Showing papers on "Amylase published in 1993"

MUTANT α-AMYLASE, DETERGENT, DISH WASHING AGENT, AND LIQUEFACTION AGENT

Abstract: In the mutant α-amylase one or more of the methionine amino acid residues is exchanged with any amino acid residue except for Cys and Met. The thus produced mutant α-amylase exhibits an improved stability in the presence of oxidizing agents and an improved thermoactivity at moderate low pH values. The mutant α-a amylase is well suited as an additive to detergents, dish washing agents and liquefaction agents.

Differentiation of the Properties of the Branching Isozymes from Maize (Zea mays).

Abstract: The multiple forms of branching enzyme (BE) from developing maize (Zea mays) endosperm were purified by modification of previous procedures such that amylase activity could be eliminated completely from the BE preparation. Three distinct assays for BE activity (phosphorylase a stimulation assay, BE linkage assay, and iodine stain assay) were used to characterize and differentiate the properties of the BE isoforms. This study presents the first evidence that the BE isoforms differ in their action on amylopectin. BEI had the highest activity in branching amylose, but its rate of branching amylopectin was less than 5% of that of branching amylose. Conversely, BEII isoforms had lower rates in branching amylose (about 9–12% of that of BEI) and had higher rates of branching amylopectin (about 6-fold) than BEI. The implication of these findings to the mechanism of amylopectin synthesis in vivo are discussed.

Salivary α-Amylase: Role in Dental Plaque and Caries Formation

Abstract: Salivary α-amylase, one of the most plentiful components in human saliva, has at least three distinct biological functions. The enzymatic activity of a-amylase undoubtedly plays a role in carbohydrate digestion. Amylase in solution binds with high affinity to a selected group of oral streptococci, a function that may contribute to bacterial clearance and nutrition. The fact that a-amylase is also found in acquired enamel pellicle suggests a role in the adhesion of a-amylase-binding bacteria. All of these biological activities seem to depend on an intact enzyme conformation. Binding of a-amylase to bacteria and teeth may have important implications for dental plaque and caries formation. a-Amylase bound to bacteria in plaque may facilitate dietary starch hydrolysis to provide additional glucose for metabolism by plaque microorganisms in close proximity to the tooth surface. The resulting lactic acid produced may be added to the pool of acid in plaque to contribute to tooth demineralization.

Purification and characterization of an extracellular amylase from Lactobacillus plantarum strain A6

Abstract: E. GIRAUD, L. GOSSELIN, B. MARIN, J.L. PARADA AND M. RAIMBAULT. 1993. Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had an estimated molecular weight of 50 kDa. It was identified as an a-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C and K,,, value of 2.38 g 1-' with soluble starch substrate. The enzyme was inhibited by N-bromosuccinimide, iodine and acetic acid. The enzyme activation energy was 30.9 kJ mol-'.

The remarkable evolutionary history of the human amylase genes

Abstract: Analysis of the structures of the human amylase genes has demonstrated that this multigene family contains at least five tandem gene copies, closely related in sequence but with distinct tissue specific expression. The structures of the genes demonstrate that the human salivary amylase gene was derived from a preexisting pancreatic amylase gene. Insertion of a retrovirus upstream of the amylase gene is responsible for the alteration in tissue specificity. A parotid specific enhancer has been identified within the retrovirus by expression studies in transgenic mice. The independent origin of salivary amylase in rodents and primates suggests that there has been strong evolutionary selection for amylase in saliva. The amylase genes demonstrate a novel mechanism for evolution of new patterns of tissue specific gene expression.

Purification and characterization of an extracellular glucoamylase from the thermophilic fungus Humicola grisea var. thermoidea

Abstract: Humicola grisea var thermoidea mycelium grown on maltose as the main source of carbon produced at least two amylases The major amylolytic component was purified to homogeneity and classified as a glucoamylase The apparent molecular mass of the purified enzyme was estimated to be 63 000 Da by SDS-PAGE and 65 000 Da by Bio-Gel P-100 filtration The purified enzyme was a glycoprotein with 18% carbohydrate content and pH and temperature optima of 50 and 55 °C, respectively The purified glucoamylase was thermostable at 60 °C with a half-life of 16 min at 65 °C In the presence of starch the purified enzyme retained 75% of its thermostability at 65 °C, while the addition of maltose failed to protect the activity The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates Starch and amylopectin were the best substrates utilized and amylose was hydrolyzed faster than maltopentaose, maltotetraose, and maltotriose Kinetic experiments suggested that maltose and starch were hyd

Construction of a model secretion system for oral streptococci.

