Showing papers on "Amylase published in 2000"

Human Milk Oligosaccharides Are Minimally Digested In Vitro

Abstract: In examining the functional aspects of human milk oligosaccharides (HMO), it is not known whether they are digested during the passage through the infant's gastrointestinal tract. HMO were prepared from individual milk samples (n = 6) and separated into neutral and acidic compounds by chromatography. These oligosaccharide fractions were studied for their digestibility by human salivary amylase, porcine pancreatic amylase and brush border membrane vesicles (BBMV) isolated from porcine small intestine; we also examined the effect of low pH on these structures. The characterization of HMO and their digestion products was performed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as TLC. It was shown that neither salivary amylase nor pancreatic amylase cleaved HMO. Only after a 2-h incubation with BBMV were slight modifications of the HMO observed. HPAEC-PAD analysis revealed two new components within the neutral oligosaccharide fractions; these were characterized by mass spectrometric analysis as lacto-N:-triose and galactose. Only lacto-N:-triose was present within digestion assays of oligosaccharides, which did not contain fucosyl or N:-acetylneuraminic acid residues. These results suggest that <5% of the HMO are digested in the intestinal tract. Hence, HMO may play a role as prebiotics or as factors influencing the local immune system of the intestine in breast-fed infants.

Amylase-resistant starch plus oral rehydration solution for cholera.

Abstract: Background Although standard glucose-based oral rehydration therapy corrects the dehydration caused by cholera, it does not reduce the diarrhea. Short-chain fatty acids, which are produced in the colon from nonabsorbed carbohydrates, enhance sodium absorption. We conducted a study to determine the effects of an orally administered, nonabsorbed starch (i.e., one resistant to digestion by amylase) on fecal fluid loss and the duration of diarrhea in patients with cholera. Methods We randomly assigned 48 adolescents and adults with cholera to treatment with standard oral rehydration therapy (16 patients), standard therapy and 50 g of rice flour per liter of oral rehydration solution (16 patients), or standard therapy and 50 g of high-amylose maize starch, an amylase-resistant starch, per liter of oral rehydration solution (16 patients). The primary end points were fecal weight (for every 12-hour period during the first 48 hours after enrollment) and the length of time to the first formed stool. Results The me...

Inhibition of Enzymic Digestion of Amylose by Free Fatty Acids In Vitro Contributes to Resistant Starch Formation

Abstract: The effect of lipids on the enzymic breakdown of starch was investigated using an in vitro assay system. Mixtures of potato amylose, amylopectin and starch and various lipids were incubated at 37 degrees C for 10 min and subjected to digestion by alpha-amylase (EC 3.2.1.1) and amyloglucosidase (EC 3.2.1.33). Lauric, myristic, palmitic and oleic acids and lysolecithin inhibited enzymic hydrolysis of amylose by approximately 35% (P < 0.05). Stearic acid and cholesterol had no effect on the enzymic breakdown of amylose. Retrograded amylose was hydrolyzed less readily (P < 0.05) than solubilized amylose, but the breakdown was not further inhibited in the presence of lauric acid. Fatty acids had no effect on the enzymic hydrolysis of amylopectin, whereas inhibition by fatty acids of the breakdown of whole starch was consistent with only the amylose fraction being affected. The possibility that interactions between starch and fatty acids in the digestive tract could contribute to the formation of resistant starch is considered.

Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana

Abstract: Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically. The highest amylase production was achieved by growing H. meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase. The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. Optimal temperature and salinity for activity were 37°C and 10% NaCl, respectively. Moreover, activity at salinity as high as 30% salts was detected. Maltose and maltotriose were the main end products of starch hydrolysis, indicating an α-amylase activity.

