Showing papers on "Amylase published in 2004"

A method for routine measurements of total sugar and starch content in woody plant tissues.

Abstract: Several extraction and measurement methods currently employed in the determination of total sugar and starch contents in plant tissues were investigated with the view to streamline the process of total sugar and starch determination. Depending on the type and source of tissue, total sugar and starch contents estimated from samples extracted with 80% hot ethanol were significantly greater than from samples extracted with a methanol:chloroform:water solution. The residual ethanol did not interfere with the sugar and starch determination, rendering the removal of ethanol from samples unnecessary. The use of phenol-sulfuric acid with a phenol concentration of 2% provided a relatively simple and reliable colorimetric method to quantify the total soluble-sugar concentration. Performing parallel sugar assays with and without phenol was more useful for accounting for the interfering effects of other substances present in plant tissue than using chloroform. For starch determination, an enzyme mixture of 1000 U alpha-amylase and 5 U amyloglucosidase digested starch in plant tissue samples more rapidly and completely than previously recommended enzyme doses. Dilute sulfuric acid (0.005 N) was less suitable for starch digestion than enzymatic hydrolysis because the acid also broke down structural carbohydrates, resulting in overestimates of starch content. After the enzymatic digestion of starch, the glucose hydrolyzate obtained was measured with a peroxidase-glucose oxidase/o-dianisidine reagent; absorbance being read at 525 nm after the addition of sulfuric acid. With the help of this series of studies, we developed a refined and shortened method suitable for the rapid measurement of total sugar and starch contents in woody plant tissues.

Starch structure and digestibility Enzyme-Substrate relationship

Abstract: Digestion of starch is effected by hydrolysing enzymes in a complex process which depends on many factors; these include the botanical origin of starch, whether the starch is amorphous or crystalline, the source of enzymes, substrate and enzyme concentration, temperature and time, as well as the presence of other substances in the multicomponent matrix in which starch occurs naturally, e.g. cereal grains. Native starch is digested ( i.e. hydrolysed) slowly compared with processed (gelatinised) starch whose crystallinity has been lost and where the accessibility of substrate to enzymes is greater and not restricted by α-glucan associations such as double helices (especially in crystallites) or amylose-lipid complexes (in cereal starches). The restriction of starch digestion (primarily in the human digestive system) due to forms which are resistant to hydrolysis has led to the concept of dietary ‘resistant-starch’. Different forms of resistance can be identified which hinder hydrolysis. With regard to digestibility, whether in the human or animal digestive tract, it is important to understand the mechanisms of enzymatic hydrolysis, and the consequence of incomplete digestion i.e. the potential loss of glucose as a valuable source of energy. This review deals with starch hydrolysis by specific amylases in model ( in vitro ) and more broadly in in vivo systems. The optimisation of starch hydrolysis and digestion is discussed in the light of modern knowledge.

Inhibitory effects of rosmarinic acid extracts on porcine pancreatic amylase in vitro.

Abstract: Porcine pancreatic alpha-amylase (PPA) was allowed to react with herbal extracts containing rosmarinic acid (RA) and purified RA. The derivatized enzyme-phytochemical mixtures obtained were characterized for residual amylase activity. These in vitro experiments showed that the amylase activity was inhibited in the presence of these phytochemicals. The extent of amylase inhibition correlated with increased concentration of RA. RA-containing oregano extracts yielded higher than expected amylase inhibition than similar amount of purified RA, suggesting that other phenolic compounds or phenolic synergies may contribute to additional amylase inhibitory activity. The significance of food-grade, plant-based amylase inhibitors for modulation of diabetes mellitus and other oxidation-linked diseases is hypothesized and discussed.

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Abstract: Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.

Digestive enzymes and metabolic profile of Pseudoplatystoma corruscans (Teleostei: Siluriformes) in response to diet composition.