Abstract: A DNA fragment corresponding to the secretory domain from the Streptococcus mutans GS-5 gtfB gene, which encodes the putative 38-amino-acid signal peptide of the glucosyltransferase I (GTF-I) enzyme product, has been constructed. This fragment was fused with the alpha-amylase structural gene from alkalophilic Bacillus sp. strain 707. This hybrid gene as well as the intact amylase gene were introduced into an Escherichia coli-streptococcus shuttle vector consisting of three components: the E. coli replicon p15Aori from pACYC177, an erythromycin resistance gene from pAM beta-1, and the streptococcal replicon from pVA838. Transformation of the oral noncariogenic bacterium Streptococcus gordonii with the chimeric plasmid harboring the hybrid amylase gene resulted in strong extracellular amylase production. By contrast, transformants containing the intact amylase gene exhibited only trace amounts of amylase activity in culture fluids. Since the two signal peptide structures of the GTF-I enzyme and the Bacillus amylase are distinct from each other, these differences might result from the inability of S. gordonii to correctly process the Bacillus signal peptide. Furthermore, culture fluids from transformants of S. mutans as well as Streptococcus milleri harboring the hybrid amylase gene showed only weak amylase activity. Deletion of the gtfB, gtfC, or ftf gene from S. mutans GS-5 did not increase amylase secretion following transformation with the hybrid amylase gene. These results suggest that in contrast to S. gordonii, the inability of S. mutans and S. milleri to secrete hybrid amylase molecules could result from incorrect interaction of the secretory components of these organisms with amylase precursor molecules.

Hydrolysis of Tropical Tuber Starches by Bacterial and Pancreatic α‐Amylases

Abstract: Action of porcine pancreatic and Bacillus subtilis α-amylases on native tuber starches of yam (Dioscorea alata), sweet potato (Ipomea batatas) and tannia (Xanthosoma sagittifolium) was studied in comparison with the well known potato and cassava starches. Large differences in enzymes susceptibilities were observed when studied on 24h. Yam starch was 3.5% hydrolysed with 2,8 μkat amylase/g starch, three times less than potato and tannia starches while sweet potato starch was 53% hydrolysed, two times less than cassava starch. Except yam, level of hydrolysis was higher with porcine pancreatic amylase than with the Bacillus subtilis amylase while initial hydrolysis rate was lesser. Microscopic observations and image analysis pointed out that the polyhedric shaped granules of tannia, sweet potato and cassava starches were much more damaged than the spherical ones. Pitting occured preferentially on the edges of the granules and the enzymes penetrated into the starch granule by pores and canals of corrosion. Conversely to other starches, hydrolysis of yam starches evidenced greater differences between action of Bacillus subtilis and pancreatic α-amylases. The enzymes acted by pitting some parts of the granules surface, the number of pores and their size being related to enzyme source. Hydrolyse von tropischen Wurzelstarken durch bakterielle und pankreatische α-Amylasen. Die Wirkung von schweinepankreatischen und Bacillus subtilis-α-Amylasen auf native Wurzelstarken von Yam (Dioscora alata), Suskartoffel (Ipomea batatas) und Tannia (Xanthosoma sagittifolium) wurden im Vergleich mit den gut bekannten Kartoffel- und Cassavastarken untersucht. Grose Unterschiede wurden beobachtet bezuglich der Enzymsuszeptibilitaten bei Untersuchungen von 24h. Yamstarke wurde zu 3,5% mit 2,8μkat Amylase/g Starke hydrolysiert, dreimal weniger als Kartoffel- und Tannia-Starken, wahrend Suskartoffelstarke zu 53% hydrolysiert wurde, zweimal weniger als Cassavastarke. Auser bei Yam war der Hydrolysegrad bei Schweinepankreas-Amylase hoher als bei der Bacillus subtilis-Amylase, wahrend die Anfangs-Hydrolysegeschwindigkeiten geringer waren. Mikroskopische Beobachtungen und Bildanalyse zeigten, das die vielflachigen Korner von Tannia-, Suskartoffel- und Cassavastarken sehr viel starker beschadigt waren als die spharischen. Lochfras trat vorwiegend an den Kanten der Korner auf, und die Enzyme durchdrangen das Innere der Starkekorner durch Poren und Korrosionskanale. Im Gegensatz zu anderen Starken wies die Hydrolyse von Yamstarken grosere Unterschiede zwischen der Wirkung von Bacillus subtilis-und pankreatischen α-Amylasen auf. Die Enzyme wirkten durch Lochfras an einigen Stellen der Kornoberflache, wobei die Anzahl der Poren und deren Grose mit der Enzymherkunft in Beziehung standen.

