Immobilization Of Phytase Research Articles

Enhanced production and immobilization of phytase from Aspergillus oryzae: a safe and ideal food supplement for improving nutrition.

Microbial phytases are potentially excellent candidates for eliminating anti-nutrient i.e. phytic acid, due to hydrolysis of phospho-monoester linkages present in the phytic acid. An average 2.29-fold increase in phytase production was obtained after statistical optimization in solid-state fermentation. Aspergillus oryzae SBS50 phytase was immobilized on a Ca-alginate matrix with an effectiveness of 53%. Immobilized-phytase retained > 50% activity after recycling for five cycles and also displayed more stability in the presence of organic solvents, metal ions, and detergents as compared to free enzyme. Values of Km and Vmax of immobilized phytase were recorded as 0.66 mM and 666.6 nmol/sec, respectively. Immobilized phytase efficiently hydrolyzed the phytate contents in wheat and pearl millet flours, exhibiting > 70% catalytic activity even after three cycles. Phytase supplementation resulted in the improved nutritional quality of these flours. Furthermore, the safety assessment of the treated and untreated samples reveals the absence of any aflatoxin in the phytase produced by the mould. The results revealed the improved stability of phytase after immobilization and as a safe and significant additive for application in the food industry.

Alginate Particles for Enzyme Immobilization Using Spray Drying.

Enzymes are important catalysts for biological processes due to their high catalytic activity and selectivity. However, their low thermal stability limited their industrial applications. The present work demonstrates a simple and effective method for enzyme immobilization via spray drying. Alginate was used as a support material. Phytase, an important enzyme in the animal feed industry, was selected to study the effect of enzyme immobilization using alginate particles on its thermal stability. The physicochemical properties of alginate particles such as size, surface morphology, and heat resistance were studied. Successful immobilization of phytase was confirmed by confocal microscopy, and the immobilized phytase retained 58% of its original activity upon heating at 95 °C, compared to 4% when the alginate support material was absent. Phytase was released promptly in a simulated gastrointestinal tract with >95% of its original activity recovered. The spray drying method for phytase immobilization is scalable and applicable to other enzymes for various applications.

Poly(vinyl alcohol)-based Electrospun Nanofibers: Characterization and Phytase Immobilization

The aim of this study is to immobilization phytase obtained from cowpea seeds into nanofiber-based on poly(vinyl alcohol) (PVA) and to investigate kinetic properties, optimal pH, and temperature of free and immobilized phytase. The structural analysis and morphological properties of the nanofibers are carried out via SEM and XRD. The results indicated that enzyme stability, pH, and thermal stability are increased after immobilizing phytase into the nanofiber. The optimum pH and temperature of the free and immobilized phytase are found as pH 5.0 and 45-65 oC, respectively. These results indicated that the immobilized phytase could be a good candidate for agriculture, animal feed, food, and medical applications.

Modification of zeolite with metallic ions improves the immobilization of phytase

Phytase is an enzyme applied in several fields, but its wider use is still limited due to its high cost and poor stability, which can be overcome using immobilization techniques. Zeolites are promising support materials for enzyme immobilization, due to their attractive properties, including the capacity for modification using a simple ion exchange procedure, enabling better chemical interaction with proteins. In this work, investigation was made of the immobilization of phytase onto zeolites modified with metallic ions (Ag, Cd, Zn, Cu, Ca, Mg, and Ni). The zeolite modified with nickel reached immobilization yield of 66%, while the recovered activity (150%) indicated activation of the catalytic activity due to the presence of the metallic ion. The immobilized phytase showed excellent performance under conditions of acidic pH, reaching a relative activity up to 7 times higher than observed for its free form. Furthermore, it was possible to recover and reuse effectively the zeolite-Ni/Phytase derivative in at least 4 consecutive cycles of phytate hydrolysis reaction. The phytase immobilized onto zeolite modified with nickel presented improved properties, expanding the range of potential applications of this important enzyme. These findings demonstrate an efficient and simple protocol for enzyme immobilization onto zeolites modified with metallic ions.

Improved thermal stability of phytase from Yersinia intermedia by physical adsorption immobilization on amino-multiwalled carbon nanotubes.

