Fructosyl peptide oxidase (FPOX, EC 1.5.3) belongs to the family of oxidoreductases, which is used as a diagnostic enzyme for diabetes mellitus. FPOX has activities toward Fru-ValHis and Fru-Lys as model compounds for hemoglobin A1c (HbA1c) and glycated albumin, respectively. However, when the concentration of HbA1c is measured, the activity toward Fru-Lys will cause interference. In this study, we focused on the substrate specificity engineering of FPOX from Eupenicillium terrenum through computational and experimental methods with characteristics more suitable for HbA1c measurement in the blood. Based on structural knowledge of E. terrenum FPOX (PDB ID 4RSL) and molecular modeling results, residues His-377, Arg-62, Lys-380, and Tyr-261 were selected as mutagenesis sites. The best mutant with lower binding energy, stronger hydrophobic interactions, and more hydrogen bonds with Fru-ValHis and higher binding energy toward Fru-Lys was selected for experimental studies. To investigate the conformational changes in FPOX due to the mutation, molecular dynamics simulation was also performed. The genes encoding of native and engineered variants were cloned into pET-22b(+) and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant enzymes were purified and their kinetic properties were studied. Substitution of Tyr261 with Trp resulted in a mutant enzyme with improved specificity for Fru-ValHis, a model compound of HbA1c. The specific activity of mutant FPOX increased by 5.1-fold to 145.2 ± 3.2 U/mg for Fru-ValHis and decreased by 13.7-fold to 1.3 U/mg ± 0.9 for Fru-Lys compared to the native variant. Kinetics analysis indicated that Tyr261Trp FPOX mutant had 11.7-fold increase in K cat /K m for Fru-ValHis compared to the wild-type enzyme, while the K cat /K m for Fru-Lys diminished by 22.4-fold. In summary, our computational and experimental results suggested that the engineered FPOX is a good candidate to efficient determination of HbA1c.

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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.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.

Tumor lysis syndrome (TLS) is a chemotherapyrelated side effect that destroys normal homeostatic mechanisms. TLS usually occurs during chemotherapy that results in hyperuricemia, hyperphosphatemia, and hypocalcemia (Hummel et al., 2005;Garay et al., 2012). On the other hand, managing hyperuricemia is crucial to controlling TLS. The most common method of hyperuricemia management to prevent acute renal failure is the use of allopurinol. The limitations of allopurinol are slow onset of action, and insufficient efficacy in high-risk patients (Cairo and Bishop, 2004). Conversion of uric acid to allantoin, a more water-soluble compound, by urate oxidase (UOX, oxygen oxidoreductase, E.C.1.7.3.3) is an alternative approach to inhibit uric acid formation (Keilin, 1959). UOX has been shown to be a more effective drug than allopurinol for the prophylaxis and treatment of TLS. This enzyme exists in many mammalians but it is inactive in humans owing to nonsense mutations in the