The effect of storage temperature on reducing sugar concentration and the activities of three amylolytic enzymes in tubers of the cultivated potato,Solanum tuberosum L.

Abstract: Reducing sugar content, and activities of three starch hydrolysing enzymes, alpha-amylase, beta-amylase and debranching enzyme were measured over several months in tubers of five cultivars stored at 4°C or 10°C. Cultivars differed in their sensitivity to storage temperature. Reducing sugar content of tubers and the activities of three starch hydrolysing enzymes increased sharply during the first weeks of storage at 4°C. At 10°C, reducing sugar content, and the activity of the three enzymes remained constant or increased only slightly.

Simultaneous kinetic determinations of lipase, chymotrypsin, trypsin, elastase, and amylase on the same microtiter plate.

Abstract: Micromethods are described to determine in 10 min the activity of the five most common pancreatic zymogens: amylase, lipase, trypsin, chymotrypsin, and elastase. Progress of the reactions is monitored at 405 nm, allowing the kinetic determination of the five enzymes on a single 96-well microtiter plate. Amylase activity is measured by the release of p-nitrophenol from a chemically defined substrate. Linearity of the assay is from 10 to 360 U/L of amylase, and activities as low as 0.4 U/L can be easily measured by extending the period of incubation up to 24 h. Chymotrypsin, trypsin, and elastase activities are monitored by the release of p-nitroanilide from specific substrates, and activities are from 25 to 6,500, 15 to 260, and 20 to 600 U/L, respectively. Finally, lipase is determined by the clearing of a commercially available stabilized emulsion of triolein. The lipase determination can be performed from 90 to 3,600 U/L. When microplate methods were compared with conventional procedures, a perfect correspondence was found between the two types of procedure. Factors necessary to convert microplate results to those of conventional assays are provided. These microassays make possible the rapid and simultaneous determination of the three main types of pancreatic hydrolases (a glycohydrolase, three proteases, and a lipase) with < 5 microliters of pancreatic juice by kinetic analysis. They could be easily adopted as routine assays in most research laboratories.

Substrate regulation and specificity of amylases from Aureobasidium strain NRRL Y-12,974

Abstract: Pullulan is an industrial biopolymer produced by the yeast-like fungus Aureobasidium, usually by direct fermentation of starch. Despite evidence that autogenous amylases produced during these fermentations are detrimental to the final molecular mass of the product, fundamental studies of these enzymes have not been reported. Total extracellular amylases were studied from the promising production strain NRRL Y-12,974. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml−1 in glucose-grown cultures to 0.03 IU ml−1 in soluble-starch-grown cultures. All cultures showed both α-amylase activity and activity against pullulan. Synthetic oligosaccharide substrates were apparently attacked by an α-glucosidase, produced in highest levels by maltose-grown cultures.