Abstract: Digestive enzyme responsiveness to feeding and associated adjustments of metabolism can be used to derive nutritionally effective diet formulations. Juvenile pintado (Pseudoplatystoma corruscans) were fed different diets. After feeding, fish were killed and blood, liver and white muscle were collected to evaluate metabolites. Stomach along with anterior, middle and posterior intestine were sampled for enzyme analysis. Non-specific protease, trypsin, chymotrypsin, amylase and lipase were assayed. Crude protein (CP) did not induce proteolytic activity; highest protease activities were observed in the stomach. Amylase was higher in the stomach in fish feeding on diets containing 13-25% starch. Lipase activity was observed along the gastrointestinal tract, with the highest activities observed in the middle section. The metabolic profile of white muscle was not affected by CP. In contrast, some plasma and liver metabolites were altered concomitant with changes in the digestive enzymes. Amino acid catabolism was increased. Digestion in pintado was responsive to cornstarch, reflected in intermediary metabolism; proteolytic activities of the digestive tract seem to be sufficient to deal with large amounts of dietary protein. As a result, we are able to recommend a balance between protein and energetic compounds, such as lipids and carbohydrates, in the diet to optimize fish growth.

Dietary supplementation with multienzyme preparations improves nutrient utilization and growth performance in weaned pigs.

Abstract: Two experiments with young pigs (25 d of age) were conducted to investigate the effect of multienzyme preparations on nutrient digestibility, growth performance, and P utilization and excretion. In Exp. 1, 24 pigs (six pigs per treatment) were used in a 28-d performance and digestibility trial using four diets: control (no enzyme) and control supplemented with enzyme preparation A, B, or C. The control diet was formulated to meet 95% of NRC (1998) nutrient specifications (except for available P, which was at 44% NRC) and composed of corn, wheat, wheat by-products, barley, soybean meal, canola meal, and peas. All three enzyme preparations contained xylanase, glucanase, amylase, protease, invertase, and phytase activities and differed in the type of plant cell wall-degrading activities; Enzyme A contained cellulase, galactanase, and mannanase; Enzyme B contained cellulase and pectinase; and Enzyme C contained cellulase, galactanase, mannanase, and pectinase. Pigs fed enzyme-supplemented diets had higher ADG (P = 0.02) and G:F (P = 0.01) than those fed the control diet. On average, and when compared with control diet, enzyme supplementation improved (P = 0.001 to 0.04) ileal digestibility of DM (60 vs. 66%), GE (62.8 vs. 70.4%), CP (62 vs. 72%), starch (86.7 vs. 94.2%), nonstarch polysaccharides (NSP; 10.1 vs. 17.6%), and phytate (59 vs. 70%). Compared with the control, total-tract digestibility of nutrients was increased (P = 0.001 to 0.01) owing to enzyme supplementation, with Enzyme C showing the highest improvement in DM, GE, CP, starch, NSP, phytate, and P utilization. Pigs fed enzyme-supplemented diets had decreased (P = 0.04) fecal P excretion. The benefit from improved nutrient utilization with enzyme supplementation was further substantiated in a 38-d growth performance study with 48 pigs. The control and Enzyme C-supplemented diets (same as Exp. 1) were assigned to six replicate pens (four pigs per pen). The study was conducted in three phases (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 38). Individual BW and pen feed disappearance were monitored. Average daily gain and G:F were 231 and 257 g (P = 0.01), and 0.56 and 0.63 (P = 0.001) for the control and enzyme-supplemented diets, respectively. It is evident from this study that the use of enzyme preparations may allow for cost-effective and environmentally friendly formulation of young pig diets.

Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

Abstract: It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42°C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making α-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, α-amylase, and 4-α-glucanotransferase.