Production and partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54

Abstract: AIM An investigation was carried out on the production of alpha-amylase by Bacillus thermooleovorans NP54, its partial purification and characterization. METHODS AND RESULTS The thermophilic bacterium was grown in shake flasks and a laboratory fermenter containing 2% soluble starch, 0.3% tryptone, 0.3% yeast extract and 0.1% K2HPO4 at 70 degrees C and pH 7.0, agitated at 200 rev min(-1) with 6-h-old inoculum (2% v/v) for 12 h. When the enzyme was partially purified using acetone (80%[v/v] saturation), a 43.7% recovery of enzyme with 6.2-fold purification was recorded. The KM and Vmax (soluble starch) values were 0.83 mg ml(-1) and 250 micromol mg(-1) protein min(-1), respectively. The enzyme was optimally active at 100 degrees C and pH 8.0 with a half-life of 3 h at 100 degrees C. Both alpha-amylase activity and production were Ca2+ independent. CONCLUSIONS Bacillus thermooleovorans NP54 produced calcium-independent and thermostable alpha-amylase. SIGNIFICANCE AND IMPACT OF THE STUDY The calcium-independent and thermostable alpha-amylase of B. thermooleovorans NP54 will be extremely useful in starch saccharification since the alpha-amylases used in the starch industry are calcium dependent. The use of this enzyme in starch hydrolysis eliminates the use of calcium in starch liquefaction and subsequent removal by ion exchange.

New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 α-amylase contributes to starch binding and raw starch degrading

Abstract: The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

Ontogeny of pancreatic enzymes in larval red drum Sciaenops ocellatus.

Abstract: The growth, survival and trypsin, lipase and amylase activities of red drum larvae were measured in two experiments. For the first trial, a group was fed live prey only (L) and another group was fed a combination of a microparticulate diet (MPD) and live food (L-MP). For the second growth trial a group fed the MPD only (MP) and a starvation group (ST) were examined in addition to the L and L-MP treatments. Enzyme activities of live prey were measured to estimate their possible contribution to larval digestion. No significant (P > 0.05) differences in final size and survival were observed between treatments L and L-MP. Larvae subjected to starvation or fed the MPD diet alone were smaller than treatments fed live prey and did not survive past days 5 and 14, respectively. Trypsin, lipase and amylase activities were detectable at hatching. No significant differences (P > 0.05) in total enzyme activities among treatments were observed before day 14. Specific activity of trypsin, lipase and amylase peaked on day 3 (prior to first feeding) and subsequently decreased. For trypsin, the percentage of enzyme activity potentially attributable to ingested prey increased with age to a maximum of 17%. For lipase and amylase this fraction was less than 5% throughout the study, except on day 8 (12% and 24%, respectively). The lack of significant differences observed in the activity of digestive enzymes among treatments suggests that dietary regime, availability of prey and possible effects of exogenous enzymes did not significantly influence enzyme activity. Therefore, the lower growth rate observed in the L-MP, MP and starved treatments cannot be attributed to low digestive enzyme production of the enzymes measured. It is more likely that the MPD failed to supply the required nutrients for adequate development.

The effect of thermal environment on the digestion of broilers

Abstract: Summary Two experiments were conducted to determine the effect of thermal environment on the digestion of broilers In both experiments the birds were exposed to three temperatures: 5, 21, and 32° C, and relative humidity was maintained at 60% In experiment one, the amount of chyme in the whole digestive tract was decreased by the cold environment (5° C, 60% relative humidity) and increased by hot environment (32° C, 60% relative humidity), compared with control environment (20° C, 60% relative humidity) The expelling of digesta from the crop or small intestine was suppressed by high temperature (32° C) (p < 001) In experiment two, the activities of trypsin, chymotrypsin, and amylase were affected significantly by different environmental temperatures (p < 001) Compared to normal environment, the activities of the three kinds of enzymes in intestinal juice were decreased by the hot environment: trypsin (p < 001), chymotrypsin (p < 005), and amylase (p < 001), respectively The activities of the three kinds of digestive enzyme were not influenced by the cold environment

Comparative characterization of complete and truncated forms of Lactobacillus amylovorus α-amylase and role of the C-terminal direct repeats in raw-starch binding.

Abstract: Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Abstract: Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA3 and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA3 nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA3 and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA3 and IAA helped in bringing the level near to those in the controls. GA3 and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA3 and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.

Protease and amylase production of Streptomyces rimosus in submerged and solid state cultivations

Abstract: The protease activity of Streptomyces rimosus TM-55 was first detected after 12 h of growth in submerged cultivation, and this activity peaked after 166 h of incubation. In solid state cultivation, protease was first secreted at 24 h, with the secretion peaking at 232 h. Amylase activity could be detected after 6 h in submerged cultivation, and it afterwards peaked after 48 h of incubation. In solid state cultivation, it began to be secreted at 24 h, and this secretion peaked after 180 h. Each gram of starch yielded 17.4 and 691.3 units of protease and amylase in submerged cultivation, respectively; whereas the values were 26.7 and 2,642.7 units in solid state cultivation. α-Amylase was the major amylase in both cultivation methods, and glucosmylase and debranching activity were minor components. Protease and amylase produced with both cultivation methods had a similar optimal pH, between 6.0 and 7.0, and optimal temperature, between 35 and 45℃. The enzyme activities produced in solid state cultivation were more stable with pH and temperature changes than those produced in submerged cultivation.