Deep-frozen, pre-proofed doughs

Abstract: The invention concerns with deep frozen, pre-proofed doughs that contain 0.5-10 wt % of a gelatin relating compound, optionally in combination with other dough improvers like ascorbic acid, vital gluten, xylanase, amylase or DATA-esters. In particular the combination with xylanase/amylase leads to unexpected results (s.v. and/or ovenspring of baked products made from the doughs). As gelatin relating compounds gelatin, hydrolysed gelatin, collagen hydrolysate and/or gelatin precursors can be used.

Microbial enzyme activities related to litter decomposition near a highway in a sub-tropical forest of North East India

Abstract: Cellulose, amylase and invertase activities were studied in extracts of leaf litters of Alnus nepalensis and Pinus kesiya during litter decomposition at a roadside (more polluted) and a non-roadside (less polluted) forest stand. Enzyme activities were considerably higher in litter at the less polluted than at the more polluted site. Cellulase and amylase activities showed a marked seasonal variation at both sites. Cellulase and amylase activities increased during litter decomposition, whereas invertase activity was higher at the begining of litter decomposition. Invertase activity correlated positively with litter soluble sugars. Cellulase and amylase activities, but not invertase activity, were correlated significantly with numbers of fungi and bacteria.

Starch‐degrading enzymes during the induction of CAM in Mesembryanthemum crystallinum

Abstract: Mesembryanthemum crystallinum plants were irrigated with 400 mol m−3 NaCl to induce CAM and levels of leaf starch, and activities of starch-degrading enzymes were measured. During Crassulacean acid metabolism (CAM) induction, daily starch turnover gradually became more pronounced and was three- to four-fold greater than in leaves of C3 plants after 3 weeks. Activities of α- and β-amylase, D-enzyme and starch phosphorylase all increased 10- to 20-fold within 3 weeks of the start of salt treatment. Activities of α- and β-amylase increased more than fourfold within the first 24 h of salt treatment, which is the fastest increase in enzyme activities so far measured during the induction of CAM with salt solution in intact plants of this species. Most enzyme activities were partially chloroplastic; however, the principal starch-degrading activity was constituted by an extra-chloroplastic β-amylase. CAM starch-phosphorylase activity, which was mainly chloroplastic, exhibited a two- to three-fold diurnal change in parallel with starch content. CAM induction in M. crystallinum is clearly associated with greater starch turnover and enhanced starch-degrading enzyme activities, which as catalysts of the initial reaction to release carbon for synthesis of phosphoenolpyruvate (PEP) appear highly significant for the functioning of the CAM pathway. The diurnal rhythm of phosphorylase activity may be of particular significance.

Changes in α- and ß-amylase during Storage of Sweetpotato Lines with Varying Starch Hydrolysis Potential

Abstract: Staple-type lines of sweetpotato (Ipomoea batatus (L.) Lam.) do not sweeten significantly upon cooking as com- pared to the traditional-type lines. Four lines exhibiting distinct differences in sweetness after cooking were evaluated for changes in α- α- and s-amylase activity and reducing sugars (by HPLC) at harvest, after curing, and at intervals during 180 days of storage. The traditional cultivar 'Jewel' and staple-type line 'Sumor' displayed high a- and s-amylase activities, which rose from low levels at harvest to peak level s ≈ ≈ 90 days into the storage period. Staple-type lines '99' and '86' displayed significantly lower a- and s-amylase activities. By using polyclonal sweetpotato s-amylase antibody and western blot following native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was confirmed that a lower level of s- amylase synthesis existed in '99' and '86'. Quantitatively, 'Jewel', 'Sumor', and an additional staple-type line, 'HiDry', had 361,374, and 365 µg s-amylase protein per gram of fresh storage root tissue, respectively, while '99' and '86' possessed <60 and 12 µg·g -1 , respectively. In raw roots, individual (glucose, fructose, and sucrose) and total sugar concentrations were significantly higher in 'Jewel' than in 'Sumor', '99', or '86'. Only trace amounts of maltose were found in raw roots of any line. Sucrose, glucose, and fructose concentrations decreased with baking in all lines except '86', in which they increased. There was substantial maltose produced by baking 'Jewel' and 'Sumor', but only trace amounts found in baked '99' and '86'. Sweetpotato germplasm can be separated into four general classes based on initial sugar concentration and changes during cooking: 1) low sugars/low starch hydrolysis, 2) low sugars/high starch hydrolysis, 3) high sugars/low starch hydrolysis, and 4) high sugars/high starch hydrolysis. At least two mechanisms may confer the lack of starch hydrolysis and subsequent sweetening in staple-type sweetpotato: 1) inhibition of s-amylase synthesis, and 2) a nonenzyme mediated mechanism.

