Increase In Phytase Activity Research Articles

Growing pigs’ diets with increased phytase activity and reduced available phosphorus resulted in similar performance and environmental impacts

The objective of this study was to evaluate, through a life cycle assessment, the environmental impacts of the postweaning growth of pigs fed with diets with reduced levels of available phosphorus and increased phytases. In Trial I (nitrogen and phosphorus balance), 25 crossbreed barrow pigs (BP 400 × BM 500 Biriba’s®), with an average weight of 23.05 ± 3.24 kg and average starting age of 60.75 ± 4.73 days, were assigned to five dietary treatments using a randomized block design, with five replications and each animal as an experimental unit. Five experimental diets were evaluated: each increasing phytase activity units (FTU) (control [0], 250, 500, 750 and 1 000 FTU) and decreasing levels of available phosphorus (0.435, 0.375, 0.315, 0.255 and 0.195%), respectively. Due to phytase supplementation, all diets meet the requirements for available phosphorus. The pigs received two daily meals. Total collection of feces was performed, adding 2 g of ferric oxide as a fecal marker. Feces were collected daily, stored in plastic bags and frozen. All excreted urine was collected in plastic buckets containing 20 mL of HCl (1:1), and a 20% aliquot was frozen for further analysis. In Trial II (performance), 40 crossbreed barrow pigs (BP 400 × BM 500 Biriba’s®) with an initial average weight of 15.11 ± 0.67 kg, average starting age of 50.63 ± 0.81 and finishing age of 72.43 ± 2.07 days were assigned using a randomized block design to five dietary treatments, with eight replications and each animal as an experimental unit. The pigs were weighed at the beginning and at the end of the trial, as were the feed supplies and refusals. These data were used to calculate the feed conversion ratio, average daily gain and average daily feed intake. The life cycle analysis was based on the CML 2001 (baseline) method V3.02, as implemented in the Simapro software, version 8.05 (PRé Consultant, 2014). These data may further serve as a reference for the study of the impact of phytase supplementation during other breeding phases, or even in other animals whose environmental impacts are being studied.

Phytase Production by Lactobacillus plantarum A1-E in Submerged and Solid-State Fermentation

Enzyme activity is influenced by several important factors, including the amount and type of substrate, solvent type, pH, temperature, presence of inhibitory and activating ions, and concentration of enzymes. Therefore, this research aimed to evaluate phytase production from Lactobacillus plantarum A1-E using submerged (SmF) and solid-state fermentation (SSF). Phytase production was determined using SmF with fructose and sucrose as the primary carbon sources at concentrations of 4.5%, 6%, and 7.5%. Additionally, SSF was conducted using three distinct substrates, including soybean Meal, rice Bran, and pollard. The results indicated that the highest phytase activity was achieved through SSF when rice bran was used as a substrate (88.48 U/mL or 4.65 U/mg). The use of 4.5% sucrose as a carbon source in the SmF technique showed the highest specific phytase activity (4.38 U/mg) compared to other carbon sources at various concentrations. The addition of metal ions showed that Fe 2+ , Mn 2+ , and Co 2+ at concentrations of 1-5 mM, Mg 2+ and Zn 2+ at concentrations of 3-5 mM, and Ca 2+ at a concentration of 3 mM acted as activators that increased phytase activity compared to control. Meanwhile, Mg 2+ and Zn 2+ at concentrations 1-2 mM were inhibitors.

Phytase production by white-rot fungi cultivated in liquid medium with particulate agro-industrial byproducts

This study aimed to screen phytase-producing basidiomycetes by submerged cultivation (SmC) with particulate agro-industrial byproducts and evaluate the enzyme's thermal and pH stability. Five among eight fungal species produced high phytase activity (> 300 U mL-1) after seven days in SmC. Ganoderma sp., the best phytase producer (1130 U mL-1), was grown in a liquid medium with particulate agro-industrial byproducts (coffee husks, citric pulp, sugarcane bagasse, and soybean hulls). Soybean hulls increased phytase activity (16%) and productivity (2.3-fold). The residual phytase activity was determined after 120 min incubation in different pH (pH 3, 5, 7, and 9) and temperatures (30, 50, and 70 oC). Phytase activity remained stable at pH ranging from 3 to 9, with 100% activity retention after 120 min at pH 7. Furthermore, Phytase retained more than 60% activity when incubated for 120 min from 30 oC to 70 oC. Our findings support future studies using Ganoderma sp. and particulate soybean hulls for phytase production for potential use in the animal feed industry.

