Protein extraction from fungi.
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Electrophoresis↗
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Results for “Polygalacturonase”
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Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum.
The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20 degrees C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH4+:NO3- ratio (total nitrogen concentration of 60 mM) and PO4(3-) did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH4+:NO3- ratio, and PO4(3-)) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.
Biotechnology↗
Growth and pectinase production by Aspergillus Mexican strain protoplast regenerated under acidic stress.
Protoplasts from Aspergillus sp. FP-180 and Aspergillus awamori NRRL- 3112 were released and regenerated at extreme acidic conditions. The best conditions for protoplast release were 0.8 M KCI, pH 5.8, and 3 h of digestion using mycelia from 12- to 16-h cultures from either Aspergillus sp. FP-180 or A. awamori NRRL-3112. The addition of fresh mycelia to an ongoing digestion after 1 h increased protoplast 4.5-5 times. A regeneration efficiency of 90% was attained at pH 6.0, and it was possible to regenerate protoplasts at pH 1.7 with a regeneration efficiency of 0.5% for Aspergillus sp. FP-180. The LpH-10 strain, derived from protoplast from Aspergillus sp FP-180, was able to regenerate at pH 1.7 and grow at pH values as low as 1.5, values at which the original strain is unable to grow. Regeneration at extreme pH improved the performance of LpH-10 strain. It showed a twofold increase in cell growth at pH 2.0 in liquid culture and a higher pectinolytic activity in relation to that produced by the original strain.
Acids↗
Enzymatic hydrolysis of ammonia-treated sugar beet pulp.
Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control.
Ammonia↗
Use of pectinase in pollen tube and pollen mother cell smears.
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Glycoside Hydrolases↗
Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.
Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.
Cell Separation↗
Use of pectinase to dissociate plant nuclei for squash preparations: effect of hydration procedures.
Scale tissues taken from cones of pendent Douglas-fir Pseudotsuga menziesii (Mirb.) Franco were treated with pectinase to dissociate nuclei for Feulgen staining. Hydration of fixed tissues with water or a graded series of ethyl alcohol before pectinase treatment resulted in distention and faint staining of nuclei and inadequate disintegration of cell walls. These problems were overcome by adopting citrate buffer hydration. Analysis of digitized scans of nuclei provided information about the distribution of the stained material in density classes and nuclear size which is usually unavailable through the conventional double wavelength DNA measuring method.
Buffers↗
A quick enzyme squash technique for detailed studies on female meiosis in Solanum.
A simple enzyme squash technique that enables detailed studies of meiosis in potato ovules has been developed. Fixation of ovules in iron-propionic-ethanol followed by enzymatic maceration and squashing in acetocarmine yielded numerous well preserved megasporocytes with nicely spread chromosomes. Resolution was sufficient, allowing detailed analysis of chromosome pairing and chiasma formation and readily permitting distinction between normal and desynaptic mutant plants. Whereas the use of previously developed ovule squash techniques has been restricted to cytogenetic analyses of plant species with relatively large megasporocytes and large chromosomes, the present technique is potentially more useful for analyses of species with small megasporocytes and small chromosomes.
Cellulase↗
Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa.
We evaluated the effects of cellulase (from Trichoderma longibrachiatum) application rates on neutral detergent fiber (NDF) concentration and fermentation products of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages harvested with decreasing dry matter (DM) digestibility. Additionally, the impacts of inoculant (Lactobacillus plantarum and Pediococcus cerevisiae), pectinase (from Aspergillus niger), or formic acid on silage composition were studied. Forages wilted to a DM content of about 320 g/kg were ensiled in laboratory silos for 60 d. Cellulase, combined with inoculant, was applied at 2, 10, and 20 ml/kg of herbage (at least 2500 IU/ml). Cellulase at 10 ml/kg was also applied alone or in combination with pectinase and inoculant or formic acid. The NDF concentration of orchardgrass silage decreased with increasing cellulase up to 20 ml/kg, at which NDF content was decreased by 30%. The NDF concentration of alfalfa silage decreased with increasing cellulase application up to 10 ml/kg, at which NDF content was decreased by 13%. Immature plants were more responsive to cellulase treatment than mature plants. Cellulase at 2 ml/kg combined with inoculant improved fermentation characteristics of the silages but generally, there was no effect on silage fermentation by higher cellulase applications, resulting in an accumulation of sugar. The improved fermentation of orchardgrass treated with cellulase and inoculant was mostly related to the effect of inoculant, whereas cellulase alone improved fermentation characteristics of alfalfa silage and this effect was enhanced by addition of inoculant. Decreased NDF and increased sugar concentrations did not improve the in vitro DM digestibility of cellulase-treated silages.
