Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Natural and recombinant production of this enzyme is one of the important issues in protein engineering field. In this study, the synthetic Phytase gene Bac phy-wild was cloned into PUC57 vector and transformed to susceptible Escherichia coli-DH5α . In order to replace the polar amino acid with non-polar amino acid within the target mutagenic protein; we used a primer designed to tryptophan (S392W) for targeted mutagenicity in the amino acid serine 392 position. After confirming the mutation at the target site, the mutated gene was registered at the NCBI gene bank. To investigate the expression and temperature stability of the mutant enzyme and compare it to WT phytase, the WT and mutated Bac Phy-Mut genes were transferred to pET26b (+) expression vector. Recombinant vector construct was transferred to a to Escherichia coli-BL21. After screening of recombinant clones, SDS-PAGE analysis and protein concentration were used to evaluate the expression of recombinant protein. The results showed the presence of protein with a molecular weight of 42 kDa. Also the study of physicochemical properties of WT and mutated phytase showed that the optimum temperature was unchanged at 55 ° C and the pH was optimized to be 5. Comparison of thermal stability of the mutated phytase in compare with WT at 40, 50, 60, 70 and 80 ° C was improved by 18, 24, 19, 9 and 6%, respectively. Hence, the results of the current research showed that by using targeted mutagenesis method we succeed to improve the mutated phytase with higher thermal stability which could be obtained and mutant phytase could be used in the agricultural and environmental industries as food and feed additive of livestock, poultry and Fish as well as in medical application.

Introduction: Phytase is an enzyme that has the ability to break down phytic acid into myoinositol and mineral phosphate, and widely uses as an additive in animal foods. The aim of this study was to achieve a high level of bacterial phytase expression in PET26b expression host. Materials and Methods: To generate the recombinant phytase enzyme, the target gene was introduced into the expression vector pET-26b-Phytase, and the recombinant vector was transferred to Escherichia coli BL21 as a host of expression after replication in E. coli DH5α, . The SDS-PAGE method was used to isolate recombinant phytase and the amount of produced recombinant protein was investigated. by electrophoresis. Results: The bacterial phytase enzyme was successfully expressed in Escherichia coli and the molecular weight of recombinant phytase produced at about 85 kDa was estimated. The optimum pH for recombinant bacterial phytase was estimated at 5. 5. Conclusion: The results showed that phytase derived from Escherichia coli due to its low production cost and high production of recombinant phytase is a desirable alternative for use in domestic animal feed industry.

Phytase (myo -inositol hexakisphosphate) is the hydrolysis enzyme of phytic acid that produces inorganic phosphate. The genephyC encoding phytase was isolated from Bacillus subtilisATCC12711 genomic library and sequenced. The nucleotide sequence of the phytase gene contained an open reading frame of 1089 bp, which codes for 90 bp signal peptide and a mature protein with a deduced molecular mass of 42 kDa. Target gene was inserted in pET32a (+) as expression vector and transformed to Escherichia coli BL21 (DE3) as host expression. The fusion phytase phyC-Trx gene was successfully overexpressed in E. coli as an active and cytoplasmic phytase. Recombinant protein production was induced with 1mM IPTG in shaking flasks at 300C in presence of 10 mM calcium. Samples were taken at 0-5 h after induction by 1 hour time intervals, and then protein electrophoresis was done.phyC -Trx protein was expressed in the cytoplasm ofE. coli successfully. Molecular weight of recombinant phytas was estimated about 64 kDa. Phytase activity was 7.44 U/ml with using standard phosphates method. Optimum pH for the degradation of phytate was 7. The results of current study showed that thermal stability and cost effective production make this enzyme attractive for using in feed supplements.