Phytate degradation and phosphorus utilisation by broiler chickens fed diets containing wheat with increased phytase activity

ABSTRACT 1. The objective of this study was to investigate wheat genotypes bred for increased intrinsic phytase activity for InsP6 disappearance and the formation of lower inositol phosphates in such wheat-fed broiler chickens. The influence of monocalcium phosphate (MCP) supplementation on these characteristics and the utilisation of P and Ca were also determined. A three-step in vitro assay and a broiler trial were performed. 2. In the 63 wheat genotypes tested in vitro, phytase activity varied from 1900 FTU/kg to 5200 FTU/kg, and InsP6 disappearance increased with higher phytase activity of wheat in a linear manner. The addition of MCP significantly reduced in vitro InsP6 disappearance by one-third, independent of the inclusion level of wheat in the feed. When exogenous phytase was added to wheat, in vitro InsP6 disappearance increased independently of the phytase activity of the wheat used. 3. In the broiler trial, four wheat genotypes with phytase activities between 2400 and 3700 FTU/kg were included at 400 g/kg in diets with and without MCP. The diets were not pelleted. Separately, wheat 1, without MCP, was tested with the addition of exogenous phytase. Unsexed Ross 308 broiler chickens were allocated to 72 metabolic units of 10 birds each and assigned one of the nine diets. Mineral utilisation was measured based on excreta collection from 20 to 23 d of age. Digesta from the crop and terminal ileum were collected on d 24. 4. In the crop and ileum, InsP6 disappearance was not affected by the wheat genotypes, but the addition of MCP significantly decreased InsP6 disappearance. Precaecal P disappearance was significantly reduced by the addition of MCP, with wheat genotypes also exerting an effect. Wheat genotypes and the addition of exogenous phytase significantly affected P utilisation. Exogenous phytase had no effect on InsP6 disappearance in the crop but did up to the terminal ileum, the precaecal InsP6 and P disappearance increased with the addition of exogenous phytase. 5. Although the intrinsic wheat phytase activity exerted distinct effects on in vitro InsP6 disappearance, no such effect was found in the broiler trial. The addition of MCP significantly inhibited InsP6 degradation in vitro and in vivo.

Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model

Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.

Changes in phytase activity, phosphorus and phytate contents during grain germination of barley (Hordeum vulgare L.) cultivars

Phytase activity, phosphorus and phytate contents in hydroponic fodder (up to 12 days of grain germination) of four Tunisian barley (Hordeum vulgare L.) cultivars (Arbi, Ardhaoui, Souihli, and Rihane) were studied. Barley cultivars differed significantly in their phytase activity (0.6–1.6 U/g), phosphorus (3.2–5.0 mg/g DM) and phytate (4.2–4.7 mg/g DM) contents. Phytase increased significantly up to 8 days of germination and decreased steadily thereafter. The phytase activity after 8 days of germination was increased up to tenfold in Arbi and Ardhaoui cultivars, eightfold in Souihli and fivefold in Rihane. The increase in phytase activity during germination was accompanied by a significant decrease in phytate content for all barley cultivars (reduced by 92–97%). The decrease in phytate content of sprouted barley by germination should improve phosphorus availability of the grain. Based on their phytase activity and phosphorus contents before germination, barley cultivars are ranked as Rihane > Ardhaoui > Souihli > Arbi. However, after germinating for 12 days the differences among varieties in phytase activity, phytate and phosphorus contents became non-significant. Further research is warranted to highlight the effect of germination on other nutritional attributes of hydroponic sprouted barley and its suitability as a feedstuff for farm animals.

