New insight for Uricase stabilization in the presence of taurine
New insight for Uricase stabilization in the presence of taurine
Parisa Shahmoradipour,1Maryam Zaboli,2masoud torkzadeh_mahani,3,*
1. Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran 2. Department of chemistry, Faculty of science, University of Birjand, Birjand, Iran 3. Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran
Introduction: The enzyme urate oxidase (UOX) or uricase (EC 1.7.3.3) is an enzyme from the group of oxidoreductases that exists in a variety of organisms but does not exist in more advanced primates, including humans. This enzyme catalyses the oxidation of uric acid to 5-hydroxy isourate and hydrogen peroxide. Uricase can be used as a medicinal enzyme to reduce the level of uric acid in plasma. Despite of the high tendency of uricase to convert the substrate into a product, the low stability of this enzyme against heat has limited its usage. So far, many efforts have been made to improve the kinetic parameters and thermodynamic stability of the enzymes, such as enzyme point mutation, enzyme immobilization, and the use of additives. One of the most widely used additives to increase the stability of enzymes is the use of osmolytes. Taurine is one of the osmolytes that is used to stabilize the structure of proteins. Taurine is an organic acid with a molar mass of 125.14 g / mol found in natural food sources, biosynthesized in the body and also produced by chemical synthesis for commercial purposes. Taurine is derived from cysteine. In this study, UOX enzyme was recombinantly expressed and purified in a bacterial host and then the effect of taurine on the activity of UOX was investigated using the optimal response surface area (RSM) method.
Methods: Enzyme expression
To express the recombinant UOX in E. coli cells of BL21 DE3 strain, the coding sequence of enzyme was subcloned in PET.28a vector and its expression was induced with IPTG (1 mM). After preparing the purified protein, the samples were loaded on 10% SDS-PAGE and the observation of a single band in the range of 35 kDa confirmed the expression of recombinant urate oxidase. The activity of urate oxidase enzyme can be measured according to the ability of the enzyme to break down uric acid and convert it to allantoin based on the reduction of uric acid absorption per minute at a wavelength of 293 nm. Response surface (RSM) method was used to achieve recommended optimal conditions including concentration of taurine, incubation time and temperature with taurine.
RSM methodology
The influence of the variables affecting the activity of the taurine-treated UOX enzyme was investigated with a full fraction design. Four operating variables viz., incubation time, the taurine concentration and temperatures were examined. To optimize the three most significant factors, 18 full factorial central composite design (CCD) of RSM using Design-Expert software version 11 was employed.
Results: The quadratic equation was used to describe the response of the system (the activity of treated-UOX). The final model was shown in the following equation to predict the value of UOX activity:
Activity= +1.49-0.0175 * A+0.1450 * B-0.1625 * C-0.1770 * AB=0.1295 * AC+0.1580 * BC+0.0683 * A2+0.0750 * B2-0.0087 * C2
where A, B, and C show incubation time, taurine concentration, and temperature, respectively. A positive sign was represented a synergistic effect, whereas the negative sign was indicated antagonistic effect on response. The optimum taurine concentration, temperature, and incubation time were found to be 450 mM, 28ºC, and 15 min, respectively, for achieving the maximum activity.
All variables (taurine concentration, incubation time, and temperature) significantly affected the enzyme activity because significant changes in enzyme activity were observed for all combinations. The high F value, low P-value (< 0.0001), R2 and Adj-R2 = 1.00 and non-significant lack of fit show that the model was highly significant.
Conclusion: The value of adjusted R2 (1.0) shows that the total variations were explained with the obtained model. The non-significant lack of fit (<0.05) presented that the quadratic model was statistically significant for the response. The best conditions for UOX stabilization were achieved at 28ºC, 450 mM taurine concentration, and 15 min incubation time. The activity of UOX was obtained about 2.10 U/ml under the optimal conditions.