Hydrolysis Of Phytic Acid Research Articles

Bacillus phytases: Current status and future prospects

Phytases catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol phosphates, myo-inositol (having important role in metabolism and signal transduction pathways), and inorganic phosphate. These enzymes have been widely used in animal feed in order to improve phosphorus nutrition and to decrease pollution in animal waste. Compared to previously described phytases, the phytase (PhyL) from Bacillus licheniformis ATCC 14580 has attractive biochemical properties which can increase the profitability of several biotechnological procedures (animal nutrition, humain health…etc). Due to its amino acid sequence with critical substitutions, the PhyL could be a model to enhance other phytases features, in terms of thermal stability and high activity. Otherwise, an engineered PhyL, with low pH optimum, will represent a challenge within the class of β- propeller phytases.

Phytic acid concentration in selected raw materials and analysis of its hydrolysis rate with the use of microbial phytases during the mashing process

Phytic acid present in the raw materials can complex with many compounds and therefore limit their availability to the yeast during the alcoholic fermentation process. An effective utilization of biogenic compounds bound in phytates requires a detailed analysis of the raw materials for their phytic acid content. The aim of this study was to characterize the major technological parameters for selected raw materials used in the distilling industry (maize, rye, wheat and triticale grain) and to determine the phytic acid content and the IP6/total phosphorus ratio. The phytic acid hydrolysis rate during the mashing process, with the use of microbial phytases, was analysed. The highest phytic acid concentrations (2.30 ± 0.20 mg/g dry matter) and the highest IP6/total P (80.42 ± 6.99%) were observed in the maize grain samples. Therefore, further studies on the phytic acid hydrolysis rate with the use of various phytases were conducted for the maize grain. The highest hydrolytic activity was observed for the Phytase 10000L preparation. This was the preparation that hydrolysed the phytic acid completely in up to 90 min. The application of a highly effective phytase, in ethanol production from maize grain, could lead to a more effective utilization of the biogenic compounds during the fermentation process. Copyright © 2015 The Institute of Brewing & Distilling

Enhanced cost-effective phytase production by Aspergillus niger and its applicability in dephytinization of food ingredients

Aspergillus niger grew well and secreted high phytase titres in solid state (SSF) and submerged (SmF) fermentations using sugarcane bagasse and cane molasses medium, respectively. Optimization of culture conditions increased the phytase production in both SSF and SmF. High enzymes production was recorded at 32°C and pH 4.0 after 48 h of incubation in SmF using 8% (v/v) cane molasses supplemented with ammonium nitrate (0.4% w/v). The mould secreted high enzyme titres using sugarcane bagasse in SSF at 32°C with a substrate to moisture ratio of 1 : 2.5 after an incubation of 48 h. Supplementation of sugarcane bagasse with starch and ammonium nitrate further increased phytase production. An overall 23- and 10-fold enhancement in phytase production was attained under SmF and SSF conditions, respectively. The crude enzyme was optimally active at pH 3.0 and 50°C that resulted in the hydrolysis of phytic acid in a time dependent manner. The enzyme resulted in dephytinization of wheat and gram flours with concomitant release of inorganic phosphate, soluble protein and reducing sugars. The enzyme being acid stable and thermostable could find application in dephytinization of food and feed ingredients of monogastric animals.

Preparation and Properties of Purified Phytase from Oakbug Milkcap (<i>Lactarius Quietus</i>) Immobilised on Coated Chitosan with Iron Nano Particles and Investigation of Its Usability in Food Industry

In this study, phytase enzyme was purified and characterized from Oakbug Milkcap mushrooms (Lactarius quietus) and the purified phytase enzyme was immobilized on the surface of modified chitosan with nano-Fe3O4 nanoparticles. The phytase was purified from Oakbug Milkcap mushrooms using ammonium sulphate precipitation in the range of 40-80%. and DEAE-sephadex ion-exchange chromatography. The purification fold was calculated by determining the activity and amount of protein for each step. The purified phytase enzyme was then immobilized on modified chitosan support material with Fe3O4 nanoparticles. The optimum pHs for the immobilized and free enzyme were 6.0 and the optimum temperatures were 60°C. The effects of some metal ions such as CuCl2, Hg2Cl2, FeCl2, MgCl2, ZnCl2, CaCl2 were investigated on both the bound and free purified phytase. The molecular weight of the enzyme was determined using the SDS-PAGE electrophoresis method. Finally, we investigated whether or not the immobilized and free enzyme could hydrolyzed the phytic acid in green lentils, red lentils, peas, pinto beans, beans, brass, corn, dried corn, oat, rye, wheat, broad bean, chickpeas and peanuts. It was concluded that the immobilized phytase was quite resistant to temperature, pH and metal ions and it could be safely used in the hydrolysis of phytic acid in the legumes in the food industry.

