Phytase Activity Research Articles

Processing Raw Rice Bran to Potential Functional Food for Human Consumption

Background: Rice bran despite its nutritious value is largely underutilized. It is partly used for edible oil extraction and as animal feed or discarded as garbage. The present study was carried out with the objective of developing rice bran into a functional food ingredient for human consumption. Methods: The collected rice bran was exposed to four different treatments that included application of edible acid for activation of phytase, followed by drying at different temperatures to inactivate lipase. Standard biochemical methods were used to analyze the nutrients, anti nutrients and acid value. Result: Processing led to the reduction of phytic acid up to 43.6% and acid value up to 85.2% in comparison to raw rice bran (control) together with observation of absence of microbial load. Moreover, the processing led to the development of potential functional product with the better retention of ash, fiber, phenolic compound and crude protein content of the raw rice bran. The organoleptic evaluation revealed that the processed product can be utilized as ready-to-cook food ingredient, which might help to improve nutrition.

Towards Improved Bioavailability of Cereal Inositol Phosphates, Myo-Inositol and Phenolic Acids

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.

Insights into the morphology-productivity relationship of filamentous fungi through small-scale cultivation and automated microscopy of Thermothelomyces thermophilus.

Filamentous fungi are a cornerstone in the biotechnological production of enzymes, proteins, and organic acids. However, challenges in understanding and controlling the relationship between morphology and productivity can limit their application. This study addresses these challenges using Thermothelomyces thermophilus, a promising thermophilic fungus known for the production of thermostable enzymes. We investigated the effects of environmental conditions on fungal morphology and enzyme production using a combination of microbioreactor cultivation, automated liquid handling, and automated microscopy. Specifically, batch and fed batch cultivations were performed at different pH levels and glucose feeding rates to study their effects on secretory phytase production, fungal growth, and morphology. Results from batch cultivations revealed a two-fold higher phytase activity at pH 5.5 compared to pH 6.5, with notably smaller fungal fragments at the end of cultivation. Conversely, fed batch cultivations at a feeding rate of 1 g (l h)-1 glucose showed a 1.6-fold higher enzyme activity at pH 5.5, accompanied by much larger fungal aggregates throughout the feeding phase. These findings suggest that large aggregates are associated with high productivity; however, their breakdown further enhances enzyme release, increasing activity in the supernatant. This study not only provides insights on the morphology-productivity relationship of T. thermophilus, but also demonstrates the efficacy of integrating microbioreactors with automated microscopy. This methodology represents a significant advance in the field of fungal biotechnology, paving the way for more efficient industrial bioprocesses.

VETOZIM MULTI - AN INNOVATIVE SOLUTION FOR THE POULTRY INDUSTRY

The materials of the article describe the role and purpose of enzymes in feed production, in particular for poultry farming. The composition of the multienzyme preparation Vetozym Multi from the company "Vetsintez" is characterized, which contains a complex of enzymatic activities: glucanase, xylanase, cellulase, protease, amylase, mannanase, phytase. The practical results of using the multienzyme preparation Vetozym Multi as part of compound feeds for broilers are presented. The recipes of the compound feed Grower for broilers are calculated and the optimal rate of enzyme introduction is established in the amount of 100 g per 1 t. The percentage of microcomponents introduced into the compound feed Grower for broilers is compared and the nutritional indicators of compound feed without and with the use of the multienzyme preparation Vetozym Multi are calculated. It was established that the introduction of the enzyme allows to reduce from 20 to 30% monocalcium phosphate in the composition of the recipe, due to the existing phytase activity. Vetozym Multi significantly increases the efficiency of feed use, an increase in average daily weight gain of broilers by 1..2.5 g is observed, the conversion rate of compound feed for poultry is reduced by 0.02…0.07 and the cost of recipes is reduced by 300-500 UAH/t, which became possible due to the optimization of the use of sunflower and grain products with a high fiber content. The presence of thermostable enzymes in the composition of the Vetozym Multi preparation allows it to be used in the production of compound feeds with advanced heat treatment at a conditioning and granulation temperature of up to +90 °C, which will contribute to a more complete breakdown and assimilation of raw material components, in particular carbohydrates, proteins and fiber. Calculation of economic indicators shows that the drug Vetozym Multi not only provides balanced nutrition for poultry, but also allows you to reduce the cost of feed due to more efficient use of less expensive components.

