Lactose inducible fermentation in Escherichia coli for improved production of recombinant urate oxidase: Optimization by statistical experimental designs

Abstract

The enzyme urate oxidase (UOX) is used as a drug for preventing and treatment of chemotherapyinduced hyperuricemia. This study deals with the statistical optimization of lactose inducible fermentation for production of soluble recombinant Aspergillus avus UOX. 10 variables were investigated by Plackett-Burman design (PBD), and the most signi cant factors were further optimized by central composite design (CCD). PBD results indicated that glycerol, yeast extract, tryptone, and lactose affected UOX activity signi cantly. The CCD results showed that the maximum enzyme activity (19.34 U/ml) could be achieved under the optimum conditions of glycerol 0.87 g/L, yeast extract 9.11 g/L, tryptone 10.29 g/L, K 2 HPO 4 1.81 g/L, and lactose 12.79 g/L. When the same induction strategy was tested at shake ask, 19.34 U/mL of UOX activity was obtained, which was 12.5 folds higher than IPTG induction protocol. Furthermore, the lower total cost (0.7 vs. 13.5 €) was additionally feature that con rmed the suitability of the lactose induction method. Collectively, our results showed that design of experiment methodology can be applied as a suitable tool for improved production of UOX using lactose as the inducer.

Optimization of Culture Conditions of Immobilized Cells for Enzyme Excretion and Cell Lysis

“…This may be due to the decrease in the rate of expression at 25 °C, which assists in the correct folding of the CGTase aggregation; hence, high CGTase excretion was observed. However, Najjari et al 15 found that 37 °C produced a high level of recombinant urate oxidase when using free cells ( E. coli ). It is suggested that a low temperature is suitable for the immobilized cell system to create high levels of the desired product.…”

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life

“…The unit of UOX activity is defined as the amount of enzyme catalyzing the reaction of 1 μmol uric acid to allantoin and H 2 O 2 per minute at 25 °C. The formation of quinoneimine dye was measured spectrophotometrically at 555 nm …”

“…Cells were harvested by centrifugation at 4 °C and 8000 rpm for 20 min, washed with normal saline (0.9%), and disrupted by sonication on ice bath for 10 min of 15 s on and 10 s off . Supernatants containing native or fusion enzymes were prepared by centrifugation (12 000 rpm for 30 min at 4 °C) and used for further analysis . For purification, a concentrated supernatant of cellular lysate of each recombinant enzyme was loaded onto an affinity column of Ni-NTA (Qiagen, Germany).…”

“… 25 Supernatants containing native or fusion enzymes were prepared by centrifugation (12 000 rpm for 30 min at 4 °C) and used for further analysis. 26 For purification, a concentrated supernatant of cellular lysate of each recombinant enzyme was loaded onto an affinity column of Ni-NTA (Qiagen, Germany). The column was washed with 3-fold volume of 50 mM Tris-HCl buffer (pH 7.0) containing 50 mM imidazole, and target proteins were eluted using a buffer containing 50 mM Tris-HCl, 50 mM NaCl, 10 mM EDTA, 500 mM imidazole, pH 7.0.…”

“…The same amounts of native UOX, PASylated variant, and rasburicase were added to 2 mL of rabbit plasma for 0, 4, 8, 16, 24, 48, 72 h at 37 °C and then assayed for activity as described earlier. 26 …”