No abstract

Phytases are enzymes that hydrolysis phytic acid and makes mineral phosphorus available to animals. Phytases face relatively extreme heating during food processing, thus thermostability plays an important role in industrial applicability of this enzyme. Herein, we report the design of a thermostable phytase with favorable biochemical properties and high enzymatic activity using molecular dynamics and rational design-based molecular engineering. Based on the crystal structure of E. coli phytase, bioinformatics analysis and docking binding energy measurement, S392F mutant was introduced by site-directed mutagenesis in order to improve thermostability of phytase through strengthen the bounding interactions. Wilde type and Mutated constructs were expressed in E. coli BL 21. The WT and manipulated phytase were purified; their biochemical and kinetic was investigated. Results revealed that recombinant WT and mutant phytase have optimum temperature of 50 °C with no significance change but optimum pH of WT and mutant was respectively 5 and 6 with a pH shift. Furthermore, S392F phytase catalytic efficiency values showed significant improve of 25.6%, compared with the WT. Analysis of the retained enzymatic activity at high temperatures, indicated that despite of phytase stability reduction at high temperatures but mutant phytase showed more stable behavior in compare with WT phytase, So that at 70 °C showed twice thermo stability and at 80 °C and 90 °C display respectively 74% and 78.4% improvement of thermostability compared to the wild-type. In conclusion, our results implied that the designed phytase could be a potential candidate for phytase manipulation research and industrial applications with improved thermostability.

Keywords: Granzyme B p28 azurin breast cancer fusion protein targeting A B S T R A C T Targeting tumor cells via multiple pathways promises the emergence of a new era in cancer therapy.Consisting of a cell-binding ligand and a cytotoxic moiety, cytolytic fusion proteins can selectively bind and kill target cells with minimal adverse effects. We designed a novel immunoproapoptotic fusion protein, p28-fur-GrB, composed of the cancer-specific azurin-derived cell penetrating peptide, p28, and a mutant version of human serine protease granzyme B. The two moieties were genetically fused by a furin sensitive linker, allowing in vivo cleavage and activation of the immunotoxin after cell entry. Synthesized coding gene of the recombinant protein was cloned and expressed in HEK293T cells, and nickel chromatography was applied for protein purification. After in vitro furin cleavage and primary analyses of SDS-PAGE, Western blotting, GrB activity and ELISA binding assay, the fusion protein was tested for its cytotoxicity on various breast cancer cell lines. Suppression of cell proliferation and viability was evaluated using the WST-1 assay. Furthermore, DNA fragmentation was measured as an indication of apoptotic effects of the fusion protein on treated cells. Based on our results, p28-fur-GrB was efficiently cleaved by furin and showed high GrB activity and binding affinity after cleavage. Following 72h of incubation with IC50 values of the fusion protein, significant cytotoxic effects of 80.6%, 77.1%, 74% and 69.6% were recorded for BT-474, MCF7, SK-BR-3 and MDA-MB-231 tumor cells, respectively. Proliferative potential of MCF 10A normal cells was not affected by the treatment. Analysis of the rate of apoptosis in treated cells confirmed our cytotoxicity results. We concluded that p28-fur-GrB is a potent anti-tumor agent with high cytotoxicity against breast cancer cells.