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.

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.

Background: In this study, PHYTASE-producing bacteria from7 cultivar of rhizosphere wheat (Chamran, Tajan, Morvarid, Pishtaz, Oroum, Sivand, Zarea) were solated and identified based on morphological and biochemical characteristics.Methods: Enzyme-producing bacteria were determined by production of clear zones around the colonies on the medium containing sodium phytate, after 24-48 h of incubation at 30oC. Because some bacteria in PHYTASE specific medium are able to create false positive results, we eliminated them by PHYTASE plate assay (cobalt chloride, ammonium molybdate / ammonium vanadate). The best PHYTASE producing isolates were recognized by its surrounding clear halo.Results: Out of 47 colonies on media plates, 19 colonies showed positive for PHYTASE production and created zone of clearing around microbial colonies, but 11 false positive colonies were detected. Finally, 8 isolates were detected as real PHYTASE-producing bacteria. Most isolated bacteria belong to two cultivars (Tajan, Chamran). One strain creating 23 mm zone was selected as the best enzyme producing isolate.Conclusion: This research provided important data about PHYTASE producing bacteria isolated from rhizosphere wheat, which will facilitate future research on the optimization of fermentatoin processes for production of high PHYTASE activity.