Bioinformatic Studies, Experimental Validation of Phytase Production and Optimization of Fermentation Conditions for Enhancing Phytase Enzyme Production by Different Microorganisms under Solid-State Fermentation

Abstract

Background: Phytase is an essential enzyme necessary for the digestive process. It is a natural enzyme found in plant materials. It prevents bad effect of phytic acid on protein and energy utilization. Phytase frees the bound minerals such as phosphorus, calcium, zinc, iron, magnesium and manganese from the phytic acid molecule providing essential minerals available for healthy nutrition. This study depends on converting food processing waste into highly valuable products. Optimizing the fermentation conditions for enhancing high phytase production with low cost was the objective of this research. Methods: A bibliographical survey was carried out to select the most fungul producers of phytase from fungal species deposited in NCBI database. Phytases of the selected organisms were analyzed in the UNIPROT database and their protein sequences were submitted to multiple sequence alignments using Clustal Omega and visualized using Jalview program. Experimental studies using five fungal strains of Aspergillus.ssp on wheat bran under Solid-State Fermentation carried out. Comparisons were made for phytase production. A. awamori NRC- F18 as the best phytase producer-strain cultured on different types of treated wastes followed by optimizing the fermentation conditions for enhancing phytase production using rice straw as the best substrate, which provides the highest phytase production. Thermostability of crude enzyme was studied. Statistical analyses were performed using SPSS at P < 0.05 or P < 0.01. Results: Bioinformatic studies predicted the most producer species and explained the difference in activity of phytases produced from different species, although they have the same function. All phytases of the selected fungal species from the database NCBI have highly conserved amino acid sequences; there are 88 identical positions; 135 similar positions, but the identity percentage was 16.858%. Experimental studies using five fungal strains of Aspergillus ssp. on wheat bran revealed optimum conditions for phytase production by A. awamori NRC- F18, which cultured on different types of treated wastes. A considerably higher phytase production was obtained using rice straw as substrate 424.66± 2.92 IU /g at pH 6 (371.883± 0.822 IU /g), after 144 hrs of incubation at 30°C. The maximum enzyme activity observed when solid: moisture was 1:4; Inoculum concentration 2mg/5g (418.363± 16.709 IU /g) and substrate concentration 4.5% (277.39± 12.05 IU /g). Glucose and Ammonium acetate were the best carbon and nitrogen sources that enhanced phytase production from A. awamori NRC- F18. The obtained phytase was found to be thermostable and the maximum temperature at which phytase still active was 80°C. Conclusion: Bioinformatic studies predicted the most producer species. Experimental study revealed that A.awamori NRC- F18 was the best Phytase -producer strain. Solid state fermentation was a good method; pretreatment of agriculture residues as rice straw was useful for less expensive phytase production, which was thermostable. A. awamori NRC- F18 can be used in the industrial production of phytase.