Showing papers on "Amylase published in 1996"

Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata).

Abstract: The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high α-amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.

Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in the oral cavity.

Abstract: Salivary α-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation. The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme. This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules. Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C. Domain A has a (β/α)8− barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure. The Ca2+ ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules. The Cl− ion is bound to Arg195, Asn298 and Arg337 and one water molecule. The highly mobile glycine-rich loop 304–310 may act as a gateway for substrate binding and be involved in a `trap-release' mechanism in the hydrolysis of substrates. Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins.

l-Arabinose selectively inhibits intestinal sucrase in an uncompetitive manner and suppresses glycemic response after sucrose ingestion in animals

Abstract: The objective of this study was to investigate the effects of L-arabinose on intestinal alpha-glucosidase activities in vitro and to evaluate its effects on postprandial glycemic responses in vivo. L-Arabinose inhibited the sucrase activity of intestinal mucosa in an uncompetitive manner (Ki, 2 mmol/L). Neither the optical isomer D-arabinose nor the disaccharide L-arabinobiose inhibited sucrase activity, whereas D-xylose was as potent as L-arabinose in inhibiting this activity. L-Arabinose and D-xylose showed no inhibitory effect on the activities of intestinal maltase, isomaltase, trehalase, lactase, and glucoamylase, or pancreatic amylase. In contrast, a known alpha-glucosidase inhibitor, acarbose, competitively inhibited (Ki, 1.1 mumol/L) sucrase activity and also inhibited intestinal maltase, glucoamylase, and pancreatic amylase. L-Arabinose suppressed the increase of blood glucose after sucrose loading dose-dependently in mice (ED50, 35 mg/kg), but showed no effect after starch loading. The suppressive effect of D-xylose on the increase of blood glucose after sucrose loading was 2.4 times less than that of L-arabinose, probably due to intestinal absorption of the former. Acarbose strongly suppressed glycemic responses in both sucrose loading (ED50, 1.1 mg/kg) and starch loading (ED50, 1.7 mg/kg) in mice. L-Arabinose suppressed the increase of plasma glucose and insulin in rats after sucrose loading, the suppression of the former being uninterruptedly observed in mice for 3 weeks. Thus, the results demonstrated that L-arabinose selectively inhibits intestinal sucrase activity in an uncompetitive manner and suppresses the glycemic response after sucrose ingestion by inhibition of sucrase activity.

Banana waste as substrate for α-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation

Abstract: Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of α-amylase when banana fruit stalk was used as substrate in a solid-state fermentation system. The effects of initial moisture content, particle size, cooking time and temperature, pH, incubation temperature, additional nutrients, inoculum size and incubation period on the production of α-amylase were characterised. A maximum yield of 5 345 000 U mg-1 min-1 was recorded when pretreated banana fruit stalk (autoclaved at 121 °C for 60 min) was used as substrate with 70% initial moisture content, 400 μm particle size, an initial pH of 7.0, a temperature of 35 °C, and additional nutrients (ammonium sulphate/sodium nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with banana fruit stalk medium compared to wheat bran.

Levels of antinutritional factors in pearl millet as affected by processing treatments and various types of fermentation

Abstract: Pearl millet (Pennisetum typhoideum) was fermented with Lactobacilli or yeasts alone and in combination, and with natural microflora after various processing treatments, as grinding, soaking, debranning, dry heat treatment, autoclaving and germination. Fermentation was carried out at 30°C for 48 hours withLactobacillus plantarum (LP) andRhodotorula (R) isolated from naturally fermented pearl millet andLactobacillus acidophilus (LA),Candida utilis (CU) and natural microflora (NF). Germination and autoclaving, and debranning and autoclaving were the most effective processing treatments to reduce the phytic acid, amylase inhibitors and polyphenols. There was a further reduction in these antinutrients due to fermentation. Phytic acid and amylase inhibitors were completely eliminated after fermentation in some of the samples especially in soaked, debranned and germinated ones. Polyphenols were altered non-significantly in general but fermentation with Lp+R and NF caused a significant increased in polyphenols.

Process for producing amylase resistant granular starch

Abstract: A resistant granular starch with high dietary fiber content and the method of preparing this product wherein a high amylose starch having at least 40% by weight amylose content and a water content of 10 to 80% by weight is heated to a temperature of from about 60 to 160°C to provide a granular starch which retains its granular structure and has a total dietary fiber content of at least 12%. Food products containing this resistant granular starch are also provided.

Cyclization reaction catalyzed by branching enzyme.

Abstract: The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.

Serum amylase and lipase in the evaluation of acute abdominal pain.

