No abstract

Nucleoside phosphorylases are valuable tools to produce modified nucleosides with therapeutic or diagnostic potential with high affinity and specificity. A wide variety of nucleoside phosphorylases are available in nature which differ in their protein sequence and show varying substrate spectra. To overcome limitations of the naturally occurring enzymes site-directed mutagenesis approaches can be used.

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.

The major barrier which restricts the enzymatic industrial application is their insufficient stability during processing. In this study, a native strain Aspergillus niger M-8, identified and deposited in GenBank under accession number MW940924, showed high chitinase production via submerged fermentation. The optimized operational conditions increased the enzyme production about 13 fold (up to 572.0 U/mg) using medium containing 0.1% untreated chitin, 0.1% starch and 0.3% tryptone at 35°C and pH 5. To increase the enzyme purification efficiency and stability, two co-solvent protein stabilizers (tryptone and yeast extract) were applied in the production medium and fractionation buffers. The results revealed that medium constituents, stabilizer type and pH greatly affected the yield and stability. Tryptone-optimized medium potentiated the enzyme specific activity (8885.0 U/mg), purification fold (16.3) and recovery (10.2%) more than the same medium supplemented with yeast extract (0.4%), under the same fractionation conditions. Furthermore, the fractionation buffer (acetate buffer, pH 5) containing 2 mM tryptone as stabilizer resulted in double the recovery (18.8%) in comparison to the yeast-containing buffer. The purified chitinase fraction using 40% (v/v) ethanol (F40) reached its optimum catalytic activity at a temperature of 30°C and pH 5. Yet, it was also stable and retained most of its initial activity at a wide range of temperature (25-70°C) and pH values (4-7). Interestingly, the more stabilized F40 chitinase had more virulence effect against mosquito larvae (LC95= 222.9 ppm) after 48 h, suggesting that it can be applied for biological control of Culex pipiens.