Screening of agroresidues for economical and sustainable Phytase Production by Bacteria

Phytase, a phytate hydrolysing enzyme has substantial application in food, feed and agriculture sector for improvising the availability of phosphorus and reduces the environmental pollution. The present study emphasizes on cost effective production of bacterial phytase. Total 25 agroresidues were screened for economic production of phytase using bacteria. A total of 30 bacterial isolates were obtained from poultry and goat shed soil and litter sample showing highest phytase activity. The efficient isolates SP-46 and SP-52 were screened for selection of efficient combination bacterial isolate and agroresidues for cost-effective production of phytase. The results showed that utilization of agroresidues not only serve as efficient substrate but also contribute to sustainable agro-waste valorisation and reduces need of expensive synthetic media for phytase production.

Fungal Phytase Increased Ileal and Total Tract Digestibility of Phosphorus of Cold-Pressed Canola Cake and Canola Meal Diets in Growing Pigs.

The co-product canola meal contains little fat and has been used in swine production as a protein source for several decades. More recently, locally produced canola cake has also become available that contains important quantities of residual oil. Both canola co-products contain a considerable quantity of phosphorus (P) with low availability. Phytase supplementation can increase P digestibility in canola meal but its efficacy remains unclear for canola cake containing high residual oil. To explore whether phytase supplementation can increase digestibility of P and other nutrients in canola cake, four diets containing 500 g of locally produced cold-pressed canola cake or canola meal, with or without supplemental phytase (Ronozyme HiPhos; Aspergillus oryzae origin)/kg, were fed to seven pigs (initial body weight; 55.9 ± 3.8 kg) in four 9-day periods following a 4 × 4 Latin square plus a 4 × 3 Youden square. Phytase supplementation increased phytase activity from below the detection limit to reach a minimum of 1000 phytase units/kg in the canola meal and cake diets; respectively. Phytase supplementation increased (p < 0.05) coefficients of ileal and total tract digestibility of P by 60% and 46% for canola meal and 76% and 57% for canola cake, respectively, but did not affect the digestibility of crude protein or gross energy. Ileal or total tract digestibility of P did not differ between the canola cake and canola meal diets in growing-finishing pigs. In conclusion, fungal phytase is effective in increasing the P digestibility of canola cake similar to in canola meal fed to growing-finishing pigs. The similar ileal or total tract digestibility of P in canola meal and cake diets either with or without phytase supplementation indicated that the residual oil in canola cake did not affect P digestion and did not affect the efficacy of phytase in increasing P digestibility.

Effect of Different Colours of Light on Chosen Aspects of Metabolism of Radish Sprouts with Phosphoromic Approach.

Among various environmental factors, light is a crucial parameter necessary for the germination of some seeds. Seed germination is an important phase in the plant life cycle, when metabolic activity is resumed and reserves are mobilized to support initial plant development. Although all nutrients are extremely important for proper physiological and biochemical development of plants, phosphorus (P) seems to play a special role, as it is an essential component of all important structural and functional substances which compose the cells of plants. We believe that transformations of the forms of phosphorus accompanying metabolic activity of germinating seeds determine the efficiency of this process, and thus the seedling's metabolic status. Therefore, we decided to study the changes in the major phosphorus-containing substances in radish sprouts during the first phase of growth. The effect of different colours of light on the quality parameters in radish, as a model plant, during germination, was evaluated. Contents of Pi, adenylates, antioxidants, pigments, phytase activity, and 31P NMR phosphorus profile were investigated. Based on the results of our study, we may propose the phosphoromic approach as an important metabolic parameter determining the physiological status of the plant.