Phytic acid is a stored form of phosphorus in cereals, 65 to 70% of phosphorus in plant sources is phytate, and broilers are only able to use part of the phosphorus in plant sources. To meet the needs of chickens, it is necessary to use other artificial resources, which not only impose part of the cost of the breeding period because of its presence in the manure but is one of the factors polluting the environment. This study aimed to use different levels of phytase enzyme to reduce dietary phosphorus levels. 600 Ross 308 broilers were used in this experiment with five treatments and six replications, and in each replication, 20 chickens were used in a completely randomized design (CRD). Experimental treatments include 1) basal diet (control) 2) basal diet with 15% less phosphorus 3) basal diet with 15% less phosphorus + 1250 (FTU) phytase enzyme 4) basal diet with 15% less phosphorus + 2500 (FTU) phytase enzyme 5) basal diet with 15% less phosphorus + 5000 (FTU) phytase enzyme. The evaluated traits included weekly feed intake, weekly weight gain, feed conversion ratio, carcass characteristics, ash, calcium, and bone phosphorus. The use of phytase enzyme in different diets had no significant effect on food intake, weight gain, and feed conversion ratio (P>0. 05). However, the use of phytase in different diets significantly affected the percentage of Gizzard, Heart, Liver, Proventriculus, and Spleen (P<0. 05). The most changes were the increase in the ratio of feed intake and weight gain in the fourth week compared to the third week so that the changes in the ratio of feed intake ranged from 1. 85 to 1. 91, and this ratio for weight gain also ranged from 3. 12 to 3. 86 was recorded, and the lowest feed conversion ratio was obtained at the same age. The percentage of raw ash in broiler chickens was significantly increased by adding dietary phytase. The lowest amount of ash, calcium, and phosphorus belonged to the second group (diets with low phosphorus and no enzyme). The difference between the other groups and the control was not significant. Feed intake, weight gain, and feed conversion ratio with the addition of phytase enzyme were not affected by phosphorus reduction and had no significant effect on carcass characteristics. Environmental pollution can be prevented by reducing the level of dietary phosphorus and reducing excreted phosphorus.

The use of genetically engineered probiotics to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivariusand Lactococcus lactis. The results showed plasmid instability, unable to express the gene. The expression of app A gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinantL. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9 U mL-1 was obtained in cell extracts of L. lactis. The growth of native and recombinantL. lactis was similar in the presence of two concentrations of ox bile.

The aim of this study was to evaluate the efficacy of Bacillus subtilis bacteria capable to produce phytase enzyme on improving broiler chicken performance and to evaluate its effect on gastrointestinal organs, and tibia strength in broiler chickens. This experiment was done using 200 one-day old broiler chicks (Ross 308), in a completely randomized design with 5 treatments, 4 replicates and 10 chicks per replicate for 42 days. The treatments include: 1) positive control diet containing sufficient phosphorus level; 2) negative control diet with 30% less phosphorus; 3) negative control diet supplemented with phytase enzyme; 4) negative control diet supplemented with a commercial probiotic; 5) negative control diet supplemented with Bacillus subtilis SH17-3. Feeding broiler chickens with a diet containing Bacillus subtilis bacteria SH17-3 significantly reduced feed intake and mean body weight gain (BWG) in the total rearing period (P<0. 05). Feeding broiler chickens with a diet containing phytase enzyme significantly increased feed intake and BWG (P<0. 05). The strength of tibia was significantly increased in birds received phytase enzyme, compared to other groups (P<0. 05). Based on the results the use of phytase enzyme in diets with phosphorus deficiency, improves performance in broilers; but Bacillus subtilis bacteria SH 17-3 could not be a good alternative for probiotic and also phytase enzyme.