Starch bioavailability in arepas made from ordinary or high amylose corn: concentration and gastrointestinal fate of resistant starch in rats.

Abstract: The purpose of the present investigation was to study the importance of the amylose/amylopectin ratio for the content and gastrointestinal fate of resistant starch in a realistic composite starchy food. Corn-based breads (arepas) from dent corn (25% amylose) and from high amylose corn (70% amylose) were used as test products. Resistant starch concentration was evaluated in vitro and in vivo using rats treated with an antibiotic drug (Nebacitin) to suppress hindgut fermentation. Experiments in rats with intact hindgut microflora allowed determination of resistant starch fermentability. The small intestinal digestibility of starch in dent corn arepas was close to 96% (total starch basis), whereas the starch in high amylose arepas was poorly digested (approximately 68%, total starch basis), as calculated from the fecal recovery of resistant starch in Nebacitin-treated animals. The main resistant starch fraction required solubilization in alkali to render it available to the analytical amylases (nonhydrated fraction). The total amount of resistant starch as well as the nonhydrated starch fraction delivered to the hindgut could be accurately predicted from analysis of starch remnants in an enzymatic gravimetric dietary fiber residue. Resistant starch present in dent corn arepas was fermented approximately 63%, whereas the fermentability of resistant starch from the high amylose product was remarkably low (< 11%).

The acetylation and enzymatic degradation of starch films

Abstract: The preparation of films from starch and their degradation by amylase enzymes is described. Starch acetate was prepared by acetylation of starch with a pyridine/acetic anhydride mixture. The resulting polymer was cast into films from solutions of 90% formic acid. A series of films with a range of acetyl content were then exposed to buffered amylase solutions and the retained tensile strength measured. It was found that with a sufficient acetyl content the wet strength of the films was maintained in the aqueous solutions, but that the acetyl content was sufficiently low enough to permit degradation by a mixture of alpha and beta amylases within a period of 1 h. These films could be useful as membranes in bioreactors, which could be degraded at will by the addition of enzymes to the system. © 1993 John Wiley & Sons, Inc.

Characteristics of Carbohydrate Metabolism in Sweet Corn (sugary-1) Endosperms

Abstract: Metabolic characteristics of developing sugary-l maize (Zea mays L.) endosperms were investigated. In the later stages of development (>30 days postpollination), sugary-1 kernels maintained higher levels of many enzyme activities and retained more moisture than normal kernels. Higher enzyme activities were attributed to moisture retention and were not associated with any increase in dry weight accumulation. Of enzyme activities measured at 20 days postpollination, that of ADP-glucose pyrophosphorylase was higher in sugary-1 kernels than in normal, whereas total amylase, a-amylase, and pullulanase activities were lower. Experiments testing the effects of zero, one, two, and three doses of the sugary-1 gene in OH43 endosperms indicated that the sugary-1 phenotype was not expressed until three doses of the sugary-1 gene were present. Decreased activities of amylases, but not of pullulanase, were attributed to an interference in detection by phytoglycogen. Increased ADP-glucose pyrophosphorylase activity is attributed to a response by the maize endosperm cells to increased sucrose concentrations. The sugary- 1 genotype of maize (Zea mays), commonly grown as sweet corn, accumulates more sugars in the endosperm than normal starchy maize. The principle difference between normal and sugary- 1 maize is that sugary- 1 endosperms accumulate the highly branched, water soluble form of starch known as phytoglycogen (Morris and Morris, 1939). There are two hypoth- eses concerning the origin of phytoglycogen . The first suggests that sugary- 1 maize kernels lack sufficient starch debranching enzyme (Erlanger, 1957). This hypothesis suggests that starch biosynthesis involves the synthesis of a highly branched interme- diate form of starch which is subsequently debranched to form the amylose and amylopectin found in normal maize starch granules. Evidence supporting this hypothesis was provided by Pan and Nelson (1984), who demonstrated significantly reduced pullulanase activity (one form of starch debranching enzyme found in devel- oping maize kernels) in sugary- 1 kernels that appeared to depend on the sugary- 1 gene dosage. The second hypothesis suggests that the sugary- 1 mutation affects a starch-branchi ng enzyme, which results in the more highly branched phytoglycogen (Boyer and Preiss, 1978). The sugary- 1 mutation has many additional effects on maize kernel development. Amyloplasts fail to form birefringent starch gran- ules and instead accumulate phytoglycogen (Boyer et al., 1977). In comparison to normal kernels, sugary- 1 kernels accumulate less dry weight (Andrew et al., 1944; Tsai et al., 1978), retain kernel moisture longer (Andrew et al., 1944), have thinner pericarp (Andrew et al., 1944) and contain altered storage protein (Tsai et al., 1978). In this study, we have investigated developmental and dosage effects of the sugary- 1 gene on carbohydrate metabolism in the

Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models.

Abstract: PURPOSE: To improve the 30-year-old "trypsin digestion" procedure for isolation of the complete retinal vasculature, which, in its time, was a revolutionary advance that allowed important discoveries about diabetic retinopathy; to provide a method that will yield more consistent results when applied to retinas representing a wide range of ages, species, and severity of vascular disease, such as that occurring in diabetes METHODS: Because the Difco trypsin preparation (Difco Laboratories, Detroit, MI) is a crude pancreatic extract, containing variable amounts of chymotrypsin, elastase, amylase, lipase, ribonuclease, collagenase, and other contaminants, an attempt was made to determine which of the major enzymes alone (using purified preparations), or what combination of enzymes, might be most effective in providing consistently clean yet intact retinal vasculatures from eyes of different origins RESULTS: Purified elastase alone (40 U/ml) in 100 mmol/l sodium phosphate buffer with 150 mmol/l sodium chloride and 5 mmol/l EDTA at pH 65 and 37 degrees C gave better results than various concentrations of purified trypsin or chymotrypsin alone, or mixtures of trypsin/chymotrypsin, trypsin/elastase, chymotrypsin/elastase, or the crude trypsin preparation CONCLUSIONS: Elastase, which exhibits broad protease activity, and not trypsin, is the most important enzyme of the standard, crude trypsin digestion procedure for removal of the nonvascular tissues of the retina

The high maltose-producing α-amylase of the thermophilic actinomycete, Thermomonospora curvata

Abstract: The alpha-amylase of Thermomonospora curvata catalyses the formation of very high levels of maltose from starch (73%, w/w) without the attendant production of glucose. The enzyme was produced extracellularly in high yield during batch fermentation in a 5-1 fermentor. Purification was achieved by ammonium sulphate fractionation, Superose-12 gel filtration and DEAE-Sephacel ion-exchange chromatography. The enzyme exhibited maxima for activity at pH 6.0 and 65 degrees C, had a relative molecular mass of 60,900-62,000 and an isoelectric point at 6.2. The exceptionally high levels of maltose produced and the unique action pattern exhibited on starch and related substrates indicate a very unusual maltogenic system. The predominance of maltose as the final end-product may be explained by the participation of reactions other than simple hydrolysis and the preferential cleavage of maltotriose from higher maltooligosaccharides. The enzyme exhibits very low affinity for maltotriose (Km = 7.7 x 10(-3) M) and its conversion to maltose is achieved by synthetic followed by hydrolytic events, which result in the very high levels of maltose observed and preclude glucose formation. This system is distinguished from other very high maltose-producing amylases by virtue of its high temperature maximum, very low affinity for maltotriose and the absence of glucose in the final saccharide mixture.

