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.

Purpose Wheat straw is an agricultural waste which can be used as a cost effective animal feed. However, high hemicelluloseand phytic acid content in wheat straw prevents it as a primary feed choice. Utilization of wheat straw in solid-statefermentation may result in wheat straw valorization and enzyme production. In this study, PHYTASE production in solid-statefermentation of wheat straw using Aspergillus ficuum and valorization of wheat straw were evaluated. Methods A two-step experimental design procedure was employed for screening and optimization of influencing factorson PHYTASE production. Effects of different nutritional and environmental factors were investigated by one factor at the timemethod (OFATM). To reach higher amounts of PHYTASE, response surface methodology (RSM) was employed to optimizePHYTASE production as a function of three of the most effective factors. Results Optimization of the significant parameters resulted in an increase in the PHYTASE activity from 0. 74 ± 0. 12 to a maximumof 16. 46 ± 0. 56 Units per gram dry substrate (U gds− 1). The high degree of the fungal PHYTASE activity on wheat strawresulted in the decrease in phytic acid content by 57. 4%, as compared to the untreated sample. Scanning electron microscopy(SEM) and FTIR structural analysis showed intensive fungal growth on wheat straw, and partial removal of hemicelluloses, lignin and phytic acid. Conclusion The study demonstrated the feasibility of wheat straw utilization in solid-state fermentation using Aspergillusficuum toward the production of PHYTASE and valorization of wheat straw as an animal feed.

Objective: Determination the effects of PHYTASE supplementation in broiler diets.Design: Randomized complete blocks design.Animals: Two hundred eighty broiler chickens.Procedure: The experimental diets were prepared based on NRC (1994) recommendations except for Prequirement. The diets, C,1P, 2P and 3P consist of 0,300,600 and 900 units of PHYTASE activity per Kg of diet (FTU /Kg) respectively, without of inorganic phosphorus supplementation. The 10,20 and 3D diets have similar composition with control diet (C) except for 1,2 and 3 g phosphorus pre Kg of diet from dicalcuim phosphate (DCP). Factors related to phosphorus nutrition were measured during the experiment.Statistical analysis: Analysis of variance, Duncan’s multiple range test of means, Linear regression equations.Results: Addition of PHYTASE had significantly effects on feed intake, body weight gain, tibia ash, tibia length, tibia phosphorus and phosphorus excretion (P<0.05). PHYTASE had no significant effects on feed conversion ratio, mortality and the cost of diet for production of I Kg of live body weight (P>0.05).Phosphorus equivalency were calculated as 700 FTU/Kg for body weight gain and 400 FTU/Kg for tibia ash equal to I g phosphorus from dicalcium phosphate.Implications: The PHYTASE enzyme is able to hydrolyze the phytate molecule and convert it to a nutritive form and destroy its negative effects. Depend on acquired results. We can conclude that supplementation of PHYTASE in broiler commercial diets is applicable and there is no need of inorganic phosphorus supplementation to diets without additional cost, and it can prevent the phosphorus loss in manure. The optimal level of PHYTASE inclusion in broiler diets is 600 FTU/Kg diet based on the results of this.

Phosphorus (P) is a macro mineral in broiler nutrition. In growing broilers, besides its requirement for proper bone development, it is also involved in almost all metabolic processes. Poor P availability results in decreased productivity and poor health status. Phosphorus availability from plant derived feeds is affected by an anti-nutritional factor “phytate”, which forms a variety of insoluble salts with most of the minerals including P, calcium (Ca), magnesium, zinc (Zn) and copper (Cu) due to its reactive anion capability. So, phytate is responsible for considerable nutrient losses as vegetable sources form a major portion in broiler diet formulations. Phytate has also been reported to form complexes with protein and proteolytic enzymes (pepsin and trypsin). Mono-gastric animals lack endogenous PHYTASE (an enzyme capable of hydrolyzing phytate bound P, Ca, protein and other nutrients), so phytate decreases the nutrient availability at the intes-tinal level in poultry. Application of PHYTASE in poultry rations may liberate cations and other nutrients bound by phytate-P complexes resulting in improved production parameters and body structure characteris-tics in broilers. However, efficacy of supplemental PHYTASE rests on its rate of application, Ca: P in ration, composition of diet, genotype and age of birds. PHYTASE could ensure the economical poultry production by the exploitation of inherent nutritional potential of feedstuffs. Some studies, however, showed that PHYTASE does not degrade dietary phytate efficiently and thus the negative influence of phytate on protein digestibil-ity is not completely removed by PHYTASE supplementation. More focused research on currently available PHYTASE feed enzymes and their potentialimproved action by the simultaneous use of other exogenous en-zymes, which complement their activity is recommended.

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.