Degradation of Anti-Nutritional Factors in Maize Gluten Feed by Fermentation with Bacillus subtilis: A Focused Study on Optimizing Fermentation Conditions

Maize gluten feed is rich in micronutrients and serves as a good source of protein and dietary fiber, but also contains anti-nutritional factors. In this study, fermentation conditions for the degradation of phytic acid and water-unextractable arabinoxylans in maize gluten feed using Bacillus subtilis were optimized. Key variables influencing the fermentation process were identified from seven potential parameters using the Plackett–Burman design. Three statistically significant factors, i.e., fermentation time, inoculum dose, and material-to-liquid ratio were further optimized through a central composite design and the efficiency of fermentation conditions was predicted. The accuracy of the predicted model was validated by subsequent experimentation. The optimum fermentation conditions were determined to be a fermentation time of 84.5 h, inoculum dose of 17.1%, and material-to-liquid ratio of 1:3.4. Under these conditions, 48% of phytic acid and 32% water-unextractable arabinoxylans were degraded. Following fermentation, the activities of protease, xylanase, phytase, and cellulase in maize gluten feed were significantly increased (p &lt; 0.001), contributing to the breakdown of phytic acid and water-unextractable arabinoxylans, which improved the protein dispersibility index, in vitro protein digestibility, and mineral bioavailability. These findings suggest that fermenting maize gluten feed with Bacillus subtilis is a practical and effective approach to reducing anti-nutrients and enhancing its nutritional quality.

Selection and immobilization of the optimal phytase-producing probiotic: a comparative in silico analyses of five autochthonous lactic acid bacteria

Phytic acid and its salts are the major storage form of both phosphate and myo-inositol, especially in cereals. Phytic acid chelates multivalent cations, which decreases the solubilized form of these nutrients and their dietary bioavailability for absorption and assimilation. Increasing micronutrient bioavailability from the economic phytate-rich food can be achieved by subjecting this food to fermentation by phytase-producing bacteria. A total of 8 lactic acid bacteria were isolated from different fermented food samples. Among the bacteria tested for their ability to produce phytase, only five isolates were selected as promising producers. The results revealed that isolate No. 4 produced the highest phytase levels of 411 U/mL. The five isolates were differentially able to tolerate acidic, alkaline, heat, surfactant, osmotic, bile, and pancreatic enzyme stresses, with the superiority of isolate No. 4, which demonstrated the most desirable probiotic potentials as confirmed by principal component analysis (PCA), heat map, and network analysis. Isolate No. 4 was identified by 16S rRNA sequencing and later confirmed as Pediococcus pentosaceus strain NMP4762Ch under the accession number MZ413646. Moreover, the active cells of P. pentosaceus were immobilized in alginate; the reusability of the immobilized active cells resulted in continuous production with an optimum phytase activity of 432.0 U/mL after 14 days, which decreased with time to reach 142.5 U/mL after 56 days. The maximum shelf stability was also observed, 557.5 U/mL, after 14 days and declined with time, reaching 133.5 U/mL by the end of the 56th day. Since humans don’t naturally produce phytase, utilizing probiotics for phytase production is important to combat mineral deficiencies.

Long story from past to present: calcium, phosphorus, and phytase

Abstract Calcium (Ca) and phosphorus (P), two main elements, have vital physiological and metabolic roles in animal bodies. Accurate comprehension of the interaction of these two elements and their value in various resources helps to obtain their optimal formulation in poultry diets. Hence, in previous studies, the hormonal axes controlling Ca and P homeostasis have been primarily investigated. However, to estimate Ca and P requirements in modern broiler chickens, in addition to growth performance, other parameters such as Ca and P digestibility, bone strength, and excretion into the environment should also be considered. Since a large amount of P in poultry feed ingredients is bound to phytate, phytases are added to poultry diets to release the P from phytate. However, many nutritionists need clarification on what dose of dietary phytase is required to release the maximum phytate P and how phytase activity will be optimized. Therefore, the present review study has attempted to explore the factors that affect the digestibility of different resources of Ca and P. In addition, the effect of excess dietary Ca on phytase activity and studies related to super dosing of phytase in broilers are provided. Finally, the values of phytate P in standard poultry feed ingredients and the latest update of the studies on determining Ca and P requirements are summarized.