Aspergillus niger↗
Additives containing bacteria and enzymes for alfalfa silage.
First-cutting alfalfa was wilted, harvested from alternate rows, left untreated or treated with additives containing lactic acid bacteria and enzymes (cellulase, amylase, and pectinase), and ensiled in bag silos. Inoculation increased lactic acid bacteria from 5 x 10(4) to 1 x 10(6) cfu/g of forage. Because treatments were bagged consecutively, the DM of treated silages was higher than that of untreated silage. However, after 4 d of ensiling, the pH of treated silage, about 4.3, was lower than that of untreated silage, 4.7, and remained lower throughout the ensiling period. After 177 d of ensiling, total lactate was about 25% higher, and ammonia N was about 40% lower, in treated silage. In addition, NDF and ADF contents were lower in treated than in untreated silage. Between 51 and 177 d of storage, glucose content increased in treated silage, but not in untreated silage, suggesting that some plant cell-wall hydrolysis occurred during prolonged storage. In vitro digestion of NDF did not differ among treatments during early incubation, but the extent of digestion after 36 and 48 h was lower in treated than in untreated silage. The microbial and enzyme silage additives used in this study improved fermentation characteristics and reduced fiber content of silage but decreased the in vitro digestibility of fiber.
Amylases↗
Effects of grain source and enzyme additive on site and extent of nutrient digestion in dairy cows.
Four lactating, cannulated Holstein cows were used in a 4 x 4 Latin square design to investigate the effects of grain source and fibrolytic enzyme supplementation on ruminal fermentation, nutrient digestion in the rumen and in the intestine, and milk production. A 2 x 2 factorial arrangement was used; two grains (barley and hull-less barley) were combined with and without enzyme. The enzyme supplement (Pro-Mote; Biovance Technologies Inc., Omaha, NE) contained primarily cellulase and xylanase activities and was applied daily to the total mixed diet. Dry matter intake was not affected by diet, but starch intake was greatest when hull-less barley was fed. Starch from hull-less barley was more digestible in the rumen and in the total tract than was starch from barley, but opposite results occurred for fiber digestion, indicating that hull-less barley depressed fiber digestion. As a result, cows fed the hull-less barley diets tended to produce more milk with a higher milk lactose content than did cows fed the barley diets. Enzyme supplementation had minimal effects on ruminal digestion but increased nutrient digestibility in the total tract and the proportion of microbial N in nonammonia N. Consequently, cows fed diets supplemented with enzyme had a higher milk protein content and tended to produce more 4% fat-corrected milk than did control cows. These results indicate that the use of hull-less barley rather than barley increased the digestible energy intake of dairy cows, resulting in higher milk production. The use of a fibrolytic enzyme mixture enhanced feed digestibility and milk production.
Animals↗
Saccharification of Okara fiber by plant dietary fiber hydrolases.
In this paper, the saccharification process of okara fiber with pectinase, xylanase and cellulase was investigated as a preliminary attempt to effectively utilize okara fiber. The solubilization of okara fiber was raised in proportion to the number of enzymes used; that is, by a single enzyme, two enzymes and three enzymes, in that order. The saccharification of okara fiber was much more completed by a combination of pectinase, xylanase and cellulase than by using individual preparations. This multi-enzyme system liberated sugars equivalent to 80% of the original okara fiber by weight. Moreover the structure of okara fiber gradually disintegrated by hydrolytic reactions with pectinase, xylanase and cellulase in turns. These results suggest that the saccharification of okara fiber progresses by the cooperative action of pectinase, xylanase and cellulase.