Lead immobilization assisted by fungal decomposition of organophosphate under various pH values

Organic phosphates (OP) account for approximately 30–90% of total soil P. However, it is too stable to be utilized by plants as available P source. Aspergillus niger (A. niger) has considerable ability to secret phytase to decompose OP. Meanwhile, mineralization of lead (Pb) is efficient to achieve its remediation. This study hence investigated Pb immobilization by A. niger assisted decomposition of OP under variable acidic environments. A. niger can survive in the acidic environment as low as pH = 1.5. However, alternation of environmental pH within 3.5–6.5 significantly changed fungal phytase secretion. In particular, weakly acidic stimulation (pH of ~5.5) increased phytase activity secreted by A. niger to 0.075 µmol/min/mL, hence elevating P release to a maximal concentration of ~20 mg/L. After Pb addition, ATR-IR and TEM results demonstrated the formation of abundant chloropyromorphite [Pb5(PO4)3Cl] mineral on the surface of mycelium at pH = 5.5. Anglesite, with a higher solubility than pyromorphite, was precipitated massively in other treatments with pH lower or higher than 5.5. This study elucidated the great potential of applying OP for Pb immobilization in contaminated water.

Multifunctionalizing the marine diatom Phaeodactylum tricornutum for sustainable co-production of omega-3 long chain polyunsaturated fatty acids and recombinant phytase

There is an urgent requirement for sustainable sources of food and feed due to world population growth. Aquaculture relies heavily on the fish meal and fish oils derived from capture fisheries, challenging sustainability of the production system. Furthermore, substitution of fish oil with vegetable oil and fish meal with plant seed meals in aquaculture feeds reduces the levels of valuable omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, and lowers the nutritional value due to the presence of phytate. Addition of exogenous phytase to fish feed is beneficial for enhancing animal health and reducing phosphorus pollution. We have engineered the marine diatom Phaeodactylum tricornutum, accumulating high levels of EPA and DHA together with recombinant proteins: the fungal Aspergillus niger PhyA or the bacterial Escherichia coli AppA phytases. The removal of the N-terminal signal peptide further increased phytase activity. Strains engineered with fcpA and CIP1 promoters showed the highest level of phytase activity. The best engineered strain achieved up to 40,000 phytase activity units (FTU) per gram of soluble protein, thus demonstrating the feasibility of development of multifunctionalized microalgae to simultaneously produce industrially useful proteins and fatty acids to meet the demand of intensive fish farming activity.

Optimization of the conditions for rice bran phytate degradation by their own phytases

The rice bran by-product of rice during milling process chemically contains moisture, ash, crude protein (12%), crude fat (15%), crude fiber 7%, crude carbohydrate (31.1%), and energy. It contains high phytate and dietary fiber contents. The cereal whole paddy after milling produces 8% of rice bran. Rice bran amino acid profile has been normally suggested to be superior to cereal grain proteins. Existing phytase within the rice bran generally improves phytate phosphorus utilizations. The present study has been conducted to find the effect of phytase activity of rice bran against five different temperatures and also the effect of phytase activity of rice bran against five different temperatures at optimum pH 5.5. The results showed that as the concentration of rice bran increases from 0 to 20%, the phytase activity also increased. The optimum phytase activity was found at 10% concentration of rice bran at 37°C. Moreover, an increase in phytase activity at the same concentration of rice bran was observed while adjusting the pH at pH 5.5. The four concentrations of rice bran showed gradual increase in activity at 50°C which was directly proportional to the concentrations of rice bran and comparatively better while adjusting the pH at pH 5.5. Around two-fold increase in activity was observed at room temperature and 50°C when the concentration of rice bran was increased from 5 to 20% with and without pH 5.5 adjustments.

Effect of citrate on Aspergillus niger phytase adsorption and catalytic activity in soil

Phytase enzymes from bacteria, fungus and plant root exudates are known to hydrolyse organic phosphorus (Po) to bioavailable inorganic orthophosphate in soil. Exploiting such biochemical functions in agricultural systems, offers the potential for alternative sustainable phosphorus sources. Phytase adsorption to soil particles and phytate metal complexation has been shown to inhibit phytate (InsP6) dephosphorylation. Organic acid anions such as citrate increase phytase catalytic efficiency towards complexed forms of InsP6, but the mechanisms are poorly understood. The aim of this work was to evaluate Aspergillus niger phytase inactivation and changes in its catalytic properties upon addition to soil, as well as the effect of citrate on phytase adsorption and activity towards free, precipitated and adsorbed InsP6.We performed a series of enzyme hydrolysis activity assays when the enzymes were free, in soil solution and adsorbed to soil under varying chemical conditions, with and without citrate. A. niger phytase showed a relatively low absorption affinity for the Cambisol test soil. Phytase activity reduced by 37.3% due to adsorption. Citrate had no effect on the rate or total amount of phytase adsorption or soil adsorbed phytase activity thereafter. Free phytases and phytases in soil solution showed optimum activity (≥80%) at pH4.5–5.5. Activity decreased slightly for soil adsorbed enzymes compared to enzymes which were free or in soil solution>pH5 and <4 in the pH dependency curve. A decrease in activity was seen only at ionic strengths (NaCl)>0.6M in adsorbed and free enzymes, while activity from enzymes in soil solution reduced in all tested ionic strengths. Citrate significantly increased phytase activity towards InsP6 adsorbed to soil when the phytases were free (p≤0.003) but not in other treatments (Na, Al and Ca-phytate). These results suggest that the effect of citrate on soil InsP6 dephosphorylation is associated with the availability of the substrate (InsP6) rather than its effect on the enzyme per se. The ionic strength and pH of soil solution has been shown to impact on phytase activity, suggesting that salinity, quality of irrigation water, wetting/drying cycles and fertilisation will have discrete impacts on the activity of phytases once released in soil and thus the ability to make Po available for uptake by plants and microbes. The optimum acidic pH of A. niger also brings into question its suitability for application in many agricultural soils.

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake.

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated To events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08-1.77, 0.02-0.67 and 0.44-2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01-1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.

Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides

The production of phytase using Aspergillus niger NCIM 563 under submerged fermentation conditions was studied using protein rich chickpea flour as substrate. Employing a hybrid statistical media optimization strategy of Plackett–Burman and Box-Behnken experimental designs in shake-flasks gave an increased phytase activity from an initial 66 IU/mL in 216 h to 160 IU/mL in a reduced time of 132 h. Productivity, thus increased by 3.97 times from 7.3 to 29 IU/mL/day. Using the optimized media, the production was successfully scaled-up further and improved up to 164 IU/mL in 96 h by studies carried out employing 2 and 10-L fermenters. The enzyme supernatant was recovered using centrifugal separation of biomass and the stability of the produced phytase was tested for animal feed applications under gastric conditions. In vitro degradation studies of water soluble monocrotophos, methyl parathion and water insoluble chlorpyrifos, pesticides used extensively in agriculture was carried out. It was observed by HPLC analysis that phytase could degrade 72% of chlorpyrifos at pH 7.0, 35 °C. Comparable results were obtained with monocrotophos and methyl parathion. With chlorpyrifos at higher temperature 50 °C as much as 91% degradation could be obtained. The degradation of chlorpyrifos was further validated by spraying phytase on harvested green chilli (Capsicum annuum L) under normal conditions of pH 7.0, 35 °C and the degradation products obtained analyzed by LCMS. Thus, the present study brings out a potentially novel application of phytase for biodegradation of organophosphorus pesticides.

Transgenic expression of phytase in wheat endosperm increases bioavailability of iron and zinc in grains.