Assessment of Potential Probiotic Yeasts Isolated from The Small Intestines of Cattle in Thailand

Y easts are widely studied and considered good candidates for probiotics due to their numerous benefits. This study aimed to investigate potential probiotic attributes of yeasts isolated from the small intestines of cattle in Thailand. Growth at body temperature, resistance to low pH, auto-aggregation capability, hydrophobicity, growth under gastrointestinal tract (GIT) conditions, safety profiles, antimicrobial activity, and extracellular enzyme production of yeast strains were evaluated in vitro. The results of sequential screening indicated that 829 out of 1,025 yeast strains could grow at both 37 and 39 °C, 682 strains could resist acidity at pH 2 for 24 h, 252 strains revealed greater than 83% auto-aggregation capability, and 97 strains showed higher than 90% hydrophobicity. Sixty strains grew well under simulated GIT conditions in the presence of pepsin, pancreatin, and bile salts. Thirteen strains of Diutina rugosa showed negative results for hemolytic, gelatinase, and phospholipase activities, while seven strains of Pichia kudriavzevii also showed negative results for the above activities, except for phospholipase. Neither D. rugosa nor P. kudriavzevii inhibited Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Additionally, 12 strains of D. rugosa and four strains of P. kudriavzevii produced phytase and cellulase, while only D. rugosa PCD3-6 exhibited protease and phytase activities. Therefore, D. rugosa (strains PCD3-6, PCD93-19, PYJ93-7, PYJ93-16, PCJ94-4, PCJ94-6, PCJ94-10, PYD100-8, CTD93-1, CTD93-2, CTJ93-5, CTJ93-6, and YTJ97-4) and P. kudriavzevii (strains PYI3-3, PCI3-4, PCJ90-7, and PYI91-15) were proposed as potential probiotic yeasts. They may be beneficially applied as feed additives to improve nutritional value and quality, thereby enhancing animal performance and health. Moreover, this work indicated useful preliminary results that could be a baseline for future research in human probiotic applications.

Sodium alginate improves phytase stability and enhances soil phosphorous utilization

A plant growth-promoting endophytic strain Bacillus sp. WR11 produces high-level phytase. Phytase degrades phytate, which is the major form of organic phosphate in soils that cannot be readily utilized by plants. To strengthen the strain or its phytase for cost-efficient biodegradation of phytate, we compared the impacts of different chemical additives on phytase production and preservation. Phytate degradation ability in soils and growth promotion effect in wheat were evaluated. To ensure safe application, potential pathogenicity of the strain was also predicted. Phytase activity assay suggests that sodium alginate (SA) significantly improves phytase production to as higher as 2.21-fold, but has little influence on the growth of WR11 during liquid fermentation. During liquid storage of cell-free supernatant (CFS) at room temperature, SA maintains 68% phytase activity indicating significantly improved stability within four weeks. Determination of water-soluble P and phytate-P indicates Bacillus sp. WR11 and CFS effectively degrade 20.17–38.55% phytate in soils. Quantification of biomass, phosphorus and iron contents shows positive effect of WR11 and its CFS on wheat growth. Particularly, the degradation ability (63.2%) and growth promoting effect of CFS is much stronger in the presence of 0.8% SA. Genomic prediction demonstrates strain WR11 has a large number of genes in the databases of Pathogen-Host Interactions, Virulence Factors of pathogenic bacteria, Antibiotic Resistance Genes, and the Comprehensive Antibiotic Research Database, respectively. In conclusion, SA significantly improves phytase production and phytate degradation. Phytase accompanied with SA may be an alternative strategy for phytate degradation and plant growth promotion, when microorganisms have safety issues.

The Effects of Single- or Mixed-Strain Fermentation of Red Bean Sourdough, with or without Wheat Bran, on Bread Making Performance and Its Potential Health Benefits in Mice Model.

