الفهرس 
Introduction and aim of workOnly 14 pages are availabe for public view
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Abstract
Uricase used as a sensitive medicinal biosensor for detection and quantification of the uric acid concentration in the blood and biological fluids. Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, is useful for enzymatic determination of urate in clinical analysis, as well as in prophylaxis.The main objective of this study was screening of some fungal strains for their ability to produce uricase and selecting the most potent isolates for medically important uricase production. 1. Twenty-five fungal strains were screened for their potentialities for uricase production by uricase plate assay method on uric acid medium. After incubation, the formation of clear zone around the fungal colony indicating its potentiality for uricase production. 2. Among the tested strains of fungi using the plate assay method, Emericella sp. strain 1-13, Asperagillus sp. strain 1-4, Aspergillus carenus and Aspergillus flavus were the most active fungal strains that able to produce uricase. The most active fungal strains were tested for uricase production under submerged fermentation conditions. 3. The highest uricase activities in liquid medium containing uric acid were obtained from Emericella sp. strain 1-13 (23.38 U/mL), Aspergillus sp. strain 1-4 (19.87 U/mL), Aspergillus carenus (16.37 U/mL) and Aspergillus flavus (10.91 U/mL).4. The optimum conditions by one-factor-at-a-time method for maximum Emericella sp.strain 1-13 and Aspergillus sp.strain 1-4 uricase production were at temperature of 30oC, uric acid concentration of 3 g/L and at pH 6.5. 5.Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1-4 is closely related to Aspergillus welwitschiae and its sequencing product was deposited in the GenBank database under accession number MG323529. 6. Phylogenetic analysis based on ITS region sequence analysis together with its phenotypic characteristics, the Emericella sp. strain 1-13 was identified as Emericella quadrilneata and sequenced product was deposited under accession number KY091263 in the GenBank database.7.Statistical screening using Plackett- Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae and Emericella quadrilneata.
8. Statistical analysis of the results of uricase production by Aspergillus welwitschiae showed that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.478, 5.26 and 4.75; respectively. 9.The optimal levels and interaction effects of these variables were further optimized using central composite design. The maximum Aspergillus welwitschiae uricase production was achieved at incubation time (7 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) which are the optimum levels for maximum uricase production (60.03 U/mL). 10. After optimization, Aspergillus welwitschiae uricase production was improved by about 3.02 fold increase as compared with that obtained from the unoptimized medium (19. 87 U/mL).11.Among fifteen screened factors through 20 runs of Plackett-Burman design; temperature, uric acid concentration and medium volume were found to be the most significant factors for uricase production by Emericella quadrilneata with the highest effects values of 9.45, 5.637 and 5.547; respectively.12.These factors were further optimized via 20 runs of central composite design (CCD) which elucidate that, temperature (35°C), uric acid concentration (5 g/L) and medium volume (50 mL) are the optimum levels for maximum uricase production (65.48 U/mL). After optimization, Emericella quadrilneata uricase production was improved by about 2.8 fold increase as compared with that obtained from the unoptimized medium (23. 38 U/mL).13.The results indicated the potentiality of purified enzyme to degrade uric acid in human serum and its possibility to use in uric acid determination in biological fluid. 14. The crude enzyme extract of Emericella quadrilneata was partially purified using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B 15.The enzyme molecular weight was determined as approximatily 70 KDa with the help of SDS-PAGE. The enzyme was purified 3.338-fold and showed a final specific activity of 30.636 U/mg protein. 16.The maximum Emericella quadrilneata uricase activity (63.91 U/mL) was obtained at 20 μg/mL of uric acid and it decreased by increasing the concentrations of uric acid. The Km and Vmax were found to be 0.021 mM and 70.81 U/mL/min; respectively.17. Emericella quadrilneata uricase activity increased gradually with increasing of pH values from 5 towards alkaline pH and reached to maximum activity at pH 8, and then activity decreased at pH 10. Also purified uricase activity was found to be higher at 37oC and after 20 minutes of incubation time and then decreased with increasing time.
18. Uricase produced by Emericella quadrilneata was stable at 40oC and it retained 81.67% of its original activity after 30 minutes. Also, retained 70.78% of its activity after exposure to 50oC for 30 minutes. The residual uricase activity showed decrease to 60.65% after 30 minutes at 60ºC and retained 24.89% of its original activity after exposure to 60ºC for 60 minutes.In conclusion, Emericella quadrilneata might be considered as a candidate source to produce extracellular uricase that can be used as uric acid biosensor and a therapeutic protein drug for minimization the urate elevated levels in human serum.