Comparison of amylolytic properties of Lactobacillus amylovorus and of Lactobacillus amylophilus

Abstract: Partially purified amylases produced by Lactobacillus amylovorus and L. amylophilus were compared and they differed in several properties. The maximum amylase activity of L. amylovorus was higher than that of L. amylophilus. As estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the molecular mass of the enzymes was 140 kDa for L. amylovorus amylase and 100 kDa for L. amylophilus amylase. Maximum enzymatic activities were obtained when the strains were grown in the presence of CaCO3, on maltose with L. amylovorus and on sucrose with L. amylophilus. Optimal activities were obtained at pH values between 5.0 and 6.0 for both amylases. The L. amylovorus amylase was stable at a higher temperature (50°C) than the L. amylophilus amylase (40°C). Of six substrates examined, greatest activity was obtained by both enzymes on soluble starch. Neither enzymes hydrolysed pullulan or α- and \-cyclodextrins. With the exception of Hg2+, which partially inhibited both enzymes, various metal ions, such as 1 mm Ca2+ and Ba2+, stimulated L. amylophilus amylase activity whereas they inhibited L. amylovorus amylase activity.

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

Abstract: The distribution and specific activities of polysaccharide-degrading enzymes were monitored during the postprandial period in the liquid-associated bacteria (LAB), liquid-associated protozoa (LAP), and solid-associated microbes (SBFP) isolated from ruminal contents of cattle fed a high-cereal diet. Polysaccharide depolymerase activities were highest in the SBFP and the LAP populations. The postprandial variations in the specific activity of amylase were similar within the subpopulations. However, carboxymethylcellulase and xylanase activities increased in the first 5 h after feeding in the LAP, but were highest at the end of the postprandial period in the SBFP. Glycosidases involved in the fermentation of soluble carbohydrates increased significantly immediately after feeding in the liquid-associated microbes. β-d-Glucosidase and β-d-xylosidase were most active in the SBFP and were maximal 23 h after feeding. The activities of the plant cell wall polysaccharide-degrading enzymes and glycosidases in the SBFP were inversely related to ruminal pH; however, the activity of enzymes in the liquid-associated populations were highest in the immediate post-feed period when ruminal pH was lowest.

Effects of dietary proteins on some pancreatic mRNAs encoding digestive enzymes in the pig

Abstract: The response of pancreatic protease syntehsis to fish meal-enriched diets was investigated in the pig, which is generally held to be a suitable model for human digestive physiology. In three sets of experiments, pigs were fed either 7, 17, 48, or 68% protein diets for 8 days (1st set), or 17 or 48% protein diets for 3, 6, or 7 days (2nd set), or 7 or 48% protein diets for 4 days (3rd set). At the end of each experiment, the pancreata were removed for biochemical and gene expression assays. The specific activities of amylase, lipase, chymotrypsin, trypsin, and elastase decreased as the result of the 7% protein diet after an 8-day feeding period (1st set). In the same experiment, all the enzyme-specific activities (those of amylase, lipase, chymotrypsin, elastase as well as carboxypeptidases A and B) increased in response to the 48% protein diet, the most strongly affected enzyme being chymotrypsin. Only chymotrypsin and carboxy-peptidase A-specific activities were further increased after feeding with the 68% protein diet for 8 days as compared with the 48% protein diet. The amylase, lipase, and trypsinogen mRNA levels remained unchanged throughout the experiments, but the mRNA encoding procarboxypeptidase A2 decreased, and that coding for chymotrypsinogen was enhanced after the animals had been fed the experimental diets for 3 days, but showed no change thereafter. Procarboxypeptidase B mRNA increased slightly only after a 6-day feeding period. When pigs were fed the 7 and 48% protein diets for 4 days, the enzymes synthesized in vitro in pancreatic lobules were correlated with the relative levels of the corresponding mRNAs, as measured by means of an in vitro cell-free reticulocyte-lysate translation system: both amylase and carboxypeptidases specific activities and their mRNA levels decreased slightly, while those of serine proteases increased. It was concluded that the biosynthesis of each serine protease was regulated separately and transiently at the pre-translational level. On the other hand, it seems very likely that amylase may be regulated at the translational level, while a multiple-level control process may take place in the case of procarboxypeptidase A2.