Abstract: The purpose of this study was to determine 1) the incidence and magnitude of elevation in admission serum amylase and lipase levels in extrapancreatic etiologies of acute abdominal pain, and 2) the test most closely associated with the diagnosis of acute pancreatitis. Serum amylase and lipase levels were obtained in 306 patients admitted for evaluation of acute abdominal pain. Patients were categorized by anatomic location of identified pathology. Logistic regression analysis was used to compare the enzyme levels between patient groups and to determine the correlation between elevation in serum amylase and lipase. Twenty-seven (13%) of 208 patients with an extrapancreatic etiology of acute abdominal pain demonstrated an elevated admission serum amylase level with a maximum value of 385 units (U)/L (normal range 30-110 U/L). Twenty-six (12.5%) of these 208 patients had an elevated admission serum lipase value with a maximum of 3685 U/L (normal range 5-208 U/L). Of 48 patients with abdominal pain resulting from acute pancreatitis, admission serum amylase ranged from 30 to 7680 U/L and lipase ranged from 5 to 90,654 U/L. Both serum amylase and lipase elevations were positively associated with a correct diagnosis of acute pancreatitis (P < 0.001) with diagnostic efficiencies of 91 and 94 per cent, respectively. A close correlation between elevation of admission serum amylase and lipase was observed (r = 0.87) in both extrapancreatic and pancreatic disease processes. Serum amylase and lipase levels may be elevated in nonpancreatic disease processes of the abdomen. Significant elevations (greater than three times upper limit of normal) in either enzyme are uncommon in these disorders. The strong correlation between elevations in the two serum enzymes in both pancreatic and extrapancreatic etiologies of abdominal pain makes them redundant measures. Serum lipase is a better test than serum amylase either to exclude or to support a diagnosis of acute pancreatitis.

Amylases of the thermophilic fungus Thermomyces lanuginosus : Their purification, properties, action on starch and response to heat

Abstract: A thermophilic fungus Thermomyces lanuginous strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ∼ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5.6 and at 65°C. It had an activation energy of 44 kJ mol-1. The apparent Km for soluble starch was 2.5 mg ml-1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species. Glucoamylase was a monomeric glycoprotein of ∼ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent Km for soluble starch was 0.04 mg ml-1 and Vmax was 660 Mmol glucose min-1 mg protein-1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol-1 Glucoamylase did not show synergistic effect with α-amylase. The properties of α-amylase and glucoamylase of Thermomyces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.

Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae.

Abstract: Sea bass (Dicentrarchus labrax) larvae were fed from day 15 to day 35 with 4 isoenergetic formulated diets that varied in protein (30, 40, 50, 60%) and carbohydrate (37, 27, 17, 7%) content. The diets were designated as P30, P40, P50 and P60, respectively. Best growth and survival were noted for P50 larvae. Poor growth and survival were observed for P30 larvae. The specific activity of amylase increased in direct relation to the dietary carbohydrate level from day 18 onwards. This increase was the result of extensive amylase synthesis. Trypsin activity was positively correlated with dietary protein level only at day 35. This study shows that the mechanisms involved in amylase regulation are efficient in very young sea bass larvae, while those related to trypsin appear later in ontogeny and thus suggests an age-dependent regulation of enzyme synthesis.

Urine trypsinogen-2 as marker of acute pancreatitis.

Abstract: We examined the clinical utility of urine trypsinogen-2 as a marker of acute pancreatitis (AP). Fifty-nine patients with AP, 42 with acute abdominal diseases of extrapancreatic origin, and 63 without evidence of acute abdominal disease were studied. Urine trypsinogen-2 was determined by a time-resolved immunofluorometric assay. As reference methods we used serum trypsinogen-2, urine amylase, and serum amylase. The diagnostic accuracy of the markers was evaluated by receiver-operating characteristic (ROC) analysis. At admission, urine trypsinogen-2 differentiated patients with AP from controls with high accuracy. The area under the ROC curve (AUC) was 0.978, which was equal to that of serum trypsinogen-2 (0.998) and serum amylase (0.969) and significantly larger than that of urine amylase. For differentiation between severe and mild AP, urine trypsinogen-2 (0.730) was equal to serum trypsinogen-2 (0.721), and clearly better than amylase in serum and urine. These results suggest that determination of urine trypsinogen-2 is a useful test to detect AP and to evaluate disease severity.

