ABSTRACT
Background: Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals' foods to hydrolyze phytate and increase absorption of phosphorus
Objectives: Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli
Materials and Methods: The Y. intermedia phytase gene was optimized according to the codon usage in E. coli. The sequence was synthesized and sub-cloned in pET-22b [+] vector and transformed into E. coli Bl21 [DE3]. The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30[degree]C. The purification of recombinant protein was performed by Ni2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures
Results: The codon optimized Y. intermedia phytase gene was sub-cloned successfully. The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme [approximately 45 kDa] was purified. Specific activity of enzyme was 3849 [U.mg-1] with optimal pH 5 and optimal temperature of 55[degree]C. Thermostability [80[degree]C for 15 min] and pH stability [3-6] of the enzyme were 56 and more than 80%, respectively
Conclusions: The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially and to be applied in animals' foods industry
ABSTRACT
Display of peptides on the surface of bacteria offers many new and exciting applications in biotechnology. Fimbriae is a good candidate for epitope display on the surface of bacteria. The potential of CS3 fimbriae of enterotoxigenic E. coli as a display system has been investigated. A novel cell surface display system with metal binding property was developed by using CS3 fimbriae. Short metal binding peptide, Gly-Cys- Gly-Cys-Pro- Cys- Gly- Cys- Gly as a cysteine rich peptide, was inserted into CS3 fimbriae and displayed on the surface of E. coli. Bacteria expressing hybrid pili with cysteine rich peptide could adsorb 392.5, 510 and 905 nmol of Ni2+, Cd2+ and Pb2+ per mg [dry weight] of cells, respectively, which are five-fold [nickel] and three-fold [cadmium] more than E. coli expressing native pili. Thus, expression of Cys-rich peptide enables bacteria to act as a metalloaffinity adsorbent. These results open the possibility for biosorption of heavy metal ions using engineered microorganisms