It was shown that any excess of substrates improves transglycosylation. Trials were conducted to obtain 5-fluoro-2′-deoxyuridine with an excess of 5-fluorouracil, an excess of thymidine, or equal-molar quantities. Conversion was 38% in 1 h when 1 : 1 molar ratio was evaluated. Using 4 : 1 molar ratio (base / nucleoside), 5-fluoro-2′-deoxyuridine production was 52% after 1 h. When an excess of thymidine (1 : 4) was used, conversion was 80% (1 h) and productivity was 0.64-fold with respect to the reaction with modified base excess (Table 2).

According to the conversions obtained for 5-fluoro-2′-deoxyuridine biosynthesis, the specificity of A. salmonicida ATCC 27013 to accept other halogenated pyrimidine bases was evaluated. Conversion was approximately 60% (3 h) in 5-chloro-2′-deoxyuridine biosynthesis using 2′-deoxyuridine, Proteasome inhibitor 2′-deoxycytidine, and thymidine as sugar donors (Table 3). Under the conditions tested, A. salmonicida ATCC 27013 accepted 5-chlorouracil but retained only SB203580 supplier residual activity (< 10%) when 5-bromouracil was used. Productivity of A. salmonicida was lower when 5-chlorouracil instead of 5-fluorouracil was assayed (5.4 and 8.2 mg L−1 min−1, respectively).

Therefore, it can be postulated that steric hindrance because of the difference in atomic radii of halogens can probably reduce reaction conversion. Aeromonas salmonicida ATCC 27013 was immobilized in agar, agarose, and polyacrylamide as previously optimized by Trelles and col. (Trelles et al., 2004). The minimum matrix percentage for

preventing undesirable microorganism release into the reaction medium was assessed, being 3% and 25% the optimal percentage for agarose and polyacrylamide, respectively. Immobilized microorganisms Rolziracetam were assayed in floxuridine biosynthesis. Conversion values within 1 h of reaction were slightly lower than those obtained with free microorganisms (60% and 65% using polyacrylamide and agarose, respectively). It is well known that this difference is related to diffusion restrictions of these matrices. Immobilization increases the biocatalyst stability. In this case, A. salmonicida ATCC 27013 was stable at 4 °C for more than 4 months without losing activity (about 90% retained activity). Besides, this immobilized biocatalyst could be used at least for 30 consecutive reactions (about 90% retained activity). Free microorganisms were stable at 4 °C for only 1 week and could not be reused for more than 10 times. Agarose was selected to perform the preliminary test for bioprocess scale-up. These trials were conducted in a 10 mL batch reactor and results were similar to those obtained at microscale (1 mL). In this report, an efficient one-pot bioprocess is described for the production of 5-fluoro- and 5-chloro-2′-deoxyuridine by transglycosylation using immobilized A. salmonicida 27013 as biocatalysts.