Correlations between total protein, lysozyme, immunoglobulins, amylase, and albumin in stimulated whole saliva during daytime

Abstract: The correlations between salivary proteins and the daytime variations are not known. The present study investigated the within-subject variation of correlations and concentrations between lysozyme, IgA, IgG, IgM, albumin, amylase, and total protein in stimulated whole saliva of healthy adults in the course of a 12-h period. After several practise sessions, unstimulated and stimulated whole saliva samples were collected five times daily (at 8 a.m., 11 a.m., 2 p.m., 5 p.m., and 8 p.m.) from 30 healthy university students. Flow rate and total protein concentration were used as covariates, and gender as a between-subject factor in the MANOVA analysis. After this adjustment, there was significant within-subject variation in salivary IgA (P < 0.001), albumin (P < 0.01), amylase (P < 0.05), and total protein (P < 0.001) concentrations. Total protein correlated significantly with amylase albumin and IgA through different samplings. In addition, IgG correlated with albumin and lysozyme in the course of 12 h. On the whole, the correlations between variables remained stable during repeated samplings. In addition, rankings of subjects for the variables tended to be maintained across different samplings (P < 0.001). However, the observed within-subject variations in salivary IgA, albumin, amylase, and total protein concentrations suggest that these proteins are subject to short-term variation.

Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate.

Abstract: Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

The α-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization

Abstract: Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425. Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose. The type II mutant was affected in the gene responsible for the synthesis of the extracellular α-amylase. This gene (amyH) was isolated by functional complementation of mutant II and sequenced. The deduced protein (AmyH) showed a high degree of homology to a proposed family of α-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals. AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids. The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli. AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential. In addition, H. meridiana and H. elongata were able to secrete the thermostable α-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes.

Amylase-resistant starch plus oral rehydration solution for cholera.

Abstract: A BSTRACT Background Although standard glucose-based oral rehydration therapy corrects the dehydration caused by cholera, it does not reduce the diarrhea. Short-chain fatty acids, which are produced in the colon from nonabsorbed carbohydrates, enhance sodium absorption. We conducted a study to determine the effects of an orally administered, nonabsorbed starch (i.e., one resistant to digestion by amylase) on fecal fluid loss and the duration of diarrhea in patients with cholera. Methods We randomly assigned 48 adolescents and adults with cholera to treatment with standard oral rehydration therapy (16 patients), standard therapy and 50 g of rice flour per liter of oral rehydration solution (16 patients), or standard therapy and 50 g of highamylose maize starch, an amylase-resistant starch, per liter of oral rehydration solution (16 patients). The primary end points were fecal weight (for every 12hour period during the first 48 hours after enrollment) and the length of time to the first formed stool. Results The mean (±SD) fecal weights in the periods 12 to 24 hours, 24 to 36 hours, and 36 to 48 hours after enrollment were significantly lower in the resistant-starch group (2206±1158 g, 1810±1018 g, and 985±668 g) than in the standard-therapy group (3251± 766 g, 2621±1149 g, and 2498±1080 g; P=0.01, P= 0.04, and P=0.001, respectively). From 36 to 48 hours after enrollment, fecal weight was also significantly lower with the resistant-starch therapy than with the rice-flour therapy (985±668 g vs. 1790±866 g, P=0.01). The mean duration of diarrhea was significantly shorter with the resistant-starch therapy (56.7±18.6 hours) than with standard therapy alone (90.9±29.8 hours, P=0.001) or the rice-flour therapy (70.8±20.2 hours, P=0.05). Fecal excretion of starch was higher with the resistant-starch therapy (32.6±30.4) than with the standard therapy (11.7±4.1 g, P=0.002) or the riceflour therapy (15.1±8.4 g, P=0.01). Conclusions The addition of a resistant starch to oral rehydration solution reduces fecal fluid loss and shortens the duration of diarrhea in adolescents and adults with cholera. (N Engl J Med 2000;342:308-13.)