Phytate is a major constituent of wheat seeds and chelates metal ions, thus reducing their bioavailability and so the nutritional value of grains. Transgenic plants expressing heterologous phytase are expected to enhance degradation of phytic acid stored in seeds and are proposed to increase the in vitro bioavailability of mineral nutrients. Wheat transgenic plants expressing Aspergillus japonicus phytase gene (phyA) in wheat endosperm were developed till T3 generation. The transgenic lines exhibited 18-99% increase in phytase activity and 12-76% reduction of phytic acid content in seeds. The minimum phytic acid content was observed in chapatti (Asian bread) as compared to flour and dough. The transcript profiling of phyA mRNA indicated twofold to ninefold higher expression as compared to non transgenic controls. There was no significant difference in grain nutrient composition of transgenic and non-transgenic seeds. In vitro bioavailability assay for iron and zinc in dough and chapatti of transgenic lines revealed a significant increase in iron and zinc contents. The development of nutritionally enhanced cereals is a step forward to combat nutrition deficiency for iron and zinc in malnourished human population, especially women and children.

Comparison of Neurospora crassa and Neurospora sitophila for phytase production at various fermentation temperatures

Kanti A, Sudiana IM. 2016. Comparison of Neurospora crassa and Neurospora sitophila for phytase production at various fermentation temperatures. Biodiversitas 17: 769-775. There is general consensus that the presence of phytate in poultry negatively influences protein in and energy utilization by poultry, and these influences would be mitigated by augmentation of hydrolytic enzymes. The objective of this study was to evaluate phytase production by Neurospora crassa and Neurospora sitophila on solid state fermentation. The isolates were isolated from oncom Bogor. Phytase production ability was first determined on submerge fermentation (SmF) using glucose as the main C-sources, and on solid state fermentation (SSF) on media containing maize 30%, soybean 30%, and rice brand 40% w/w. Maximum enzyme activities were observed at 96 h incubation. SSF produce higher phytase than SmF. Optimum temperature for phytase production was 35°C. Highest phytase production by Neurospora crassa was 45.25 Unit per g substrate, while 40.78 Unit per g substrate was produced by Neurospora sitophila. Peptone and yeast extract were good N-sources for both isolates. Starch supplement increased phytase activity. Increased amylase activity was also observed when starch supplement was added on SSF. This study proves that Neurospora crassa and Neurospora sitophila can be used to produce phytase for better poultry nutrition. Keywords: Neurospora crassa, Neurospora sitophila, phytase, solid state fermentation

A novel phytase characterized by thermostability and high pH tolerance from rice phyllosphere isolated Bacillus subtilis B.S.46

In this study, an extracellular alkali-thermostable phytase producing bacteria, Bacillus subtilis B.S.46, were isolated and molecularly identified using 16S rRNA sequencing. Response surface methodology was applied to study the interaction effects of assay conditions to obtain optimum value for maximizing phytase activity. The optimization resulted in 137% (4.627U/mL) increase in phytase activity under optimum condition (56.5°C, pH 7.30 and 2.05mM sodium phytate). The enzyme also showed 60–73% of maximum activity at wide ranges of temperature (47–68°C), pH (6.3–8.0) and phytate concentration (1.40–2.50mM). The partially purified phytase demonstrated high stability over a wide range of pH (6.0–10.0) after 24h, retaining 85% of its initial activity at pH 6 and even interestingly, the phytase activity enhanced at pH 8.0–10.0. It also exhibited thermostability, retaining about 60% of its original activity after 2h at 60°C. Cations such as Ca2+ and Li+ enhanced the phytase activity by 10–46% at 1mM concentration. The phytase activity was completely inhibited by Cu2+, Mg2+, Fe2+, Zn2+, Hg2+ and Mn2+ and the inhibition was in a dose dependent manner. B. subtilis B.S.46 phytase had interesting characteristics to be considered as animal feed additive, dephytinization of food ingredients, and bioremediation of phosphorous pollution in the environment.

