Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. amiga)

Three phytases were purified about 14200-fold (LP11), 16000-fold (LP12), and 13100-fold (LP2) from germinated 4-day-old lupine seedlings to apparent homogeneity with recoveries of 13% (LP11), 8% (LP12), and 9% (LP2) referred to the phytase activity in the crude extract. They behave as monomeric proteins of a molecular mass of about 57 kDa (LP11 and LP12) and 64 kDa (LP2), respectively. The purified proteins belong to the acid phytases. They exhibit a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 80 microM (LP11), 300 microM (LP12), and 130 microM (LP2) and k(cat) = 523 s(-1) (LP11), 589 s(-1) (LP12), and 533 s(-1) (LP2) at pH 5.0 and 35 degrees C. The phytases from lupine seeds exhibit a broad affinity for various phosphorylated compounds and hydrolyze phytate in a stepwise manner.     

Hydrolysis of precipitated phytate by three distinct families of phytases

While genetically modified plants that secrete histidine acid phosphatases (HAPs), β-propeller phytases (BPPs) and purple acid phosphatases (PAPs) have been shown to assimilate soluble phytate, little is known about whether these plants have the ability to hydrolyze precipitated phytate. In this study, the ability of representative members of these three classes of phytases to hydrolyze metal-phytate salts and to hydrolyze phytate adsorbed to aluminum precipitates was compared. All three phytases were able to hydrolyze Ca²⁺-, Mg²⁺-, and Mn²⁺-phytates, but were unable to hydrolyze Al³⁺-, Fe²⁺-, Fe³⁺-, Cu²⁺-, and Zn²⁺-phytates. When these ions were present, the hydrolysis of Ca²⁺-phytate was prevented. Citrate was more potent than malate and oxalate in solubilizing some of these phytate salts for enzyme hydrolysis. Phytate adsorbed to aluminum precipitates was resistant to all three enzymes, except when organic acids were added (citrate>oxalate>malate). While increasing concentrations of organic acids were inhibitory to enzyme activity (oxalate >citrate>malate), PAP was more resistant to citrate than HAP. As desorption of phytate from a solid surface by organic acids is essential for phytase activity, the genetic engineering of plants that enhances the secretion of both citrate and phytases from the root may be a feasible approach to improving soil phytate assimilation.     

Nitrogen losses from two grassland soils with different fungal biomass

Nitrogen losses from agricultural grasslands cause eutrophication of ground- and surface water and contribute to global warming and atmospheric pollution. It is widely assumed that soils with a higher fungal biomass have lower N losses, but this relationship has never been experimentally confirmed. With the increased interest in soil-based ecosystem services and sustainable management of soils, such a relationship would be relevant for agricultural management. Here we present a first attempt to test this relationship experimentally. We used intact soil columns from two plots from a field experiment that had consistent differences in fungal biomass (68 ± 8 vs. 111 ± 9 μg C g⁻¹) as a result of different fertilizer history (80 vs. 40 kg N ha⁻¹ y⁻¹ as farm yard manure), while other soil properties were very similar. We performed two greenhouse experiments: in the main experiment the columns received either mineral fertilizer N or no N (control). We measured N leaching, N₂O emission and denitrification from the columns during 4 weeks, after which we analyzed fungal and bacterial biomass and soil N pools. In the additional ¹⁵N experiment we traced added N in leachates, soil, plants and microbial biomass. We found that in the main experiment, N₂O emission and denitrification were lower in the high fungal biomass soil, irrespective of the addition of fertilizer N. Higher ¹⁵N recovery in the high fungal biomass soil also indicated lower N losses through dentrification. In the main experiment, N leaching after fertilizer addition showed a 3-fold increase compared to the control in low fungal biomass soil (11.9 ± 1.0 and 3.9 ± 1.0 kg N ha⁻¹, respectively), but did not increase in high fungal biomass soil (6.4 ± 0.9 after N addition vs. 4.5 ± 0.8 kg N ha⁻¹ in the control). Thus, in the high fungal biomass soil more N was immobilized. However, the ¹⁵N experiment did not confirm these results; N leaching was higher in high fungal biomass soil, even though this soil showed higher immobilization of ¹⁵N into microbial biomass. However, only 3% of total ¹⁵N was found in the microbial biomass 2 weeks after the mineral fertilization. Most of the recovered ¹⁵N was found in plants (approximately 25%) and soil organic matter (approximately 15%), and these amounts did not differ between the high and the low fungal biomass soil. Our main experiment confirmed the assumption of lower N losses in a soil with higher fungal biomass. The additional ¹⁵N experiment showed that higher fungal biomass is probably not the direct cause of higher N retention, but rather the result of low nitrogen availability. Both experiments confirmed that higher fungal biomass can be considered as an indicator of higher nitrogen retention in soils.     

Pathway of dephosphorylation of myo-inositol hexakisphosphate by phytases of legume seeds

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential removal of phosphate groups via D-Ins(1,2,3,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2) to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final degradation product Ins(2)P via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2).     

Effects of two grass species on the composition of soil fungal communities

Many studies have shown effects of plants species on fungal communities, but these are often confounded with soil effects. Thus, the specific role of plant species in structuring rhizospheric and soil fungal communities is poorly described. Our study used microcosms in which plants were grown under artificial conditions to bridge this gap. Two perennial grasses dominating subalpine grasslands, Festuca paniculata and Dactylis glomerata, were grown at two levels of fertilization on standard soil. Fungal communities were determined by 454 pyrosequencing of the internal transcribed spacer 1 region. Among the fungal communities characterized by the primers used, original communities were associated to each plant species and also diverged between rhizosphere and bulk soils within each plant species, though there were no significant fertilization effects. Differences regarded global composition of the fungal communities and abundant molecular operational taxonomic units (MOTUs). Both plant species and location effects were reflected more in the abundance than in the composition of MOTUs. The observed differences in fungal communities coincide with differing strategies of plant root growth, with D. glomerata having greater root mass, length, and area than F. paniculata. Our study, by dissociating soil effects from plant effects, demonstrated that plant species exert a key control on soil fungi. We suggest that such effects may be linked to inter-specific differences in root traits and their consequences on nitrogen uptake.     

Colorimetric characterization for comparative analysis of fungal pigments and natural food colorants

Exogenous pigments produced by ascomycetous filamentous fungi belonging to the genera Penicillium, Epicoccum, and Monascus, preselected based on chemotaxonomic knowledge, have been extracted and characterized by quantitative colorimetry. The color characteristics of the fungal extracts were compared to water soluble natural colorants derived from sources currently in use. The tested fungal extracts also included some commercially available Monascus colorants. The a values for the fungal extracts were found to be both positive and negative, the b values were found to be positive, while the hue angles of the fungal color extracts ranged from 40 to 110 indicating the color distribution of fungal extracts over the red-orange-yellow region of the CIELAB color space. The fungal extracts exhibited additional color hues in the red spectrum and similar hues in the yellow spectrum as compared to the reference natural colorants. They were also found to be similar or brighter in terms of chroma to some of the reference natural colorants. Principal component analysis was performed to group and distinguish different colors based on the a and b values. The fungal color extracts could be grouped in accordance with the similarity or difference in the color to those of the existing natural colorants. The diversity of colors was not only found among different fungal genera and/or species but also within the same species on changing the media. There was a marked change in the color composition of the extracts resulting in relatively different hues. Our results, thus, indicate that there exists pigment-producing genera of ascomycetous fungi other than Monascus that produce color shades in the red and the yellow spectra in addition or similar to reference colorants. These color shades could add to the color palette of the natural colorants currently in use. In addition, the multivariate approach in distinguishing and classifying the colorants was shown to be a very useful tool in colorimetric comparison of colorants.     

Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes

The activity of wheat and Aspergillus niger phytases was determined following preincubation for 60 min at 37 degrees C alone or in the presence of pepsin or pancreatin to examine their ability to survive in the gastrointestinal tract. At pH 3.5 both phytases were stable, but at pH 2.5 wheat phytase rapidly lost activity. Following preincubation at pH 3.5 in the presence of 5 mg of pepsin/mL, A. niger phytase retained 95% of its original activity, whereas only 70% of the wheat phytase activity was recovered. The stability of A. niger phytase in the presence of pepsin was the same at pH 2.5 as at pH 3.5. Results similar to those with pepsin at pH 3.5 were obtained following preincubation of the phytases in the presence of pancreatin at pH 6.0.     

Salt effect on the pH profile and kinetic parameters of microbial phytases

The pH profiles of two microbial phytases were determined using four different general purpose buffers at different pH values. The roles of calcium chloride, sodium chloride, and sodium fluoride on activity were compared in these buffers. For Aspergillus niger phytase, calcium extended the pH range to 8.0. A high concentration of sodium chloride affected the activity of fungal phytase in the pH 3-4 range and shifted the pH optimum to 2.0 from 5.5 in Escherichia coli phytase. As expected, both of the microbial phytases were inhibited by sodium fluoride at acidic pH values. Because the Km for phytate increased nearly 2-fold for fungal phytase while Vmax increased about 75% in a high concentration of sodium chloride, it is possible that salt enhanced the product to dissociate from the active site due to an altered electrostatic environment. Modeling studies indicate that while the active site octapeptide's orientation is very similar, there are some differences in the arrangements of alpha-helices, beta-sheets, and coils that could account for the observed catalytic and salt effect differences.     

Effect of the herbicide fluazifop-butyl on fungal populations and activity in soil

The side effects of fluazifop-butyl on soil fungal populations and oxygen uptake were studied by incubating soil samples with a range of fluazifop-butyl concentrations (0, 0.6, 3 and 6 μg g^?1) over 8 weeks. Cellulose decomposition in soil was also studied in laboratory experiments with the herbicide which was either incorporated in soil or sprayed onto calico squares which were buried in soil. The mycelial dry weight of six fungal species under the effect of the herbicide was also examined. Fluazifop-butyl had no significant effect on total fungal propagule populations at 0.6 μg g^?1. At 3 and 6 μg g^?1, it caused temporary reduction in fungal populations observed after 1 and 2-wk of incubation. The herbicide had no significant effect on OZ uptake. The decay of calico buried in herbicide-treated soil was generally stimulated, while the decomposition of herbicide-treated calico, buried in untreated soil, was temporary delayed. The mycelial dry weight yields of Aspergillus favus (at 2 and 12 μg mL^?1 of fluazifop-butyl) and Cunninghamella echinulata (at 12 μg mL^?1) were significantly increased. At 24 μg mL^?1 the mycelial dry weight of A. flavus and Alternaria alternata was significantly reduced.     

Kinetic characterization of the enzymatic and chemical oxidation of the catechins in green tea

The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.     

Changes in spatial distribution of fungal propagules associated with invertebrate activity in soil

Soil fungal species density and aggregation had changed 6 months after spring burning and raking in a Wisconsin prairie. Fungal species density had increased by 29% in burned relative to raked or undisturbed plots; species density was highest at the soil surface in burned and raked plots, and lowest at the surface in undisturbed plots. Fungal propagules of the same species were less aggregated in burned and raked plots than in undisturbed. These changes imply greater mixing of fungal propagules in soil of burned and raked plots than in undisturbed plots. The physical effects of burning and raking and the properties of fungal spores do not account for the changes. It is argued that soil invertebrates are responsible for the mixing because: (1) at any depth in soil, density of mites and collembola increased concurrently with fungal species density; and (2) path analysis shows that fungal species density is equally associated with microarthropod density and root biomass after burning, but after raking, fungal species density was more strongly associated with root biomass than microarthropods.     

Effects of solarization of soil on nematode and fungal pathogens at two sites in victoria

The effect of covering soil with transparent polyethylene sheets, known as soil solarization, on the viability of plant pathogens was determined. The treatment was tested in mid-summer on sandy loams in N.W. and S. Victoria. Columns of moist soil were inoculated with one of a variety of pathogens, viz. Fusarium oxysporum, Pythium irregulare, Plasmodiophora brassicae, Sclerotium cepivorum, S. rolfsii, Sclerotinia minor, Verticillium dahliae and the nematodes Macroposthania xenoplax, Meloidogyne javanica, Pratylenchus penetrans and Tylenchulus semipenetrans. Columns were placed vertically in soil, and then treated either for 4 weeks in N.W. Victoria, or 6 weeks in S. Victoria.Preliminary laboratory tests showed that pathogens were killed by temperatures within the range 38–55°C. The relative sensitivities of pathogens to fluctuating soil temperatures were similar at both sites. The most sensitive were the nematodes, and the fungi V. dahliae, S. cepivorum and S. minor, while F. oxysporum, P. irregulare and P. brassicae were the least sensitive. In N.W. Victoria treatment effects were apparent to 26 cm and most pathogens were not recovered from 0 to 11 cm. In S. Victoria treatment effects were apparent to a depth of 16cm and most pathogens were not recovered from 0 to 6cm.     

Kinetic studies of proteolytic enzyme activity of arctic soils under varying toluene concentrations

The original focus of this study was an analysis of proteolytic enzyme activity of Alaskan arctic tundra soils, however initial results raised questions regarding the method (Watanabe and Hayano, 1995). Thus, the goals of the study changed to 1) an investigation of the method, and 2) a comparison of enzyme activities of two different soil layers from the arctic tundra. Methodological examination included the impact of toluene, used to prevent immobilization of the product, and blank correction on enzyme activity, and a search for a true 6-h linear rate of activity during a 48-h incubation. We measured native and potential, using casein as an artificial substrate, activities as net amino acid production in mineral and organic soil layer samples. Varying toluene concentration had no clear effect on activity; omitting toluene resulted in zero native activity and reduced potential for the organic samples, but not for the mineral. Comparison of activities with and without blank correction indicated, particularly for potential activity of samples with low native rates, that correction was required for accuracy. Native and potential activity of the organic samples, and native of the mineral were linear during the first 6 h of incubation; linearity was found during the 6-24 h incubation for potential activity of the mineral. Soil layer activity data indicated that native activity was higher in organic soils as compared with mineral. The organic layer potential activity was ten-fold greater than the native, suggesting substrate limitation; potential and native activities did not differ in the mineral layer, indicating substrate sufficiency. Casein addition changed the kinetic pattern for both layers from hyperbolic to sigmoidal for the mineral and linear for the organic, implying different enzyme pools or behavioral changes of existing pools. Native activity based on total soluble protein was higher for the mineral samples relative to the organic, reiterating substrate capacity differences and variations in enzyme/substrate interactions.     

Isolation and characterization of two endoxylanases from Fusarium graminearum

This paper reports the first isolation from cultures of two endoxylanases secreted by Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch]. When F. graminearum is grown on wheat bran hydrated with a modified synthetic medium, high xylanase activity can be extracted. The two endoxylanases were identified by LC-MS/MS as the products of genes FGSG_6445 (Genbank gene id 2788192 ) (xylanase 1) and FGSG_3624 (GenBank accession no. AJ863566 ) (xylanase 2) with 61 and 51% sequence coverage, respectively. Both enzymes showed a pH optimum at pH 6, with xylanase 1 exhibiting a wider active pH range (5.5-9) than xlylanase 2 (5.5-7.5). Their temperature dependences were similar, >60% between 35 and 60 °C, with optimal temperatures of 45 °C for xylanase 1 and 50 °C for xylanase 2. Kinetic studies found that both enzymes had a lower K(m) for linear beachwood xylan than arabinoxylan. For xylanase 2, the V(max) increased with arabinoxylan, but decreased for xylanase 1.     

Interaction of phytases with minerals and availability of substrate affect the hydrolysis of inositol phosphates

The stability and activity of phytases in the soil environment may be affected by their sorption on soil particle surfaces and by substrate availability with important consequences for P cycling and nutrient bioavailability. This work evaluated the interaction of phytases with goethite, haematite, kaolinite, montmorillonite and two oxisol clays and investigated how this interaction is affected when myo-inositol hexakisphosphate (InsP₆) was sorbed on the mineral surfaces. phyA histidine acid phosphatases of fungal origin were used and their ability to release orthophosphate from the InsP₆-saturated minerals was evaluated.The phytases showed a high affinity for the mineral surfaces, with a loss of enzyme activity generally being observed over 24 h (up to 95% of the initially added activity). The loss of phytase activity was dependent on the type of mineral, with kaolinite and montmorillonite showing the greatest effect. Retention of enzyme activity was higher with the two oxisol clays, suggesting that the heterogeneous nature of clay surfaces and the presence of endogenous organic matter may limit the inhibition caused by interaction with minerals.In the presence of mineral surfaces saturated with InsP₆, the partitioning of enzyme activity between the solution and the solid phase was shifted more towards the solution phase, presumably due to the mineral surfaces being occupied by the substrate. However, phytases were not able to release any orthophosphate directly from InsP₆-saturated goethite and haematite, and hydrolysed InsP₆ that was desorbed from haematite. Conversely, in the case of kaolinite and of the oxisol clays, where desorption was limited, phytases appeared to be able to hydrolyse a small fraction of the InsP₆ adsorbed on the surfaces. These findings suggest that the bioavailability of P from inositol phosphates is governed to a large extent by the mineral composition of soil and by competitive effects for sorption on reactive surfaces among inositol phosphates and phytases.     

Isoflavone characterization and antioxidant activity of ohio soybeans

Seventeen Ohio soybeans were screened for isoflavone content and antioxidant activity. Isoflavone content was determined by C(18) reversed phase high-performance liquid chromatography coupled with a photodiode array detector. Antioxidant activities of soybean extracts were measured using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and photochemiluminescence (PCL) methods. The highest and lowest total isoflavone contents were 11.75 and 4.20 micromol/g soy, respectively, while the average was 7.12 micromol/g soy. Antioxidant activities of soybean extracts ranged from 7.51 to 12.18 micromol butylated hydroxytoluene (BHT) equivalent/g soy using the DPPH method. Lipid and water soluble antioxidant activities of soybean extracts ranged from 2.40 to 4.44 micromol Trolox equivalent/g soy and from 174.24 to 430.86 micromol ascorbic acid equivalent/g soy, respectively, using the PCL method.     

Contribution of microbial phytases to the improvement of plant growth and nutrition: A review

Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.     

Elemental,functional infrared and free radical characterization of humic acid-type fungal polymers (melanins)

Summary Humic acid-type polymers (melanins) synthesized in culture media by the fungi Aspergillus glaucus, Eurotium echinulatum, Hendersonula toruloidea, Stachybotrys atra and Aspergillus sydowi were analysed for elemental composition, functional group content, infrared (IR) and electron spin resonance (ESR) properties. Results were discussed in comparison with range values referred for soil humic acids. The fungal polymers showed significant differences in carboxyl and nitrogen content and C/H atomic ratios, reflecting a different degree of condensation (aromaticity) among the various samples. IR analysis gave evidence of: (a) the predominant aromatic character of melanins from A. glaucus, E. echinulatum and H. toruloidea; (b) the high content of aliphatic and olefinic components of S. atra melanin; (c) the typical presence of amide bonds in the nitrogen-richest melanins from A. sydowi and H. toruloidea; and (d) the generally low amount of free carboxyl groups, which often appeared involved in hydrogen bonds. ESR spectra showed that all the melanins studied contained appreciable concentrations of organic free radicals of prevailing semiquinonic nature and of the same order of magnitude commonly measured in humic acids from soil and other sources. The free electron concentration was shown to be directly related to the C/H atomic ratio and to the degree of aromaticity shown by IR analysis. This indicated that the highest free radical content in the melanins from E. echinulatum and A. glaucus was associated with the highest presence of condensed aromatic structures. Humic acid-type polymers synthesized by soil fungi may, therefore, contribute to the total free radical content of soil humic substances and play important roles in all reactions involving free radicals in soils and related environments.     

Purification and characterization of two novel beta-galactosidases from Lactobacillus reuteri

The intracellular beta-galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess beta-galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri beta-galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.