Phytase is used in poultry diets to hydrolyze and release of phytate-bound phosphorus. Immobilization on nanomaterials optimizes enzyme's thermal stability and reusability. This study aimed to immobilize the recombinant phytase from Yersinia intermedia on the surface of amino-multi-walled carbon nanotubes (amino-MWCNTs) by physical adsorption. For this, zeta potential measurement, FTIR spectroscopic analysis, scanning electron microscope (SEM), kinetic as well as thermodynamic parameters were used to characterize immobilized phytase on amino-MWCNTs. According to results, the optimum temperature of the immobilized phytase increased from 50 to 70°C and also thermal and pH stability improved considerably. Moreover, immobilization led to an increase in the value of Km and kcat from 0.13 to 0.33mM and 2220 to 2776s-1, respectively. In addition, the changes in activation energy of thermal inactivation (ΔE#a (D)), the free energy of thermal inactivation (ΔG#D) and the enthalpy of thermal inactivation (ΔH#D) for immobilized phytase increased by +11.05, +24.7 and +11.4kj/mole, respectively, while the value of the change in the entropy of thermal inactivation (ΔS#D) decreased by -0.04 kj/mole.K. Overall, our results showed that adsorption immobilization of phytase on amino-MWCNTs increases thermal, pH and storage stability as well as some of kinetic parameters.

Immobilization of Phytase from Rice Bean (Vigna umbellata Thunb.) on Glutaraldehyde Activated Chitosan Microspheres

Immobilization of Phytase from Rice Bean (Vigna umbellata Thunb.) on Glutaraldehyde Activated Chitosan Microspheres

Immobilization of phytase on zeolite modified with iron(II) for use in the animal feed and food industry sectors

Here, we investigated the feasibility of immobilizing phytase onto zeolite modified with iron(II) ions, for application in the feed and food sectors. Zeolites have excellent physical-chemical characteristics, ion exchange capacity, and can act as dietary supplements. The phytase immobilization yield was up to 73 % at pH 7 and ionic strength of 100 mM, with recovered activity higher than 100 %, representing a gain in catalytic activity. The immobilized phytase could be reused, retaining 80 % of its activity in the seventh cycle of phytate hydrolysis, indicating its potential for use in industrial processes. For use in animal feed, the immobilized phytase hydrolyzed the phytate of soybean meal and wheat bran, retaining >50 % of its activity in gastric simulation tests with pH varying from 2 to 7. The results highlighted the innovative combined benefits of phytase, zeolite, and iron for animal nutrition. For application in the food industry, the immobilized phytase could hydrolyze the phytate in soybean milk and be reused in the reaction, retaining ∼50 % of its catalytic activity after 6 cycles. These results indicated the excellent potential of zeolite/iron-immobilized phytase for use in animal feed and the food industry.

Fabrication of PVA-chitosan-based nanofibers for phytase immobilization to enhance enzymatic activity

In the present paper, polyvinyl alcohol (PVA)-chitosan (CS) nanofibers were fabricated by using the electrospinning method under a constant voltage (23 kV) and the distance (12 cm). Phytase was purified from cowpea seeds and it was immobilized into the PVA-CS nanofibers. The effects of the electrospinning parameters such as enzyme concentration and flow rate on the catalytic activity of the free and immobilized phytase were also studied. The fabricated nanofibers were characterized via FT-IR, SEM, and XRD techniques. PVA-CS nanofiber and phytase containing nanofiber (phytase-PVA-CS) were exhibited a homogenous morphology with an average diameter of about 43.6 ± 13.7 and 65.3 ± 26.0 nm and fiber diameter of nanofiber containing phytase was increased after enzyme immobilization into PVA-CS nanofiber. The optimum pH and temperature of the free and immobilized phytase were also investigated. The obtained results showed that the optimum pH and temperature values of the immobilized enzyme were shifted to higher pH and temperature values after the immobilization of phytase into PVA-CS nanofiber. According to these results of the immobilized phytase, it could be used in some industrial applications such as animal feed, agriculture, pharmaceutical area, and food industry.

Langmuir and Langmuir-Blodgett Films of Poly[(9,9-dioctylfluorene)-co-(3-hexylthiophene)] for Immobilization of Phytase: Possible Application as a Phytic Acid Sensor.

In this work, the copolymer poly[(9,9-dioctylfluorene)-co-(3-hexylthiophene)] was employed as a matrix for immobilizing phytase, aiming at the detection of phytic acid. The copolymer was spread on the air-water interface forming Langmuir monolayers and phytase adsorbed from the aqueous subphase. The interactions between the copolymer and the enzyme components were investigated with surface pressure and surface potential-area isotherms, Brewster angle microscopy, and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). The enzyme could be incorporated in the monolayers from the aqueous subphase, expanding the copolymer films and maintaining its secondary structure. The polymeric films presented a morphological heterogeneous pattern at the air-water interface because of the ability of their chains to fold and entangle, causing inherent defects in the organization as well as unbalanced lateral distribution at the air-water interface because of the formation of aggregates. The interfacial films were transferred to solid supports as Langmuir-Blodgett films and characterized by PM-IRRAS and scanning electronic microscopy, which showed not only the co-transfer of the enzyme but also the maintenance of their heterogeneous morphological pattern. The enzymatic activity of the blended film was analyzed by UV-vis spectroscopy and allowed the estimation of the value of the Michaelis constant (13.08 mM), demonstrating the feasibility of the system to selectively detect phytic acid for biosensing purposes.

