Theoretical and experimental study of the trehalose and myo-inositol mixed osmolytes on thermal stability of recombinant urate oxidase enzyme
Theoretical and experimental study of the trehalose and myo-inositol mixed osmolytes on thermal stability of recombinant urate oxidase enzyme
Reihaneh Samareh-Hoseinalinejad,1,*Maryam Zaboli,2Masoud Torkzadeh-Mahani,3
1. Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, Graduate University of Advanced Technology 2. b Department of chemistry, Faculty of science, University of Birjand 3. a Department of Biotechnology, Institute of Science, High Technology and Environmental Sciences, Graduate University of Advanced Technology
Introduction: Today, due to the wide application of enzymes in various industries, the stabilization of enzymes in order to maintain their activity under different conditions has attracted a lot of attention. Enzyme urate oxidase or uricase (EC: 1.7.3.3) is an enzyme of the oxidoreductases family that catalyzes the oxidation of uric acid to 5-hydroxy isovarate and releases CO2 and H2O2. 5. Hydroxy isovarate is an intermediate compound that is converted to allantoin through several stages of enzymatic or chemical reaction, which is 5 to 10 times more soluble in water than uric acid. Uricase is present in most organisms and microorganisms, but some primates and Reptiles have lost uricase gene activity during the development of their genomes due to mutations. The concentration of uric acid in human serum is 6-1.5 for women and 7-2.5 for men. Any increase in purine catabolism or a decrease in uric acid excretion leads to the formation of monosodium urate (MSU) crystals in the joints or soft tissues, leading to hyperuricemia (a common feature of Tumor Lys Syndrome (TLS) and gout). Gout presents with painful inflammation of the joints, deposition of sodium urate crystals, and uric acid kidney stones. Uric acid is naturally the product of the catabolism of purine nucleotides.
Osmolytes are small organic compounds found naturally in living cells that protect the cell from osmotic stress. Osmolytes contain amino acids, methylene amines and polyols. Polyols also contain glycerol, sucrose and trehalose and other sugars such as inositol. Osmolytes stabilize the protein structure and protect their natural form from being disturbed.
Methods: First, the urate oxidase coding sequence was subcloned into PET.28a expression vector and after induction with IPTG inducer, the recombinant protein was purified by affinity chromatoghraphy. In order to prove the protein expression, the samples were loaded on 10% SDS-PAGE gel and single protein bands in the range of 35 kDa was observed and also the concentration of the purified enzyme was determined using Bradford method.
To measure the activity of the enzyme, we used its ability to break down uric acid molecules and reduce its absorption at 293 nm. we applied response surface methodology for prediction of the optimal incubation temperature, the concentration of trehalose and myo-inositol and the incubation time of mixed osmolytes with uricase enzyme. In the following, the activity of treated enzyme measured under recommended condition.
Results: The best enzyme activity in the different treated condition was observed in a mixture of 12.5 mM myo-inositol and 1.125 mM trehalose at 21.25 ° C and 35 minutes incubation time .
Conclusion: To increase stability, many approaches can be take into account, such as mutations or genetic manipulation, chemical modifications, stabilization, and the use of additives, one of which is osmolytes. The results showed that the addition of 12.5 mM myo-inositol and 1.125 mM trehalose had the maximum effect on uricase enzyme stability