Accumulation of Fermentable Sugars and Metabolic Acids in Food Particles that Become Entrapped on the Dentition

Abstract: Earlier studies (Kashket et al., 1991) showed that particles of high-starch snack foods remained longer on the teeth than those of high-sucrose, low-starch foods. The question arose whether the prolonged presence of food particles enhances cariogenicity. A study was undertaken to measure sugars, starches, and metabolic acids in retained food particles. Subjects consumed portions of different foods, and particles were removed from all bicuspids and first molars at defined times after swallowing. Dry weights, sugars, and short-chain carboxylic acids were determined. High-sucrose foods were cleared rapidly from the teeth, while high-starch foods were retained for up to 20 min. Sucrose, glucose, and fructose persisted in the retained particles. Particles of high-starch foods accumulated maltose and maltotriose, presumably from the breakdown of starch by salivary amylase. At maximum, maltose plus maltotriose constituted 94% of total sugars in particles of potato chips; corresponding values in doughnuts, peanut butter cookies, and salted crackers were 43, 51, and 61%, respectively. Total fermentable sugars in the particles of high-starch foods were similar to those for the high-sucrose confectionery products. Carboxylic acids accumulated within the particles, presumably due to the fermentation of the sugars by entrapped salivary micro-organisms. At maximum (5 to 7 min), acetic, formic, lactic, and propionic acids rose 17-, 30-, 15-, and 1.3-fold, respectively, in doughnuts, and to smaller degrees in potato chips, salted crackers, and chocolate-caramel-peanut bars. In summary, the study demonstrated the persistence of sugars, the progressive accumulation of starch breakdown products, and the fermentation of the accumulated sugars in retained food particles. The findings support the view that high-starch foods contribute to the development of caries lesions.

Amylase gene structures in primates: retroposon insertions and promoter evolution.

Abstract: Amylase transcription in the human salivary gland results from the evolutionary juxtaposition of two inserted elements, a gamma-actin pseudogene and an endogenous retrovirus, to create an unusual salivary-specific promoter. We utilized these structures as molecular tags to characterize the amylase genes in extant primates by polymerase chain reaction amplification of promoter fragments from genomic DNA. Six distinct amylase promoter structures were identified, which allowed us to infer the structures of common ancestors and trace the evolution of the modern human amylase promoters. Our data show that integration of the pseudogene and retrovirus were evolutionarily recent events. The gamma-actin pseudogene integrated after the divergence of the New World monkeys from the primate ancestral tree, and the retrovirus integrated later, after the divergence of the Old World monkeys. The New World monkey amylase promoter represents the mammalian amylase precursor structure before integration of the two retroposons. Two distinct amylase genes were identified in the Old World monkeys, one with a complete gamma-actin pseudogene insert and another novel structure with a truncation of the gamma-actin sequences. We demonstrated abundant amylase expression in the saliva of an Old World monkey, indicating that the endogenous retrovirus is not required for amylase transcription in the primate salivary gland.

The mechanism of porcine pancreatic alpha-amylase. Kinetic evidence for two additional carbohydrate-binding sites.

Abstract: Kinetics of inhibition of the two porcine pancreatic alpha-amylase components (PPA I and PPA II) by acarbose were performed using reduced DP18-maltodextrin and amylose as substrates. Similar Line-weaver-Burk primary plots were obtained. Two mixed non-competitive models are proposed. X-ray crystallographic data [Qian, M., Buisson, G., Duee. E., Haser, R. & Payan, F. (1994) Biochemistry 33, 6284-6294] are in support of the mixed non-competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DP18-maltodextrin hydrolysis gives straight-lines plots while amylose gives parabolic curves. These results, confirmed by Dixon-plot analyses, allow us to postulate that, in inhibition of reduced DP18-maltodextrin hydrolysis, one molecule of acarbose is bound/ amylase molecule. In contrast, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surface sites found by X-ray crystallography [Qian, M., Haser, R. & Payan, F. (1995) Protein Sci. 4, 747-755]; the glucose site close to the active site and the maltose site, 2 nm away. In conclusion, no significant difference between PPA I and PPA II has been observed, either from molecular mass or from kinetic behaviours; this suggests multiple forms of the enzyme. A general mechanism of PPA action is proposed; in addition to the active site, long-chain substrate hydrolysis requires the glucose-binding site and the maltose-binding site, while only one site is necessary for the hydrolysis of short chain substrate.

A peracid based dishwashing detergent composition

Abstract: A warewashing composition for a machine dishwasher and a method of using it is described. The composition comprises effective amounts of an organic peroxy acid, and an amylase enzyme which, when incubated at 55 °C in a solution of 2mM sodium citrate, 1mM epsilon phthalimidoperoxyhexanoic acid in 36 ppm water at pH 8.0, has a half-life of two minutes or greater based on an activity vs. time plot obtained via monitoring color development at 405nm of solution samples incubated with p-nitrophenyl-α-D-maltoheptaoside as substrate and gluco amylase and α-glucosidase as coupled enzymes; and 1 % to 75 % by weight of a builder. A 1 % aqueous solution of the composition must have a pH of less than 10.