Background: Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity).Objective: In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator.Material and Methods: phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking.Results: The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called “allosteric site”.Conclusions: The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

Background & Objectives: Phytate is the primary storage form of phosphorus in plant seeds. It has an anti-nutritive effect in both human and animals. Phytase is a subgroup of phosphatases which catalyzes the hydrolysis of phytate. Microbial phytases have potential biotechnological application in various fields, such as agriculture, human and animal foods. This study was aimed to investigate the optimal conditions for the production of phytase by Bacillus subtilis isolated from the soil.Materials & Methods: Samples were collected from Arak area, where the soil was contaminated with animal faces. Samples were incubated in PSM medium at 30oC for 48 hours. The screening of phytase - producing bacteria on PSM media was performed based on the formation of the clear halo. The most suitable bacterial strain was identified according to its biochemical and morphological characteristics. The level of enzyme production and phosphate- solubilising activity of this strain was assessed in different pH range, and on various media types including PSM, PVK, NBRIP, as well as NBRIY.Results: In this study, maximum enzyme production by the bacterial isolates has been observed following 36-48h incubation in PSM medium.12S phytase- producing bacterial strain was identified as Bacillus subtilis. Our results showed that the optimum pH for phytase production in PSM medium is pH of 7. Furthermore, investigating different media, phytate- containing PVK was recognised as the most appropriate medium for enzyme production.Conclusion: B. Subtiliseisolate can provide an opportunity to introduce new phytase to food as well as environmental industries. Moreover, PVK can be used as an effective medium to produce phytase enzyme and to screen phytase- producing bacterial starins.

We investigated the effect of Phytase enzyme on phosphorus, protein, fat, dry matter and Energy digestibility of four vegetative food comprised of wheat meal (WM), corn meal (CM), wheat bran (WB), rice bran (RB) in rainbow trout feeding. A base diet including fish, soybean and meat flour, was prepared. Four diets were prepared using a mixture of 75% base diet and 25% WM or CM and 85% base diet and 15% WB or RB with similar gross energies. Chromic oxide was added as 0.5% of the diet and used as an indigestibility marker. A phytase solution was sprayed on all diets at a minimum concentration of 1000 phytase units per Kg (FTU/Kg) of dry diets. Experiments were carried out in two cubic meter tanks that were filled with 500 liters of water. Maximum and minimum of temperature, oxygen and pH were 18 and 15°C;8 and 6.8mg/l and 7.7 and 7.5 respectively.Results showed no significant effect of Phytase on the amount of bone phosphorus and protein digestibility (P>0.05), while there was an increase in these indexes with Phytase supplementation. Moreover, Phytase significantly improved digestibility of dry matter.Results also showed that kind of diets had different effects on the nutritional digestibility. The diet containing of WM was the best with those containing CM, WB and RB gradually losing their suitability in terms of the nutritional digestibility. Due to their availability and suitability, we suggest that these diets, especially that containing WM be used in Rain bow trout culture.

An experiment was conducted to evaluate the effects of organic acids and phytase enzymesupplementation on immune system and carcass characteristics of broiler chickens. Theexperiment was done in factorial arrangement 2×2×2 based on completely randomized designwith 8 treatments, 5 replicates and 12 chicks in each to 42 d of age. Diets including naturalvinegar (0 and 2%), citric acid (0 and 1%) and phytase enzyme (0 and 500 U phytase/kg offeed). All the birds were vaccinated against Newcastle disease by lasota vaccine via aye drop in11 and 23 days. In order to study the immune system situation, blood samples of two birds ofeach replicate was taken in 42 day. After serum separation, the IgG of NDV was detected by HItest. One birds of each replicate (five birds of each treatment) randomly selected and were killedto evaluate lymphoid organs weigh and carcass characteristics at 42 day. The analysis of resultsshowed that addition of vinegar and phytase significantly increased the relative weight of bursa(P<0/05). Interaction effect of citric acid with vinegar and with phytase enzyme significantlyincreased the relative weight of bursa (P<0/05) also vinegar significantly increased the relativeweight of spleen (P<0/05). Phytase significantly increased the abdominal fat (P<0/05) whilecitric acid and interaction effect of citric acid with vinegar significantly decreased theabdominal fat (P<0/05). In current study treatments hasn’t any effect on internal organs relativeweight (P>0/05).