Correction to: Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

Inthe original version of this article, under Calculation of Immobilization Parameters heading, the presentation of the equations are incorrect. The correct presentation of the equations are given below.

Phytase Immobilization on Hydroxyapatite Nanoparticles Improves Its Properties for Use in Animal Feed.

The enzyme phytase has important applications in animal feed, because it favors the bioavailability of phosphorus present in phytate, an antinutritional compound widely found associated with plant proteins. However, for feed applications, the phytase must withstand high temperatures during the feed pelleting process, as well as the gastrointestinal conditions of the animal. This work evaluates the feasibility of immobilizing phytase on hydroxyapatite (HA) nanoparticles, in order to improve its properties. HA is a material with excellent physicochemical characteristics for enzyme immobilization, and it can also act as an inorganic source of phosphorus and calcium in animal feed. The strong affinity of the phytase for the support resulted in rapid adsorption, with total immobilization yield and recovered activity greater than 100%. After immobilization, the phytase showed a broader activity profile in terms of pH and temperature, together with considerably higher thermoresistance at 80 and 90°C. As a proof of concept, it was shown that the phytase immobilized on HA presented good resistance to acidic conditions and resistance to proteolysis when passing through simulated gastrointestinal conditions of fish. The findings showed that phytase immobilized onto HA presents suitable properties and has great potential for use in animal feed.

Covalent immobilization of phytase on the multi-walled carbon nanotubes via diimide-activated amidation: structural and stability study

Phytase is the most widely used feed enzyme in the world. This enzyme inactivates under pelleting conditions such as temperatures above 80 °C. The present study reported covalent attachment of phytase onto functionalized multi-walled carbon nanotubes (F-MWNT). The optimum enzyme loading and immobilization efficiency were 110 µg/mg of functionalized multi-walled carbon nanotubes and 62%, respectively. Characterization, structural changes and immobilization of phytase were studied by scanning electron microscopy (SEM), circular dichroism (CD), zeta potential measurement and Fourier transform infrared (FTIR) spectroscopy. Immobilized phytase exhibited improved stability towards temperature than the free phytase. The free phytase retain 3% and 27% of relative activity at 90 and 80 °C, respectively after 2 min of incubations. While immobilized phytase retained about 33% and 51% at the same condition. Also the results showed that the presence of small quantities of NaCl (<1 M) in the reaction media increased enzyme activity of immobilized phytase up to 78% but free phytase activity was not significantly changed until 1 M NaCl which increased by 30%. Higher concentrations of 1 M NaCl drastically reduced both free and immobilized phytase activities.

Investigation of the effects of starch on the physical and biological properties of polyacrylamide (PAAm)/starch nanofibers

Here, we report the development of a new polyacrylamide (PAAm)/starch nanofibers’ blend system and highlight its potential as substrate for efficient enzyme immobilization. PAAm was synthesized and blended with starch. The final blend was then electrospun into nanofibers. The response surface methodology was used to analyze the parameters that control nanofiber’s diameter. Electrospun mat was then modified either by cross-linking or phytase immobilization using silane coupling agent and glutaraldehyde chemistry. Physico-chemical properties of blends were investigated using spectroscopic and thermal studies. The evaluation of immobilized enzyme kinetics on both pure and the starch blended PAAm nanofibers was performed using Michaelis–Menten kinetic curves. Fourier transform infrared spectroscopy results along with differential scanning and X-ray diffraction confirmed that blending was successfully accomplished. TGA analysis also demonstrated that the presence of starch enhances the thermal degradability of PAAm nanofibers. Finally, it was shown that addition of starch to PAAm increases the efficacies of enzyme loading and, therefore, significantly enhances the activity as well as kinetics of the immobilized enzyme on electrospun blend mats.

Protected activity of a phytase immobilized in mesoporous silica with benefits to plant phosphorus nutrition

Phosphorus (P) is one of the main nutrients required for plant growth and is absorbed and metabolized in the form of orthophosphate ions (Pi). In agriculture, P is a major component of fertilizers. Forecast shortages in P mine stocks within the coming decades call for alternative sources of this element for agricultural use. In the present study, we explore the possibility of incorporating phytase near the plant root system. Phytase is an enzyme capable of mobilizing the main organic form of P as phytate, which can represent up to 90 % of the total P stock in soils. We report the immobilization of phytase in KIT-6 silica mesoporous materials with large pores of 8.6 nm. The enzymatic activity is localized in the porous network, and the main kinetic features of the free phytase appear to be maintained within the functional material (pHopt = 5–5.5 and T opt = 55 °C). Most importantly, the enzyme immobilized in the pores shows a higher temperature stability and appears to be protected from protease degradation. Finally, phytase-loaded KIT-6 proved efficient at hydrolyzing phytate and delivering inorganic P to growing Medicago truncatula plants, which accumulated this newly available P in both roots and shoots.