Assessment on proximate composition, dietary fiber, phytic acid and protein hydrolysis of germinated Ecuatorian brown rice.

Germinated brown rice (GBR) is considered healthier than brown rice (BR) but its nutritive value has been hardly studied. Since nutritive quality of GBR depends on genetic diversity and germination conditions, six Ecuadorian BR varieties were germinated at 28 and 34 ºC for 48 and 96 h in darkness and proximate composition, dietary fiber fractions, phytic acid content as well as degree of protein hydrolysis and peptide content were studied. Protein, lipids, ash and available carbohydrate ranged 7.3-10.4%, 2.0-4.0%, 0.8-1.5% and 71.6 to 84.0%, respectively, in GBR seedlings. Total dietary fiber increased during germination (6.1-13.6%), with a large proportion of insoluble fraction, while phytic acid was reduced noticeably. In general, protein hydrolysis occurred during germination was more accused at 28 ºC for 48 h. These results suggest that GBR can be consumed directly as nutritive staple food for a large population worldwide contributing to their nutritional requirements.

Fungal phytases: characteristics and amelioration of nutritional quality and growth of non-ruminants.

Fungal phytases are histidine acid phosphatases, a subclass of acid phosphatases, which catalyse the hydrolysis of phytic acid resulting in the release of phosphate moieties and thus mitigate its antinutritional properties. The supplementation of feed with phytases increases the bioavailability of phosphorus and minerals in non-ruminant animals and reduces the phosphorus pollution due to phosphorus excretion in the areas of intensive livestock production. Although phytases are reported in plants, animals and micro-organisms, fungal sources are used extensively for the production of phytases on a commercial scale. Phytases have been produced by fungi in both solid-state fermentation (SSF) and submerged fermentation (SmF). The fungal phytases are high molecular weight proteins ranging from 35 to 500kDa. They are optimally active within pH and temperature ranges between 4.5 and 6.0, and 45 and 70°C respectively. Phytate degradation leads to amelioration in the nutritional status of foods and feeds by improving the availability of minerals, phosphorus and proteins in non-ruminant animals and human beings and thus mitigates the environmental phosphorus pollution. Our article focuses on the role of fungal phytases in improving nutritional value of foods and feeds with concomitant increase in growth of non-ruminant animals and mitigating environmental phosphorus pollution.

Molecular and Biochemical Characteristics of Recombinant β-Propeller Phytase from Bacillus licheniformis Strain PB-13 with Potential Application in Aquafeed

Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase 'rPhyPB13' was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other β-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0-6.5 and 60 °C in the presence of 1 mM Ca(2+) and was highly active over a wider pH range (pH 4.0-8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 μmol/min/mg and 27.46 s(-1), respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca(2+) and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a β-propeller phytase that has application potential in aquaculture feed.

Analyses of Species of Phosphorus in Four Selected Streams within Abakaliki, Metropolis, Nigeria

This work investigates the speciation of phosphorus in four selected streams within Abakaliki, Nigeria using phytase hydrolysis of phytic acid as an indicator. Phytic acid is an important organophosphoruscompound that can be hydrolysed by phytases occurring in soil and water. It is the main form in which phosphorus is stored in seeds hence its availability in natural environment like water and soil. Different species of phosphorus exist in nature as a res ult of the interconversion, hydrolysis and oxidation occurring between organic phosphate molecules. Investigation of these species of phosphorus (PHP, FRP, TDP and DOP) is crucial to the understanding of the biogeochemical processes prevalent in any aquati c ecosystem. Water samples were collected from four streams (Presco campus, Ama, Udele and Iyi-okwu streams) in Abakaliki, Nigeria within a period of 5 days in October, 2012. The samples were digested with 0.04M peroxydisulphate and 0.01M H2SO 4 at 121∫C for 45 minutes in an autoclave. After Original ResearchArticle

Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsoniiZJ0702

BackgroundPhytic acid and phytates can interact with biomolecules, such as proteins and carbohydrates, and are anti-nutritional factors found in food and feed. Therefore, it is necessary to remove these compounds in food and feed processing. Phytase can hydrolyze phytic acid and phytates to release a series of lower phosphate esters of myoinositol and orthophosphate. Thus, the purification and characterization of novel phytases that can be used in food and feed processing is of particular interest to the food and feed industries.ResultsA novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702 was purified to homogeneity with a yield of 5.7% and a purification fold of 44. The molecular weight of the purified phytase obtained by SDS-PAGE was 43 kDa. The homology analysis based on N-terminal amino acid and DNA sequencing indicated that the purified phytase was different from other known phytases. The optimal thermal and pH activity of the phytase was observed at 55°C and 7.5, respectively. Seventy-three percent of the original activity of the phytase was maintained following incubation at 90°C for 10 min. The phytase was stable within a pH range of 6.0 − 8.0 and showed high substrate specificity for sodium phytate. Cu2+, Co2+, Zn2+, Mn2+, Ba2+ and Ni2+ ions were found to inhibit the activity of the phytase.ConclusionsA novel phytase purified from B. nealsonii ZJ0702 was identified. The phytase was found to be thermally stable over a wide temperature range at neutral pH. These properties suggest that this phytase is a suitable alternative to fungal phytases for the hydrolysis of phytic acid and phytates in food and feed processing industries.

Effects of inositol 1,2,6-triphosphate on human liver cancer SMMC-7721 cells

Phytic acid (myo-inositol hexaphosphoric acid) is present substantially in plant kingdom, and has striking anticancer effects. Phytic acid can be hydrolyzed into lower inositol phosphates by partial dephosphorylation during food processing. Inositol 1,2,6-triphosphate [Ins(1,2,6)P3] is the derivative of phytic acid prepared by partial hydrolysis of phytic acid with Aspergillus terreus phytase. The effects of Ins(1,2,6)P3 on the cell proliferation rate, cell cycle distribution, immunohistochemical staining of proliferating cell nuclear antigen (PCNA), and α-fetal protein (AFT) in human liver cancer SMMC-7721 cell line were investigated. Ins(1,2,6)P3 inhibited cell proliferation of SMMC-7721 cells in a dose and time-dependent manner. Ins(1,2,6)P3 induced an accumulation of cells in the G0/G1 phase of the cell cycle. AFT test and PCNA test indicated that the malignant expression of SMMC-7721 cells was reduced and the malignant cells differentiated toward normal cells. Results of these studies demonstrated that Ins(1,2,6)P3 has obvious anticancer activities.

Phytase에 의한 phytic acid의 분해 반응속도 연구

Phytases are hydrolytic enzymes that catalyze the sequential hydrolysis of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) to myo-inositols with lower numbers of phosphate groups. Two types of phytases have been identified which initiate hydrolysis of the phytic acid at either the 3- or 6- position of the inositol ring. In the present investigation, a mathematical model was proposed and computed to estimate maximum enzyme reaction rate constants which fit the experimental data obtained by other authors. Although the data points were scattered to some extent, good agreement was found between the model and the experiment data. It appears that the maximum rate constants of removal of the first, second, and third phosphate groups were not equal. Also there was neither a steady trend upward or downward in the rate constants with the stepwise hydrolysis reactions.

Purification and Biochemical Characterization of Thermostable Phytase from Newly Isolated Bacillus subtilis CF92

Bacillus subtilis CF92, an isolate from cattle feces, produces phytase, which catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates. Phytase from B. subtilis CF92 was purified via ethanol precipitation, anion-exchange chromatography, and gel filtration chromatography. Molecular weight of the purified phytase was estimated to be 46 kDa by SDS-PAGE. Purified phytase exhibited optimal activity at 60°C. The enzyme retained 40% of its original activity after 30 min incubation at 80°C. Optimum pH was 7.0, although activity remained fairly stable over pH range of 4.0 to 8.0. The enzyme was activated in the presence of EDTA and significantly inhibited by metal ions. Phytase exhibited substrate-specificity on polyphosphate compounds such as adenosine triphosphate, sodium tripolyphosphate, and sodium phytate. Km and Vmax values for sodium phytate were 0.42 mM and 4.35 μmol/min, respectively.

Chromobacterium sp. From the tropics: detection and diversity of phytase activity.

Phytases are a group of enzymes that catalyze phytic acid hydrolysis with release of phosphorus (P). The ability of Chromobacterium sp. to produce phytase was detected in 115 out of 118 candidate bacteria isolated from different Brazilian biomas. This is the first report revealing the genus Chromobacterium as phytase producer.

Heterologous expression and functional characterization of a plant alkaline phytase in Pichia pastoris

Phytases catalyze the sequential hydrolysis of phytic acid ( myo-insositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytic acid constitutes 3–5% of the dry weight of cereal grains and legumes such as corn and soybean. The high concentration of phytates in animal feed and the inability of non-ruminant animals such as swine and poultry to digest phytates leads to phosphate contamination of soil and water bodies. The supplementation of animal feed with phytases results in increased bioavailability to animals and decreased environmental contamination. Therefore, phytases are of great commercial importance. Phytases with a range of properties are needed to address the specific digestive needs of different animals. Alkaline phytase (LlALP1 and LlALP2) which possess unique catalytic properties that have the potential to be useful as feed and food supplement has been identified in lily pollen. Substantial quantities of alkaline phytase are needed for animal feed studies. In this paper, we report the heterologous expression of LlALP2 from lily pollen in Pichia pastoris. The expression of recombinant LlALP2 (rLlALP2) was optimized by varying the cDNA coding for LlALP2, host strain and growth conditions. The catalytic properties of recombinant LlALP2 were investigated extensively (substrate specificity, pH- and temperature dependence, and the effect of Ca 2+, EDTA and inhibitors) and found to be very similar to that of the native LlALP2 indicating that rLlALP2 from P. pastoris can serve as a potential source for structural and animal feed studies.