The breakdown of starch in leaves

Abstract: This review describes recent progress in discovering the pathway of starch breakdown in leaves. The synthesis of starch from photo-assimilated carbon is one of the major biochemical fluxes in plants. Despite this, the pathway through which this starch is remobilized has not been defined. Numerous enzymes that could participate in starch breakdown are present in leaves, but until recently, the relative importance of each had not been determined. Through studies using model species such as Arabidopsis and potato, significant progress has now been made in determining the roles of known enzymes, and in the discovery of novel proteins necessary for breakdown. These data allow a tentative pathway for starch breakdown to be mapped out, involving hydrolysis primarily to maltose and subsequent maltose export to the cytosol. This provides a framework for complete discovery of the pathway and for the analysis of its regulation. Contents Summary 247 I. Introduction 247 II. Structure of the starch granule 248 III. Initial attack on the granule and the role of glucan, water dikinase 249 IV. Debranching of branched glucans 250 V. The metabolism of linear glucans 251 VI. Export of starch catabolites 254 VII. Metabolism of glucose and maltose 255 VIII. The emerging pathway of starch breakdown and its regulation 256 Acknowledgements 258 References 258.

Alpha amylase from a fungal culture grown on oil cakes and its properties

Abstract: Solid-state fermentation was carried out for the production of α -amylase using Aspergillus oryzae . Different oil cakes such as coconut oil cake (COC) sesame oil cake (SOC), groundnut oil cake (GOC), palm kernel cake (PKC) and olive oil cake (OOC) were screened to be used as substrate for the enzyme production and also compared with wheat bran (WB). GOC was found to be the best producer of the enzyme among these. Combination of WB and GOC (1:1) resulted higher enzyme titres than the individual substrates. Maximum amount of enzyme (9196 U/gds) was obtained when SSF was carried out using WB + GOC, having initial moisture of 64% and supplemented with lactose and ammonium nitrate (1% each) at 30 0 C for 72h using 2 mL spore suspension (6x10 7 spores/ml). Partial purification of the enzyme using ammonium sulphate fractionation resulted in 2.4-fold increase in the activity. The enzyme showed molecular weight of 68 KDa by SDS-PAGE. Except Mn, all other metal ions such as Ca, K, Na, Mg were found to be inhibitory for the enzyme activity. The enzyme was optimally active at 50

Effect of C:N ratio on alpha amylase production by Bacillus licheniformis SPT 27

Abstract: Bacillus licheniformis SPT 27 is an isolate which produces extra cellular alpha (α-) amylase exhibiting activity at a wide pH range and was relatively stable. The B. lichenformis isolate, however, produces low yields of the amylase. Our results show that the amylase production is higher in the presence of starch, with Amarantus peniculatus starch producing the highest amount of amylase. Amongst sugars, fructose supported maximum amylase production. Of the nitrogen sources tested, peptone and ammonium hydrogen phosphate where the best organic and inorganic sources respectively. The C:N ratio found to be the optimum was 1:1. Key Words: Amarantus manipulates , alpha (α-) amylase, Bacillus licheniformis SPT 27, C:N ratio. African Journal of Biotechnology Vol.3(10) 2004:519-522

Inhibitory effect of clonal oregano extracts against porcine pancreatic amylase in vitro.

Abstract: Oregano (Origanum vulgare) is a rich source of natural phenolic antioxidants and has potential to be a source of nutritional ingredients for functional foods. Herbs such as oregano have long been used in food preservation and in traditional medicine in the treatment of common ailments and have potential for positive modulation of oxidation-linked diseases such as diabetes. One of the potentially important components of anti-diabetic activity by oregano extract is mild amylase inhibition by phenolic antioxidants to help contribute towards management of hyperglycemia. Previously, we reported the ability of rosmarinic acid, one of the principal phenolic components of oregano, to inhibit porcine pancreatic amylase (PPA) activity. Here, we investigated the effect of 50% ethanol extracts of eleven phenolic antioxidant-rich oregano clonal lines on the activity of PPA in vitro. To this end, we analyzed extract total soluble phenolic content by the Folin-Ciocalteu reagent method, rosmarinic acid (RA), protochatechuic acid (PA), quercetin, and p-coumaric acid (pCA) contents by HPLC, antioxidant activity as 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging, and PPA-inhibitory activity by incubation of the enzyme with clonal oregano extracts and characterization of the activity of the phenolic-bound enzyme. Clonal oregano extracts inhibited the activity of PPA in vitro by 9-57%. Amylase inhibition by oregano extract was associated with extract total phenolic content and RA, quercetin, PA, and pCA content, as well as extract antioxidant activity and protein content. Our finding that clonal oregano extracts can inhibit PPA supports a potential new functionality for oregano as an anti-hyperglycemic agent. This provides an opportunity for a food-based strategy for modulation of starch breakdown to glucose, which could contribute to the management of hyperglycemia and diabetes complications in the long term.

Resistant starch: Classification, structure, production

Abstract: Starch is a high-molecular carbohydrate composed of linear (amylose) and branched (amylopectin) chains of glucose residues. In water, at increased temperatures, it undergoes gelatinisation followed by amylase-induced hydrolysis. Owing to this, it is completely digested in the gastrointestinal tract of humans. Also raw starch of some plant species, e.g. cereals, is subject to complete but slow digestion. In addition, starch may occur in the form incapable of enzymatic hydrolysis, referred to as “resistant starch” (RS). Resistant starch is a sum of starch and products of its degradation undigested in the small intestine of humans. There are four types of resistant starch. Type I RS – physically unavailable starch. Amylolytic enzymes have no access to starch accumulated in undamaged plant cells as the gastrointestinal tract lacks enzymes capable of degrading the components of plant cell walls. Type II RS – raw starch of some plant species, e.g. potato. Type III RS – retrograded starch, i.e. spontaneously- or artificially-precipitated from starch paste, occurring in the form of water-insoluble semi-crystalline structures. As a result of retrogradation, more thermostable structures are formed by amylose rather than by amylopectin. The amount of resistant starch produced this way increases along with the increasing amylose content of starch. Type IV RS – chemically- or physically-modified starch. Resistant starch has been reported to reduce the caloric value of food products and to decrease glucose level in blood. In the large intestine, it is fermented by gut-colonising bacteria. Products of its fermentation include gases and short-chain fatty acids – acetic, propionic and butyric, which are responsible for favourable selection of intestinal microflora, reduce the levels of cholesterol, triglycerides, and urea in blood, as well as prevent the formation of gut cancer. Commercial preparations of resistant starch are also produced to be used as food additives. Their resistance to the activity of enzymes, as well as physiological effects of their application, are determined with in vitro and in vivo methods. Results of those analyses are not always unequivocal, especially with the application of resistant starch with artificially-increased resistance. Resistant starch may serve as dietary fibre and exert a health-promoting effect on the human organism. Therefore further studies into its formation and results of its application in food production are still necessary.

Biochemical characterization of α-amylase from the yeast Cryptococcus flavus

Abstract: During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The Km value for the pure enzyme was 0.056 mg ml−1 with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50°C. The enzyme retained 90% of the activity after incubation at 50°C for 60 min and was inhibited by Cu2+, Fe2+ and Hg2+.

Fermentative production of DL-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Abstract: Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.

Digestive enzyme profiles reveal digestive capacity and potential energy sources in fed and starved spiny lobster (Jasus edwardsii) phyllosoma larvae.

Abstract: The impact of starvation on the digestive enzyme (protease, trypsin, lipase and amylase) activities of Stage I and IV Jasus edwardsii phyllosoma larvae was used to identify the nutrients metabolised or conserved during food deprivation, highlighting the most critical energy reserves. Protease activities increased significantly in both Stages I and IV phyllosoma, suggesting that protein catabolism provided energy during food deprivation. Lipase activity decreased significantly in starved Stages I and IV larvae indicating that lipid may be spared for fuelling later developmental moults. The use of protein, while sparing lipid, may provide immediate energy but not at the expense of long-term lipid energy stores which are known to be important during their lengthy larval phase. The preferential use of protein during short-term periods of starvation suggests that particular attention must be given to providing sufficient protein in artificial diets at all times. Amylase activity in starved Stage I larvae was lower than in fed animals, suggesting that the starved animals are not gaining sufficient carbohydrate. However, amylase activity was similar in fed and starved Stage IV larvae, possibly due to the catabolism of accumulated glycogen stores that were not sufficiently developed in Stage I animals.