Fabrication of phospho-phytase/heteroatomic hierarchical Fe-ZSM-5 zeolite (HHFeZ) bio-conjugates for eco-sustainable utilization of phytate-phosphorus

The originally fabricated phytase/HHFeZ bio-conjugates could appropriately enhance enzyme stabilization after heteroatomic hierarchical Fe-ZSM-5 zeolite (HHFeZ) was developed as a novel matrix for affinitive immobilization of Escherichia coli (E. coli) phytase. The immobilization strategy was primarily dependent on the affinitive coordination between heteroatomic iron atoms anchored into the HHFeZ frameworks with multiple sites of phosphorylation in phytase. In addition, the ordered multiporous structures of HHFeZ contributed to phytase immobilization and stabilization. The conditions for phytase immobilization were optimized under a solution pH of 4.5 in a batch mode, and the maximum phytase immobilization capacity was determined to be 2.01 mg mg−1. Compared to those of free phytase, the thermal and proteolytic resistance of phytase/HHFeZ bio-conjugates were effectively improved, and the enzymatic activity was maintained over a broad pH range. The phytase/HHFeZ bio-conjugates would be an innovative choice for eco-sustainable exploitation and utilization of phytate-phosphorus, especially with respect to eutrophication control.

Polypyrrole/phytase amperometric biosensors for the determination of phytic acid in standard solutions

Amperometric biosensors based on the physical immobilization of phytase (PhyA) into polypyrrole (PPy) films were prepared in aqueous medium. The PPy/PhyA films were characterized by cyclic voltammetry, and surface and structural characterization techniques, SEM and FTIR. Both voltammetric and amperometric transduction methods were used in order to detect phytic acid in acetate buffer at pH 5.5 at room temperature. The biosensors exhibited a detection limit of 0.15 mmol L −1 and a linear range of phytic acid content from 0.5 to 2.0 mmol L −1, which are adequate values for typical analyses of phytic acids in most seeds, grains, and vegetables.

Activity stabilization of Aspergillus niger and Escherichia coli phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays

The aim of this work was to study the stabilization of the activity of two commercial microbial phytases (Aspergillus niger and Escherichia coli) after immobilization on nanoclays and to establish optimal conditions for their immobilization. Synthetic allophane, synthetic iron-coated allophanes and natural montmorillonite were chosen as solid supports for phytase immobilization. Phytase immobilization patterns at different pH values were strongly dependent on both enzyme and support characteristics. After immobilization, the residual activity of both phytases was higher under acidic conditions. Immobilization of phytases increased their thermal stability and improved resistance to proteolysis, particularly on iron-coated allophane (6% iron oxide), which showed activation energy (E(a)) and activation enthalpy (ΔH(#)) similar to free enzymes. Montmorillonite as well as allophanic synthetic compounds resulted in a good support for immobilization of E. coli phytase, but caused a severe reduction of A. niger phytase activity.

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60°C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65°C. The Km and Vmax values were measured as 5mM and 0.63U/mg for free enzyme and 12.5mM and 0.71U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7h at 60°C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.

Production, purification and properties of microbial phytases

Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyse the release of phosphate from phytate (myco-inositol hexakiphosphate). Several cereal grains, legumes and oilseeds, etc., store phosphorus as phytate. Environmental pollution due to the high-phosphate manure, resulting in the accumulation of P at various locations has raised serious concerns. Phytases appear of significant value in effectively controlling P pollution. They can be produced from a host of sources including plants, animals and micro-organisms. Microbial sources, however, are promising for their commercial exploitations. Strains of Aspergillus sp., chiefly A. ficuum and A. niger have most commonly been employed for industrial purposes. Phytases are considered as a monomeric protein, generally possessing a molecular weight between 40 and 100 kDa. They show broad substrate specificity and have generally pH and temperature optima around 4.5–6.0 and 45–60°C. The crystal structure of phytase has been determined at 2.5 Å resolution. Immobilization of phytase has been found to enhance its thermostability. This article reviews recent trends on the production, purification and properties of microbial phytases.

Aspergillus ficuum Extracellular Phytase Immobilization on Glutaraldehyde‐activated Silicate

Technique d'immobilisation de l'enzyme 3-phytase de Aspergillus ficuum, et construction d'un bioreacteur a lit de garnissage. Caracteristisation des cinetiques de l'enzyme immobilisee