The effects of single- (Lactobacillus fermentum) or mixed-strain (Lactobacillus fermentum, Kluyveromyces marxianus) fermentation of red bean with or without wheat bran on sourdough bread quality and nutritional aspects were investigated. The results showed that, compared to unfermented controls, the tannins, phytic acid, and trypsin inhibitor levels were significantly reduced, whereas the phytochemical (TPC, TFC, and gallic acid) and soluble dietary fiber were increased in sourdough. Meanwhile, more outstanding changes were obtained in sourdough following a mixed-strain than single-strain fermentation, which might be associated with its corresponding β-glucosidase, feruloyl esterase, and phytase activities. An increased specific volume, reduced crumb firmness, and greater sensory evaluation of bread was achieved after mixed-strain fermentation. Moreover, diets containing sourdough, especially those prepared with mixed-strain-fermented red bean with wheat bran, significantly decreased serum pro-inflammatory cytokines levels, and improved the lipid profile, HDL/LDL ratio, glucose tolerance, and insulin sensitivity of mice. Moreover, gut microbiota diversity increased towards beneficial genera (e.g., Bifidobacterium), accompanied with a greater increase in short-chain fatty acid production in mice fed on sourdough-based bread diets compared to their controls and white bread. In conclusion, mixed-strain fermentation's synergistic effect on high fiber-legume substrate improved the baking, sensory quality, and prebiotic effect of bread, leading to potential health benefits in mice.

The AtMINPP Gene, Encoding a Multiple Inositol Polyphosphate Phosphatase, Coordinates a Novel Crosstalk between Phytic Acid Metabolism and Ethylene Signal Transduction in Leaf Senescence.

Plant senescence is a highly coordinated process that is intricately regulated by numerous endogenous and environmental signals. The involvement of phytic acid in various cell signaling and plant processes has been recognized, but the specific roles of phytic acid metabolism in Arabidopsis leaf senescence remain unclear. Here, we demonstrate that in Arabidopsis thaliana the multiple inositol phosphate phosphatase (AtMINPP) gene, encoding an enzyme with phytase activity, plays a crucial role in regulating leaf senescence by coordinating the ethylene signal transduction pathway. Through overexpressing AtMINPP (AtMINPP-OE), we observed early leaf senescence and reduced chlorophyll contents. Conversely, a loss-of-function heterozygous mutant (atminpp/+) exhibited the opposite phenotype. Correspondingly, the expression of senescence-associated genes (SAGs) was significantly upregulated in AtMINPP-OE but markedly decreased in atminpp/+. Yeast one-hybrid and chromatin immunoprecipitation assays indicated that the EIN3 transcription factor directly binds to the promoter of AtMINPP. Genetic analysis further revealed that AtMINPP-OE could accelerate the senescence of ein3-1eil1-3 mutants. These findings elucidate the mechanism by which AtMINPP regulates ethylene-induced leaf senescence in Arabidopsis, providing insights into the genetic manipulation of leaf senescence and plant growth.

Responses of crops for diverse soil phosphorus conditions and potential crops for phytoextraction of enriched soil phosphorus

Phytoextraction is an attractive method to extract excess soil phosphorus (P) builds up from the soils. The crops potential to extract an enriched P in soil is still lacking. Therefore, four crops were grown in three different soils with variable P levels (0 to 200 mg P/kg soil) and screened for P acquisition potentials. Total P accumulation in roots and shoot was high with enriched P levels in all soils, for example in soil A at 200 mg P/kg soil, the values were 0.44% for cucumber (Cucumis sativus), 0.40% for sunflower (Helianthus annus), and ∼0.30% for cabbage (Brassica oleracea) and 0.20% for Indian mustard (Brassica juncea). The cucumber and sunflower showed a greater biomass production (>6.0 g/pot) with enriched P levels and exhibited more prominent root characteristics, suggests more favorable for P acquisition from the P enriched soils. More prominent enhanced phosphomonoesterase and phytase activities in these crops possibly contributing to their P accumulation ability under high soil P conditions. This study shows that, cucumber and sunflower can generate high dry matter content and can accumulate greater quantity of P from the soils through different mechanisms and they can be potential candidates for phytoextraction of P from the soil P enriched areas.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Biochemical properties of phytase immobilized and its effect on growth parameters of tomato