Kinetic Characterization of O-Phospho-l-tyrosine Phosphohydrolase Activity of Two Fungal Phytases

Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The Km for phosphotyrosine ranged from 465 to 590 microM as opposed to 135 to 160 microM for phytate. The Vmax, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 x 10(6) to 1.6 x 10(7) M(-1) s(-1)); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5 degrees C higher to 70 degrees C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.

Phytate degradation by Bifidobacterium on whole wheat fermentation

Whole cereal-based products have a beneficial effect on health, but they also contain high levels of phytate, defined as anti-nutrient. The possible use of different strains of bifidobacterial species (B. catenulatum, B. longum and B. breve) with phytate degrading activity as starters in the fermentation process was investigated. Fermentative parameters of doughs (pH, volume, total titrable acidity [TTA] and acetic and lactic acids production) and the hydrolysis profile of the phytic acid were determined during whole wheat dough fermentation. During fermentation in the presence of different bifidobacterial strains, the concentration of phytic acid showed a progressive decrease, leading to a higher release of hydrolysis products than in the control, within short fermentation time. According to the fermentative parameters bifidobacterial strains showed a good adaptation to the dough ecosystem. The range of TTA and lactic acid production was dependent on the strain. Among the tested bifidobacterial strains, those belonging to the species B. breve and B. longum induced a higher hydrolysis of phytic acid with simultaneous production of lower inositol phosphates. In addition, dough containing those strains had high pH and minor acidity than those containing a commercial starter (L. plantarum). Therefore, the tested bifidobacterial strains could be used as breadmaking starters contributing to different acidification degrees and promoting simultaneously the degradation of phytic acid in the whole wheat dough.

Cereal‐based biorefinery development: Integrated enzyme production for cereal flour hydrolysis

Restructuring the traditional fermentation industry into viable biorefineries for the production of fuels, chemicals and plastics is essential in order to replace (petro)chemical processing. This work presents engineering aspects of Aspergillus awamori submerged fermentation for on-site production of an enzymatic consortium that contains glucoamylase, protease and phosphatase. The crude broth filtrate was used for the production of wheat flour hydrolysates. Improvements on traditional starch hydrolysis carried out in two stages (liquefaction and saccharification) were attempted through integration of unit operations and reduction of processing temperature and reaction duration. An initial increase of temperature to 68 degrees C and a subsequent decrease to 60 degrees C for the rest of the enzymatic hydrolysis resulted in a starch to glucose conversion yield of 94 and 92% when a wheat flour concentration and commercial starch concentration of 225 g L(-1) was used, respectively. The use of crude broth filtrates resulted in the simultaneous hydrolysis of wheat protein and phytic acid, as was indicated by the increase in free amino nitrogen and phosphorus concentration, respectively.

Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP)

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1–5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares <25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.

Alkaline phytase from Lilium longiflorum: Purification and structural characterization

Phytases catalyze the hydrolysis of phytic acid ( myo-inositol hexakisphosphate), the most abundant inositol phosphate in cells. Phytases are of great commercial importance because their use as food and animal feed supplement has been approved by many countries to alleviate environmental and nutritional problems. Although acid phytases have been extensively studied, information regarding alkaline phytases is limited. Alkaline phytases with unique catalytic properties have been identified in plants, however, there is no report on the purification or structural properties. In this paper, we describe the purification of alkaline phytase from plant tissue. The purification was challenging because of contamination from non-specific phosphatases and acid phytases and low endogenous concentration. The purification of alkaline phytase from pollen grains of Lilium longiflorum involved selective precipitation by heat and ammonium sulfate followed by anion exchange and chromatofocusing chromatography and, finally, gel electrophoresis. Alkaline phytase was purified ∼3000-fold with an overall recovery of 4.2%. The native molecular mass was estimated to be in the range of 118 ± 7 kDa by Ferguson plot analysis and M r of denatured protein in the range of 52–55 kDa by SDS–PAGE suggesting that the enzyme is a homodimer. Separation by 2-D gel and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis of separated proteins indicates the presence of multiple mass and charge isoforms with p I values between 7.3 and 8.3. To our knowledge, this is the first alkaline phytase to be purified from plant sources. The unique properties suggest that the enzyme has the potential to be useful as a feed and food supplement.