing the cariogenic potential of starch-containing foods such as crackers and cakes is supported.
Abstract: Tea decoctions prepared from a number of black and green teas inhibited amylase in human saliva. Black teas gave higher levels of inhibition than green teas, and removal of tea tannins with gelatin led to the loss of inhibitory activity from all decoctions. Streptococcal amylase was similarly inhibited by tea decoctions. Fluoride was without effect on amylase. Since salivary amylase hydrolyzes food starch to low molecular weight fermentable carbohydrates, experiments were carried out to determine whether tea decoctions would interfere with the release of maltose in food particles that became entrapped on the dentition. Subjects consumed salted crackers and rinsed subsequently for 30 s with black or green tea decoctions, or water. Maltose release was reduced by up to about 70% after rinsing with the teas. Black tea decoction was significantly more effective than green tea, in agreement with the in vitro data. The observations supported the hypothesis that tea consumption can be effective in reducing the cariogenic potential of starch-containing foods such as crackers and cakes. Tea may reduce the tendency for these foods to serve as slow-release sources of fermentable carbohydrate.
164 citations
TL;DR: The binding of RBI to TMA constitutes a new inhibition mechanism for α -amylases and should be general for all α-amylase inhibitors of the cereal inhibitor superfamily.
146 citations
TL;DR: Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles in cultivated barley and an additional allele in an accession of wild barley, suggesting that increased thermostability results in more efficient starch degradation.
134 citations
TL;DR: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant and throws light on the strict specificity of BASI for AMy2, and shows that domain B of AMY 2 contributes greatly to the specificity of enzyme-inhibitor recognition.
131 citations
TL;DR: The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and theEnvironmental stress responses of the plant.
Abstract: Expression of alpha-amylase genes during seedling development plays a key role in production of sugar from the starch stored in the cereal seed. Rice alpha-amylase Amy3D promoter/GUS constructs in transgenic rice cell lines were studied to identify cis elements in the promoter of this metabolite-regulated gene. Three sequences having the greatest effects on Amy3D gene expression included the amylase element (TATCCAT), the CGACG element, and a G box-related element (CTACGTGGCCA). These promoter cis elements are needed for high-level expression of Amy3D under conditions of sugar starvation. The involvement of G box cis-elements in environmental stress responses suggest a link between the nutrient stress and the environmental stress responses of the plant.
129 citations
TL;DR: A comprehensive picture of changes in carbohydrates, carbohydrate hydrolases, cell structure and texture in banana fruit during ripening is described in this paper, where insoluble carbohydrates were separated into seven fractions based on differential solubility.
127 citations
TL;DR: The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize as discussed by the authors, but there were no differences among the cooked pure starches.
Abstract: The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize. Four sorghum cultivars that represent different types of endosperm were used. Starch digestibilities of 4% cooked sorghum flour suspensions, measured as reducing sugars liberated following α-amylase digestion, were 15–25% lower than for cooked maize flour, but there were no differences among the cooked pure starches. After the flours were predigested with pepsin to remove some proteins, the starch digestibility of cooked sorghum flours increased 7–14%, while there was only 2% increase in normal maize; however, there was no effect of pepsin treatment on starch digestibility if the flours were first cooked and then digested. After cooking with reducing agent, 100 mM sodium metabisulfite, starch digestibility of sorghum flours increased significantly while no significant effect was observed for maize. Also, starch solubility of sorghum flours at 85 and 100°C was lower than in maize, and sodiu...
122 citations
TL;DR: It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol as increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein.
Abstract: Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U Sonnewald, 1992, Plant J 2: 571–581) The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L cv Desiree) tubers Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase (ii) The transformants contained higher pools of uridine nucleotides As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein Possible ways in which PPi hydrolysis could affect these processes are discussed
TL;DR: The gibberellin‐inducible rice α‐amylase gene, RA my1A, was demonstrated to be sugar repressed in rice embryos and functional dissection of the promoter of RAmy1A in relation of its sugar‐modulated expression was performed.
TL;DR: The new data presented here clearly show that unlike exoamylases, Novamyl® does not require a non-reducing end and attacks amylose, Indp5 and cyclodextrins in an endo-like manner.
Abstract: The thermostable -amylase Novamyl® is used in the baking industry as an antistaling agent due to its ability to reduce retrogradation of amylopectin We have studied its enzymatic properties at pH 50 We make two main conclusions: (1) Novamyl® shows sequence homology to cycloglycosyl transferases (CGTases); like these enzymes, Novamyl® cleaves cyclodextrins, forms transglycosylation products and is subject to product inhibition by maltose Novamyl® has 5 subsites in the active site and is also subject to substrate inhibition (2) Novamyl® is clearly different from exoglucanases like β-amylase and glucoamylase It is able to hydrolyse a pentasaccharide with bulky substituents at both ends (INdp5) and is inhibited by the α-amylase inhibitor Trestatin A Although Novamyl® appears unable to hydrolyse α-1,6-linkages, it is able to degrade amylopectin to a greater extent than β-amylase as well as β-limit dextrins Novamyl® degrades amylose in such a manner that initially the molecular weight is drastically reduced while β-amylase does not show any detectable effect on the molecular weight of this substrate Products of the degradation of amylopectin and amylose by Novamyl® are maltose and oligosaccharides, whereas β-amylase and glucoamylase produce only maltose and glucose, respectively This was shown in baking experiments as well The new data presented here clearly show that unlike exoamylases, Novamyl® does not require a non-reducing end and attacks amylose, Indp5 and cyclodextrins in an endo-like manner Based on these results Novamyl® should be reclassified
TL;DR: The members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.
Abstract: A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69,627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47-70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) 1-5 from an alkalophilic Bacillus 1-5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an alpha-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The beta-cyclodextrin-hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.
TL;DR: Pigeonpea (Cajanus cajan L) seeds were analysed quantitatively for amylase inhibitor (AI) activity and qualitatively, by an in-gel-detection method on polyacrylamide gels, to develop strategies to strengthen defense mechanisms in pigeonpea against H. armigera.
TL;DR: Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase.
Abstract: Corticium rolfsii AHU 9627, isolated from a tomato stem, is one of the strongest producers of a raw-starch-digesting amylase. The amylase system secreted by C. rolfsii AHU 9627 consisted of five forms of glucoamylase (G1–G5) and a small amount of α-amylase. Among these amylases, G1, G2 and G3 were able to hydrolyze raw starch. Five forms of glucoamylase were separated from each other and purified to an electrophoretically homogeneous state. The molecular masses were: G1 78 kDa, G2 78 kDa, G3 79 kDa, G4 70 kDa, and G5 69 kDa. The isoelectric points were: G1 3.85, G2 3.90, G3 3.85, G4 4.0, and G5 4.1. These glucoamylases showed nearly identical characteristics except that G4 and G5 were unable to hydrolyze raw starch.
TL;DR: These two experiments showed that abomasal supplementation of EPDE did not successfully supply cellulases and amylases to the intestine, due partially to their limited resistance to low pH and pepsin proteolysis.
Abstract: The effect of site of supplementation of a mixture of two crude preparations (Enzyme C and Enzyme X) of exogenous polysaccharide-degrading enzymes (EPDE) was studied in vivo using four ruminally and duodenally cannulated heifers (Exp. 1). The treatments were as follows: control (no EPDE), EPDE supplied through the diet (EF, 47.0 g/d), and EPDE infused continuously into the abomasum (EA, 41.6 g/d). Enzyme treatment increased the concentration of soluble reducing sugars (P < .05) and decreased NDF content (P < .05) in the treated feed, but this did not increase the rate or extent of in sacco disappearance of DM from the feed. Compared with control, ruminal fermentation was not affected by EF, but abomasal infusion increased (P < .05) rumen ammonia levels and shifted ruminal VFA patterns. Ruminal carboxymethylcellulase (CMCase) and xylanase activities were not affected by treatment. Abomasal infusion increased (P < .05) duodenal xylanase activity as compared with control and EF, but apparent digestion of DM, NDF, and CP were not affected by treatment. Negligible levels of CMCase and amylase reached the duodenum. During an in vitro experiment (Exp. 2), abomasal stability of the two EPDE was studied over a range of pH from 3.39 to .85, with or without pepsin. Carboxymethylcellulase activity (in Enzymes C and X) and beta-glucanase activity (in Enzyme C) were largely unstable against pepsin proteolysis (P < .001) and low pH (P < .001). Xylanase and amylase activities were resistant to pepsin but irreversibly inactivated at low pH. These two experiments showed that abomasal supplementation of EPDE did not successfully supply cellulases and amylases to the intestine, due partially to their limited resistance to low pH and pepsin proteolysis. Although EPDE significantly increased the level of xylanase activity at the duodenum, this did not significantly improve total tract digestion.
TL;DR: In this paper, a simple, non-aseptic, one-stage process was successfully carried out by using an air lift bioreactor under the optimised growth conditions at pH 5·0 and 35°C.
TL;DR: The results indicate that repression is also triggered efficiently by endogenous carbohydrates, and a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene is identified.
Abstract: We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization.
TL;DR: In this article, the location and distribution of modifying groups on amylopectin and amylose were determined by enzyme-catalyzed hydrolysis, using various combinations of isoamylase, β-amylases, α-amymylase and Amyloglucosidase.
TL;DR: A series of recommendations on measurements of catalytic concentrations of enzymes are presented in this paper, which form part of a series of recommended methods for enzyme measurements, including methods for aspartate aminotransferase, g-glutamyltransferase, and alanine aminothransferases.
Abstract: This paper forms part of a series of recommendations on measurements of catalytic concentrations of enzymes. Others deal with: Part 1. General considerations. Approved recommendation, 1979 (1). Part 2. Method for aspartate aminotransferase. Approved recommendation, 1985 (2). Part 3. Method for alanine aminotransferase. Approved recommendation, 1985 (3). Part 4. Method for g-glutamyltransferase (4). Part 5. Method for alkaline phosphatase (5). Part 6. Reference materials for enzyme measurements (Stage 1, draft 1989 – copy available from Committee Chairman). Part 7. Method for creatine kinase. Approved recommendation, 1991 (6). Part 8. Method for lactate dehydrogenase. Approved recommendation, 1993 (7).
TL;DR: It is concluded that the simultaneous determination of serum lipase and amylase marginally improved the diagnosis of acute pancreatitis in patients with acute abdominal pain, however, the sensitivity of the assay with samples collected 4-5 days after onset of the disease remained low.
Abstract: The clinical value of amylase and lipase measurement for the diagnosis of acute pancreatitis was evaluated in 253 patients presenting with acute abdominal pain. Acute pancreatitis was detected in 32 patients by computed tomography or ultrasound. In the serum samples collected on days 0-1 after the onset of symptoms, lipase was elevated in 100% and amylase in 95%. A 95% sensitivity/specificity was reached at a lipase cutoff near twofold above normal. The receiver-operating characteristics (ROC) showed similar curves for both enzymes, lipase being slightly superior to amylase. The ROC curves from days 2-3 demonstrated a much lower sensitivity/specificity of both enzymes. Lipase, however, was notably superior to amylase: at a sensitivity of 85% the specificity of lipase (amylase) was 82% (68%). In samples from days 4-5 the accuracy of the enzyme assays was even worse; at a sensitivity of 60% the specificity did not increase above 70%. The diagnostic value of simultaneous measurement of amylase and lipase was tested at different cutoffs in two groups: the OR group, in which one of the two parameters had to be elevated, and the AND group, in which both parameters had to be above normal. Combination of both parameters mainly improved the specificity of the assay (from 91 to 98% on days 2-3 and from 93 to 97% on day 4-5) but only when, in the OR group, twofold elevated amylase was combined with lipase. We conclude that the simultaneous determination of serum lipase and amylase marginally improved the diagnosis of acute pancreatitis in patients with acute abdominal pain, however, the sensitivity of the assay with samples collected 4-5 days after onset of the disease remained low.
TL;DR: GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions and IAA further inhibited both theGermination and growth of stressed seedlings.
Abstract: The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.
TL;DR: A novel amylase was isolated from the Mucor sp.
TL;DR: In this paper, two commercial preparations of polysaccharide-degrading enzymes (E1 and E2) were tested in vitro and in vivo for rumen stability, and the tested enzyme preparations were remarkably resistant to microbial fermentation in in vitro conditions.
TL;DR: In this paper, the effect of ball-mill treatment on physical properties and molecular change of maize starch granule was investigated, and the results showed that starch granules lost smoothness on surface and became rough, even though their changing speed was different among the three species.