AbstractBackgroundPhosphorus (P) is one of the nonrenewable resources of critical importance in agricultural production. P is present in soil in organic and inorganic forms. Phytate constitutes the majority of organic P in soil. Phytate binds strongly to the solid phase of the soil and becomes unavailable for use by plants. Therefore, the soluble phytate‐P ratio in soil is mostly at very low levels. Plants and associated microorganisms secrete organic acids and hydrolyzing enzymes such as phytase to dissolve phytate in the soil. Both the solubility of phytate and phytase activity are limiting properties for the uptake of phytate‐P by plants.AimsOur aim was to evaluate the effects of phytase immobilized on zinc oxide nanoparticles (ZnO Np) on tomato plant (Solanum lycopersicum) growth parameters. In this study, seedling period was analyzed.MethodsIn the study, phytase activity of 13 different bacteria was investigated, and phytase was purified from Lactobacillus kefiri, showing the highest activity, and its biochemical properties were determined. Phytase was immobilized on zinc oxide (ZnO) nanoparticles and characterized by X‐ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopes analysis. The effects of ZnONps, immobilized phytase, and free phytase on the growth parameters of tomato plant were investigated. Tomato seeds were soaked with ZnONps, immobilized and free phytase for 30 min at room temperature and sown in pots containing suitable growing medium. Vegetative development of tomato plant, plant height, number of lateral branches, main stem diameter, distance between nodes, number of nodes, main root, and shoot length were determined.ResultsPhytase was partially purified with 7.60% recovery and specific activity of 1758.5 (EU mg−1 protein). Molecular mass of partially purified phytase was approx.72 kD, optimum pH and temperature values were determined as pH 5.0 and 70–80°C, respectively. Immobilized phytase caused a significant increase of 41.1% in plant height, 64.1% in main root, and 36.1% in shoot length in tomato plants compared to the control. In addition, a significant increase was observed in the number of side branches, main stem diameter, distance between nodes, number of nodes, and vegetative growth of the plant.ConclusionsThe results showed that the immobilized phytase enzyme has a positive effect on seedling growth in tomato and can be used in tomato cultivation in the future.

Biochemical characterization of purified phytase produced from Aspergillus awamori AFE1 associated with the gastrointestinal tract of longhorn beetle (Cerambycidae latreille)

ABSTRACT The need for industrially and biotechnologically significant enzymes, such as phytase, is expanding daily as a result of the increased use of these enzymes in a variety of operations, including the manufacture of food, animal feed, and poultry feed. This study sought to characterize purified phytase from A. awamori AFE1 isolated from longhorn beetle for its prospect in industrial applications. Ammonium sulfate precipitation, ion-exchange chromatography, and gel-filtration chromatography were used to purify the crude enzyme obtained from submerged fermentation using phytase-producing media, and its physicochemical characteristics were examined. The homogenous 46.8-kDa phytase showed an 8.1-fold purification and 40.7% recovery. At 70 C and pH 7, the optimum phytase activity was noted. At acidic pH 4–6 and alkaline pH 8–10, it likewise demonstrated relative activity of 88–95% and 67–88%, respectively. It showed 67–70% residual activity between 30 and 70 C after 40 min, and 68–94% residual activity between pH 2 and 12 after 2 h. The presence of Hg+, Mg2+, and Al3+ significantly decreased the enzymatic activity, whereas Ca2+ and Cu2+ enhanced it. Ascorbic acid increased the activity of the purified enzyme, whereas ethylenediaminetetraacetic acid (EDTA) and mercaptoethanol inhibited it. The calculated values for Km and Vmax were 55.4 mM and1.99 μmol/min/mL respectively. A. awamori phytase, which was isolated from a new source, showed unique and remarkable qualities that may find use in industrial operations such as feed pelleting and food processing.

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Production of cleavage-resistant phytase transgenic pigs by handmade cloning

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry.