Phytase activity in lichens

BackgroundThe degree to which storage conditions and duration affect phytase activity and inositol phosphates (InsP) was evaluated in wheat. Three wheat cultivars were stored as whole or ground kernels at 20, 7, and −18°C for 13, 48, 118, 188, 265, and 349 days. Samples were analyzed for InsP3‐6 concentration and phytase activity.ResultsThe InsP6 concentration of whole wheat kernels stored at −18 and 7°C did not differ from that at time point 0. Storage of whole kernels at 20°C significantly reduced the InsP6 concentration at days 48, 188, 265, and 349, by up to 8.4%. The InsP6 concentration of ground wheat samples was reduced by up to 23.6% until day 349. The lower InsP isomers Ins(1,2,3,4,5)P5 and Ins(1,2,4,5,6)P5 were detected only at a storage temperature of 20°C and at day 118 or later. Phytase activity did not change over time in ground kernels stored at −18 and +7°C but was decreased in ground kernels at 20°C and in whole kernels independent of temperature. Probable cultivar effects were found regarding phytase activity over time, dependent on grinding and storage temperature.ConclusionTo avoid loss of InsP6 content, wheat samples should be stored as whole kernels at or below 7°C. Phytase activity is more stable in ground samples than in whole kernels. However, some cultivars likely are more prone than others to storage effects.

Changes in phytic acid content, activity of phytase and α-amylase in rye breads were determined during rye bread making. The activity of phytase is highest in grain and flour whereas the activity in the sourdoughs is almost the half of the activity in the flour. The activity was unchanged in the dough after mixing and proofing. Degradation of phytic acid (IP6) into lower inositol phosphates and free phosphate is almost completed during the production of rye bread with long fermentation time whereas the degradation is less completed when whole grains are included in the recipe. In rye bread made from milled rye (DB00), 99% of IP6 is degraded and IP3 becomes the dominating inositol phosphate in this bread type presumably resulting in a high level of bioavailable minerals. In rye bread made with 30% grains (SB30), 94% of the IP6 content was reduced with IP4 and IP3 being the dominating lower inositol phosphates. In rye bread made with 50% whole grains (KB50) the degradation of IP6 was 82%, and the three inositol phosphates IP5, IP4 and IP3 were found in equal amount in this bread type. Due to significant amounts of phytic acid and remaining IP5, some of the minerals might not be available for human absorption after consumption of this bread type.

Purification and properties of a phytase from Candida krusei WZ-001

Phytic acid is an antinutritional factor that forms 1–2% of most of the seeds and their co-products representing more than 60% of their total phosphorus. Monogastric and agastric animals are unable to utilize phytate phosphorus either due to lack of or insufficient amount of phytate degrading enzymes. Phytases (myo-inositol hexakisphosphate-phosphohydrolase) are a special class of phosphatases that catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol phosphates, myo-inositol and inorganic phosphate. Phytases are found naturally in plants and microorganisms and a sizeable number of phytases have been purified and characterized from various fungi, yeasts and bacteria. The present investigation involves studies on the effect of moisture content, pH value and different media ingredients such as carbon, nitrogen, and surfactants on the production of phytase by the fungus Aspergillus ficuum DSM 932 in solid-state fermentation (SSF) using rice bran as substrate. The production of phytase by SSF was favored, when the fungus was grown at a moisture content of 60% and pH 7.0, resulted in a phytase activity of 5.2 units/g dry substrate. There was a 20% increase in phytase yield in the presence of sucrose in SSF medium, while glucose and fructose were not effective in enhancing the phytase activity when used individually. Yeast extract was found to be a favorable nitrogen source for phytase production by SSF, which resulted in a 20% increase in phytase activity. There was no significant effect in increasing phytase production with the use of either soy peptone or tryptic soy as nitrogen source. Approximately 30% inhibition in phytase activity was shown in the presence of the surfactant Tween-80 or Triton X-100 in the SSF. By supplementing rice bran with sucrose and yeast extract, and performing the SSF in tray bioreactors, a phytase activity of 6.76 units/g dry substrate could be obtained. Keywords: phytase, solid-state fermentation, Aspergillus ficuum , nutritional factors, rice bran

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle–antibody–phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

ABSTRACTThe effect of diets differing in enzyme supplements, mineral phosphorus (P) and microwave wheat treatment on phytate hydrolysis and lower inositol phosphate isomers (InsPs) appearance in broiler crops was studied. The broilers (16- and 15-day-old) were assigned to 48 pens of 15 or 20 birds each (n = 8 pens per treatment) in Experiments 1 and 2, respectively. In Experiment 1, birds received a low-P wheat-soybean meal diet where the wheat was either microwave treated or not. These diets were offered without further supplementation or with added phytase (500 FTU/kg diet), alone or in combination with a xylanase (16,000 BXU/kg diet). In Experiment 2, two maize-soybean meal-based diets were fed, without or with monocalcium phosphate supplementation. Furthermore, these diets were offered without further supplementation or with phytase at 500 or 12,500 FTU/kg diet. On day 23 or 24 (Experiments 1 and 2, respectively), crop digesta were pooled per pen, freeze-dried and analysed for InsPs and the marker TiO2. Microwaving reduced the intrinsic phytase activity and InsP6 hydrolysis, but increased the concentration of Ins(1,2,3,4,5)P5 and Ins(1,2,4,5,6)P5 in the digesta of crop (Experiment 1). Microwave treatment significantly interacted with enzyme supplementation for Ins(1,2,5,6)P4 concentration, indicating a synergistic effect of intrinsic and supplied phytase in the crop. Xylanase tended to support phytase hydrolysis in diets with microwave-treated wheat. Phytase addition increased InsP6 hydrolysis up to 79% (Experiment 2). Thus, wheat phytase activity can cause high InsP6 hydrolysis in the crop. Treatment differences in lower InsPs indicated that hydrolysis of the first InsP6 phosphate group is not the only step in the degradation cascade in the crop of broilers that is influenced by dietary factors.

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.

Induced modification of plant gene expression is of both fundamental and applied importance. Cis-acting regulatory elements (CREs) are major determinants of the spatiotemporal strength of gene expression. Yet, there are few examples where induced genetic variation in predetermined CREs has been exploited to improve or investigate crop plants. The digital PCR based FIND-IT technology was applied to discover barley mutants with CRE variants in the promoter of the nutritional important barley grain phytase (PAPhy_a) gene. Mutants with higher or lower gene expression and ultimately higher or lower mature grain phytase activity (MGPA), respectively, were discovered. Field trials and inositol phosphate profiling during germination showed that PAPhy_a does not influence agronomic performance under the trial conditions but it does shorten the lag time of phosphate mobilization during germination. Higher endogenous MGPA is an improvement of grain quality for feed use as it improves the phosphate bioavailability for monogastric animals. Moreover, as the targeted CRE motifs of the PAPhy_a promoter are shared with a range of seed expressed genes like key cereal and legume storage genes, the current results demonstrates a concept for modulating individual gene expression levels of a range of seed genes.

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis".

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10-14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.

Phytoextraction of excess soil phosphorus

Atmospheric nutrient deposition plays a crucial role in supplying nutrients to forests on poor soils, making it a key factor in maintaining nutrient stocks and forest productivity. We compared total atmospheric deposition in production forests of European beech (Fagus sylvatica), Douglas fir (Pseudotsuga menziesii), and Scots pine (Pinus sylvestris) by measuring bulk deposition and throughfall while accounting for canopy exchange. We assessed the differences in total deposition resulting from forest management practices such as high-thinning, shelterwood and clearcutting, on forest structure for both macronutrients and micronutrients in areas exposed to high nutrient deposition. We demonstrate that total nutrient deposition is highest in Douglas fir stands and lowest in Scots pine stands, primarily due to differences in dry deposition. The total deposition in forest exceeds national estimates because nutrient deposition in forests was higher than expected compared to more open areas. Canopy openness strongly influences the total deposition of all nutrients except phosphorus (P). Total deposition increases by a factor of 2.2 when moving from clearcuts to closed forests, with the extent of the increase varying among tree species and nutrients. Additionally, total deposition fluctuates between seasons, revealing clear seasonal patterns in both throughfall and canopy exchange. Our results suggest that the effective capture of potentially growth-limiting nutrients (such as K, Ca, Mg and Mn) in closed and thinned production forests significantly contributes to the resilience of forests on nutrient-poor, acidified soils. Our results underscore the importance of considering the effects of harvest intensity on canopy openness and forest structure and, to a lesser extent, tree species when calculating nutrient inputs from atmospheric deposition.

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cereals are among the foods rich in myo-inositol hexakisphosphate (phytic acid, IP6), lower myo-inositol phosphates (IPx), a wide range of phenolic compounds, as well as vitamins, minerals, oligosaccharides, phytosterols and para-aminobenzoic acid, and are attributed with multiple bioactivities, particularly associated with the prevention of metabolic syndrome and colon cancer. The bran fraction of wheat, maize, brown rice and other cereals contains high levels of phytate, free and total phenolics, and endogenous enzymes such as amylases, phytase, xylanase, β-glucanase and feruloyl esterase, whose activities can be increased by germination. The preliminary steps of digestion begin in the oral cavity where substrates for the action of endogenous cereal and salivary enzymes start to be released from the food matrix. IP6 released from phytate complexes with arabinoxylans, starch and protein bodies would eventually enhance the absorption of nutrients, including phenolics, by regulating tight junctions and, together with ferulic acid (FA), would maintain cell barrier integrity and epithelial antibacterial immunity. In addition, both IP6 and FA exert potent and complementary antioxidant effects, while FA together with IPx generated through advanced hydrolysis of IP6 by endogenous and microbial phytases may affect digestive enzyme activity and incretin secretion, resulting in modulated insulin and glucagon release and prevention of various diabetic complications. Contrary to widespread negative attitudes towards phytate, in this review, we present the strategy of selecting cereals with high phytate and phenolic content, as well as high endogenous phytase, feruloyl esterase and endoxylanase activities, to produce value-added health-promoting foods. The advanced hydrolysis of phytate and phenolic compounds by cereal and/or microbial enzymes would generate substantial amounts of "enzymatically generated inositol" (EGI), including IP6, IPx and myo-inositol, the compounds that, together with free FA, provide enhanced bioavailability of cereal nutrients through multiple synergistic effects not previously realised.

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.