Abstract: Effect of ball-mill treatment on physical properties and molecular change of maize starch granule was investigated. Ball-mill treatment was done by rotary type mill, and species of maize starch are normal, waxy and high amylose (amylo). Running time of the treatment is 0–320 h. Starch granules loss smoothness on surface and became rough, even though their changing speed was different among the three species. But, they retained whole figure and size after 320 h treatment in the all cases. Amylase susceptivity and water absorption activity were measured. Structural change of starch components was compared among the three species with X-ray diffraction, DSC and GPC. High Performance An-ion Exchange Chromatography (HPAEC) pattern of debranched sample treated with ball-mill for 320 h showed that formation of very short chain in amylopectin is little. 13C solid-NMR spectra suggest that disruption of molecules of amylopectin and amylose with ball-mill might occur at their glycosidic linkage. However, very slight radical was observed by Electron Spin Resonance spectroscopy (ESR) in the case of 320 h sample.
TL;DR: The inhibitory effect of maltose was found to be of the non-competitive type, while that of maltotriose was competitive, and it can be concluded that the first reaction product to be released upon maltopentaose hydrolysis is maltose, and that the second product is maltotRIose.
Abstract: A kinetic study was carried out on the inhibitory effects of acarbose, maltose, and maltotriose on porcine pancreatic alpha-amylase (PPA), using maltopentaose as the substrate. Lineweaver-Burk plots showed that the inhibitory action of acarbose is of the mixed non-competitive type. The secondary plots gave straight lines. A model involving abortive complexes accounts for these results. Dixon plot analysis led to the same conclusion. According to the proposed model, one molecule of acarbose per amylase molecule binds either directly to free enzyme at the active site or to the enzyme-substrate complex at a secondary carbohydrate-binding site, which becomes functional after the substrate has bound to the enzyme molecule at the active site. Kinetic analysis of the inhibition exerted by either the maltose or maltotriose reaction products of maltopentaose hydrolysis were then performed. The inhibitory effect of maltose was found to be of the non-competitive type, while that of maltotriose was competitive. It can therefore be concluded that the first reaction product to be released upon maltopentaose hydrolysis is maltose, and that the second product is maltotriose. This indicates that after hydrolysis of the maltopentaose chain, the reducing side fragment is released first.
TL;DR: In this paper, the α-amylase from malt barley and wheat flours was found to be slightly more sensitive to pressure than α-AMylase at higher pressure levels.
TL;DR: Analysis of the maltooligosaccharide composition of bread suggest that maltotriose and maltotetraose produced by the enzyme reaction are responsible for retarding bread retrogradation.
Abstract: For better understanding of the antistaling effect of starch-hydrolyzing enzymes, maltose-, maltotriose-, or maltotetraose-producing enzymes were applied to bread mix and the retrogradation rate of the bread was determined using differential scanning calorimetry. A new amylase isolated from Bacillus subtilis SUH 4-2, which selectively produces maltose and maltotriose from starch solution (amylase II), and another amylase from Streptomyces albus KSM-35, mainly producing maltotetraose and maltotriose (amylase IV), were cloned, characterized, and evaluated as antistaling agents for bread. Addition of amylase II or amylase IV significantly reduced the bread staling rate during 7 days of storage (p < 0.05), and especially amylase IV was as effective as a commercial enzyme, Novamyl. Analyses of the maltooligosaccharide composition of bread suggest that maltotriose and maltotetraose produced by the enzyme reaction are responsible for retarding bread retrogradation.
TL;DR: The α-amylase was rich in both basic and hydrophobic amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys- Ile-Asn-Phe, and exhibited Michaelis-Menten kinetics for starch, which enhanced thermostability at temperatures above 80°C and increased the half-life of activity to more than 8 h.
Abstract: An extracellular alpha-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5-6.0 and 95 degrees C, and the enzyme was stable in the pH range of 4.0-8.0. Calcium enhanced thermostability at temperatures above 80 degrees C, increasing the half-life of activity to more than 8 h at 85 degrees C, 80 min at 90 degrees C, and 19 min at 95 degrees C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The alpha-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and beta-mercaptoethanol activated the enzyme. The alpha-amylase exhibited Michaelis-Menten kinetics for starch, with a K(m) of 5.0 mg.ml-1 and kcat/K(m) of 5.2 x 10(5) ml.mg-1s-1. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an alpha-amylase from a strain of the genus Thermus.