Cellulase↗
Effects of protein level and enzyme supplementation upon growth and rate of digesta passage of male turkeys.
An experiment was conducted to determine the effect of feeding enzyme supplements (Avizyme and protease) at two levels of dietary crude protein (24 and 28%) to male turkeys from 0 to 5 wk of age. The Avizyme-protease enzyme mixture was fed at five concentrations within each protein level in an effort to determine an optimal level of supplementation. Enzymes were added to the 24% protein diet to determine whether the supplements would enhance the growth performance of birds fed the lower protein diet to the level of those fed the 28% protein diet. Rate of passage was evaluated to determine whether an ANF effect corresponded to enzyme concentration or protein level. Dietary protein level significantly influenced growth. Birds fed the 28% protein diet had improved body weight gain, feed consumption, and feed efficiency of 11.5, 6.5, and 4.4%, respectively, when compared with birds fed the 24% protein diet. Enzyme addition to the 24% protein corn-soybean meal diet produced a graded response but did not improve growth over the control. Although enzyme supplementation improved poult utilization of the 24% protein diet, growth and feed utilization were not equal to the performance of the poults fed the 28% protein diet. When the enzyme mixture was added to the 28% diet, performance was not consistently altered. Rate of digesta passage was not different between the levels of dietary protein or among levels of enzyme supplementation, indicating that the applied protein levels did not deleteriously affect nutrient utilization.
Animal Feed↗
[Biosynthesis of cellulolytic enzymes and xylanase during submerged cultivation of the fungus Aspergillus terreus 17P].
The fungus Aspergillus terreus 17P--producer of cellulolytic enzymes--was cultivated in the Biotec 10 l fermenter on the medium containing minced and heated (at 200 degrees) wheat straw aerated with a different rate. At the mixing rate of 350 rpm and aeration rate of 0.7 r/rpm on the fourth day the culture liquid was obtained whose filtrate contained an active complex of cellulolytic enzymes and xylanase: CI--3.4; APB--1.1, Cx--35.7, cellobiase--0.23, xylanase--73.8 units/ml. The fractionation of the culture liquid filtrate with ammonium sulphate showed that the fraction precipitated at an interval of saturation of 0.3--0.7 contained the largest portion of cellulolytic enzymes and xylanase. The isolated enzymic preparations had a cellulolytic and xylanase activity and contained lipase, pectinase, laminarinase. They also contained low quantities of amylase, protease, beta-1,4- and beta-1,6-glucanase. Enzymic hylrolysis by the Asp. terreus 17P preparation of straw yielded glucose and xylose, of cotton, Na-KMC, cellobiose--glucose, Xylane hydrolyzate contained xylose and arabinose.
Amylases↗
pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger.
The nucleotide sequence data for pgaA and pgaB have been deposited with the EMBL, GenBank and DDBJ Databases under accession numbers Y18804 and Y18805 respectively. pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillus niger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB were characterized with respect to pH optimum, activity on polygalacturonic acid, and mode of action and kinetics on oligogalacturonates of different chain length (n=3-7). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 mu+kat.mg(-1) and 8.3 mu+kat.mg(-1) respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.
3' Untranslated Regions↗
[Type of interactions between agarose and Aspergillus niger endopolygalacturonases].
Endopolygalacturonase has been purified by binding on Sepharose 6 B gel; studies on interactions between agarose and the enzymes have shown that endopolygalacturonase chromatography on Sepharose 6 B was on ion-exchange chromatography and did not involve biospecific interactions.
Aspergillus niger↗
Pectin: cell biology and prospects for functional analysis.
Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.
Cell Wall↗
[Formation of an extracellular system of enzymes during growth of Geotrichum candidum 3C on cell walls isolated from cereal grain capsules].
The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum 3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain capsules, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, alpha-L-arabinan, and 1,4-beta-D-galactan), as well as beta-D-galactosidase and alpha-L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl-alpha-D-glucurono-beta-D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52-56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8-28 h of cultivation. Enzymes degrading alpha-D-galacto-beta-D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.
Cell Wall↗