Redistribution of mineral elements in wheat grain when applying the complex enzyme preparations based on phytase

Biogenic minerals play an important role in the whole human nutrition, but they are included in the grain of the phytates that reduces their bioavailability. Whole wheat bread is generally considered a healthy food, but the presence of mineral elements in it is insignificant, because of weak phytate degradation. From all sources of exogenous phytase the most productive are microscopic fungi. To accelerate the process of transition hard mineral elements are mobilized to implement integrated cellulolytic enzyme preparation based on the actions of phytase (producer is Penicillium canescens). Phytase activity was assessed indirectly by the rate of release of phosphate from the substrate. It has been established that the release rate of the phosphoric acid substrate is dependent on the composition of the drug and the enzyme complex is determined by the presence of xylanase. The presented experimental data shows that a cellulase treatment of the grain in conjunction with the β-glucanase or xylanase leading to an increase in phytase activity could be 1.4 - 2.3 times as compared with the individual enzymes. As a result of concerted action of enzymes complex preparation varies topography grain, increase the pore sizes in seed and fruit shells that facilitate the penetration of the enzyme phytase in the aleurone layer to the site of phytin hydrolysis and leads to an increase in phytase activity. In terms of rational parameters of enzymatic hydrolysis, the distribution of mineral elements in the anatomical parts of the grain after processing complex enzyme preparation with the help of X-ray detector EMF miniCup system in a scanning electron microscope JEOL JSM 6390 were investigated. When processing enzyme preparation wheat trend in the distribution of mineral elements, characteristic of grain - the proportion of these elements in the aleurone layer decreases, and in the endosperm increases. Because dietary fiber and phytate found together in the peripheral layers of fiber-rich grains, it is difficult to separate the effects of degradation processes nonstarch polysaccharides and fiberphytate redistribution of polyvalent metal ions. However, studies have shown that phytase - an effective mechanism for regulating mineral nutrient diet. Application of phytase in grain bakery technology will increase the biological value of the product.

Plant uptake of phosphorus from sparingly available P- sources as affected by Trichoderma asperellum T34

The contribution of Trichoderma asperellum T34 to the plant uptake of phosphorus (P) from sparingly phytoavailable forms such as insoluble calcium (Ca) phosphates and phytates was studied. Two experiments with cucumber (Cucumis sativus L.) on siliceous sand were performed involving two factors, namely: (i) P source, viz., KH2PO4, phytate (Ins6P), and phosphate rock (PR), and (ii) inoculation with T34. Liquid pure cultures of T34 were also used. T34 increased the total content in P of cucumber roots irrespective of the particular P form and enhanced total P uptake by plants with P supplied as Ins6P or PR. The increased phytase activity observed with T34 contributes to explain its favourable influence on the uptake of P supplied as Ins6P. Solubilization of Ca phosphates from PR was favoured by the slightly acidifying effect and the increased organic anion concentration promoted by the fungus in the plant growth media. It can be concluded that T34 can improve P nutrition in plants grown on media containing phytates or insoluble Ca phosphates as dominant P forms.

Phytase-Producing Bacteria from Extreme Regions in Indonesia

In this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genusBacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity toB. cereus, while isolate MC8 exhibited 99% sequence identity toB. aryabhatti and D6 99% sequence identity toB. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

Optimization of Phytase Production from Escherichia coli by Altering Solid-State Fermentation Conditions

Cultivation of Escherichia coli on wheat-bran substrate under various Solid-State Fermentation (SSF) conditions was evaluated for phytase yield along with the enzyme activity profile as a potential, low-cost alternative to submerged-liquid fermentation. The maximum phytase activity achieved by E. coli was 350 ± 50 SPU of phytase activity per gram of bran, incubated for 96 h with a substrate bed moisture content of 70% (w/v) at 37 °C with a relative air humidity of 90%, and supplemented with 10% (w/w bran) Luria-Bertani broth powder which translates into a 300% increase in phytase activity compared with an un-supplemented culture. The greatest improvements in phytase yield were associated with nutrient supplementation and the optimization of initial substrate moisture content. E. coli production of phytase utilizing solid-state fermentation technology was shown to be feasible utilizing the low-cost agro-residue wheat bran as substrate. Furthermore, the effect of pH and temperature on phytase activity was monitored from pH 2.5 to pH 7.5, and for temperatures ranging from 20 °C to 70 °C. Optimal phytase activity was at pH 5.5 and 50 °C when produced under the SSF optimized conditions.

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria.

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria.